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Quantitative proteomics using stable isotope labeling with amino acids in cell culture
Abstract
Stable isotope labeling with amino acids in cell culture (SILAC) is a simple in vivo labeling strategy for mass spectrometry-based quantitative proteomics. It relies on the metabolic incorporation of nonradioactive heavy isotopic forms of amino acids into cellular proteins, which can be readily distinguished in a mass spectrometer. As the samples are mixed before processing in the SILAC methodology, the sample handling errors are also minimized. Here we present protocols for using SILAC in the following types of experiments: (i) studying inducible protein complexes, (ii) identification of Tyr kinase substrates, (iii) differential membrane proteomics and (iv) studying temporal dynamics using SILAC 5-plexing. Although the overall time is largely dependent on the rate of cell growth and various sample processing steps employed, a typical SILAC experiment from start to finish, including data analysis, should take anywhere between 20 and 25 d.
... The major difference between this approach and others is that the labeling of the proteins is performed metabolically, and also the mixing of the samples to be compared is performed in the first steps of sample preparation leading to less variability in the results (Figure 3) [30]. Other advantages of the use of SILAC is its ease of use and implementation and also the possibility of multiplexing (up to 5 samples per experiment) [9,30]. ...
... The major difference between this approach and others is that the labeling of the proteins is performed metabolically, and also the mixing of the samples to be compared is performed in the first steps of sample preparation leading to less variability in the results (Figure 3) [30]. Other advantages of the use of SILAC is its ease of use and implementation and also the possibility of multiplexing (up to 5 samples per experiment) [9,30]. ...
... While it was proposed initially that dialyzed serum should be used to avoid the presence of non-labeled amino acids, this fact posed as a challenge for some cell culture types. In contrast, many studies have already been performed with regular serum, proving that this extra caution may not be necessary [30]. ...
... [17,24] In this report, we have devised biosynthesis of isotopically labeled clickable glutathione by feeding heavy and light derivatives of azido-Ala to cells, which enablet oq uantify glutathionylated peptidesw ith isotopicm ass difference. While SILAC [34] or isobaric tagging [35] could be used instead of isotopic clickable glutathione, our approach may hold several advantages, such as eliminating an eed to extensively label aw hole proteome as in SILAC [34] or introducing an isotopic-labeling step at the beginningo fe xperiment, as opposed to isobaric tagging. [35] We have demonstrated the use of light and heavy derivatives of azido-Ala for proteomic identification and quantification of glutathionylated peptides upon addition of H 2 O 2 , especially estimating R H/L values of 797 glutathionylated cysteines. ...
... [17,24] In this report, we have devised biosynthesis of isotopically labeled clickable glutathione by feeding heavy and light derivatives of azido-Ala to cells, which enablet oq uantify glutathionylated peptidesw ith isotopicm ass difference. While SILAC [34] or isobaric tagging [35] could be used instead of isotopic clickable glutathione, our approach may hold several advantages, such as eliminating an eed to extensively label aw hole proteome as in SILAC [34] or introducing an isotopic-labeling step at the beginningo fe xperiment, as opposed to isobaric tagging. [35] We have demonstrated the use of light and heavy derivatives of azido-Ala for proteomic identification and quantification of glutathionylated peptides upon addition of H 2 O 2 , especially estimating R H/L values of 797 glutathionylated cysteines. ...
Protein S‐glutathionylation is one of the important cysteine oxidation events that regulate various redox‐mediated biological processes. Despite several existing methods, there are few proteomic approaches to identify and quantify specific cysteine residues susceptible to S‐glutathionylation. We previously developed a clickable glutathione approach that labels intracellular glutathione with azido‐Ala by using a mutant form of glutathione synthetase. In this study, we developed a quantification strategy with clickable glutathione by using isotopically labeled heavy and light derivatives of azido‐Ala, which provides the relative quantification of glutathionylated peptides in mass spectrometry‐based proteomic analysis. We applied isotopically labeled clickable glutathione to HL‐1 cardiomyocytes, quantifying relative levels of 1398 glutathionylated peptides upon addition of hydrogen peroxide. Importantly, we highlight elevated levels of glutathionylation on sarcomere‐associated muscle proteins while validating glutathionylation of two structural proteins, α‐actinin and desmin. Our report provides a chemical proteomic strategy to quantify specific glutathionylated cysteines.
... A mass spectrometry-based quantitative proteomic method, stable isotope labeling by amino acids in cell culture (SILAC), is an approach that utilizes amino acids with substituted stable isotopic nuclei to metabolically label whole cellular proteomes during cell culture [11][12][13]. Using this strategy, we can label the entire proteomes of different cells with stable isotope-containing amino acids, culture the cells together and still be able to distinguish the specific cellular source of proteins. SILAC labeling strategy has been used to identify bidirectional cell-specific signaling events initiated between HEK293 cells either expressing the Eph receptor, EPHB2 or its transmembrane ephrin ligand, ephrin-B1 [14]. ...
... Only non-transduced MDA-MB-231 and 82T cells were used for SILAC labeling and phosphoproteomics analysis. Three-state stable isotopic labeling by amino acids in cell culture (SILAC) of MDA-MB-231 and 82T was performed as described earlier [12,20]. Briefly, to facilitate the incorporation of medium and heavy labels for MDA-MB-231 cells, MDA-MB-231 were cultured in DMEM-F12 SILAC labeling media supplemented with medium ( 2 H 4 -Lys and 13 C 6 -Arg) or heavy amino acids ( 13 C 6 15 N 2 -Lys and 13 C 6 15 N 4 -Arg). ...
Background
Cancer-associated fibroblasts (CAFs) are one of the most important components of tumor stroma and play a key role in modulating tumor growth. However, a mechanistic understanding of how CAFs communicate with tumor cells to promote their proliferation and invasion is far from complete. A major reason for this is that most current techniques and model systems do not capture the complexity of signal transduction that occurs between CAFs and tumor cells. Methods
In this study, we employed a stable isotope labeling with amino acids in cell culture (SILAC) strategy to label invasive breast cancer cells, MDA-MB-231, and breast cancer patient-derived CAF this has already been defined above cells. We used an antibody-based phosphotyrosine peptide enrichment method coupled to LC–MS/MS to catalog and quantify tyrosine phosphorylation-mediated signal transduction events induced by the bidirectional communication between patient-derived CAFs and tumor cells. ResultsWe discovered that distinct signaling events were activated in CAFs and in tumor epithelial cells during the crosstalk between these two cell types. We identified reciprocal activation of a number of receptor tyrosine kinases including EGFR, FGFR1 and EPHA2 induced by this bidirectional communication. Conclusions
Our study not only provides insights into the mechanisms of the interaction between CAFs and tumor cells, but the model system described here could be used as a prototype for analysis of intercellular communication in many different tumor microenvironments.
... The extracted protein pellet was dried and resuspended in 50 mM triethylammonium bicarbonate buffer and quantified using a bicinchoninic acid assay. The methods used for protein reduction, alkalyzation 34 , trypsin digestion 35 , purification and estimation are described in SI. Reverse phase fractionation for peptides was carried out using an in-house prepared stage tip column-based protocol (See SI). ...
Several bacteria of environmental and clinical origins, including some human-associated strains secrete a cross-kingdom signaling molecule indole-3-acetic acid (IAA). IAA is a tryptophan (trp) derivative mainly known for regulating plant growth and development as a hormone. However, the nutritional sources that boost IAA secretion in bacteria and the impact of secreted IAA on non-plant eukaryotic hosts remained less explored. Here, we demonstrate significant trp-dependent IAA production in Pseudomonas juntendi NEEL19 when provided with ethanol as a carbon source in liquid cultures. IAA was further characterized to modulate the odor discrimination, motility and survivability in Drosophila melanogaster. A detailed analysis of IAA-fed fly brain proteome using high-resolution mass spectrometry showed significant (fold change, ± 2; p ≤ 0.05) alteration in the proteins governing neuromuscular features, audiovisual perception and energy metabolism as compared to IAA-unfed controls. Sex-wise variations in differentially regulated proteins were witnessed despite having similar visible changes in chemo perception and psychomotor responses in IAA-fed flies. This study not only revealed ethanol-specific enhancement in trp-dependent IAA production in P. juntendi, but also showed marked behavioral alterations in flies for which variations in an array of proteins governing odor discrimination, psychomotor responses, and energy metabolism are held responsible. Our study provided novel insights into disruptive attributes of bacterial IAA that can potentially influence the eukaryotic gut-brain axis having broad environmental and clinical implications.
... Only phosphopetides and proteins present and quantified in at least 2 out of 3 repeats were considered as positively identified in a sample (ACKR2_T0, ACKR2_T3, ACKR2_T30, CCR5_T0, CCR5_T3, and CCR5_T30) and used for further analyses. Geometric mean of biological replicate SILAC ratios were used to assess phosphorylation sites and protein relative quantification (Harsha, et al., 2008). Phosphorylation sites and proteins were considered up-or down-regulated if the geometric mean of SILAC ratio of replicates was >1.5 or <0.67, respectively (>50% change in level) (Kim, et al., 2013;Storvold, et al., 2013). ...
ACKR2 is an atypical chemokine receptor which is structurally uncoupled from G proteins and is unable to activate signaling pathways used by conventional chemokine receptors to promote cell migration. Nonetheless, ACKR2 regulates inflammatory and immune responses by shaping chemokine gradients in tissues via scavenging inflammatory chemokines. To investigate the signaling pathways downstream to ACKR2, a quantitative SILAC-based phosphoproteomic analysis coupled with a systems biology approach with network analysis, was carried out on a HEK293 cell model expressing either ACKR2 or its conventional counterpart CCR5. The model was stimulated with the common agonist CCL3L1 for short (3 min) and long (30 min) durations. As expected, many of the identified proteins are known to participate in conventional signal transduction pathways and in the regulation of cytoskeleton dynamics. However, our analyses revealed unique phosphorylation and network signatures, suggesting roles for ACKR2 other than its scavenger activity. In conclusion, the mapping of phosphorylation events at a holistic level indicated that conventional and atypical chemokine receptors differ in signaling properties. This provides an unprecedented level of detail in chemokine receptor signaling and identifying potential targets for the regulation of ACKR2 and CCR5 function.
... SILAC is an in vivo labeling strategy for mass spectrometry-based quantitative proteomics (38,85). HEK293T cells stably expressing either WT 122 or 1(A322D)22 GABAA receptors were labeled with heavy media (SILAC DMEM media (Pierce, #88420) plus 10% ...
Gamma-aminobutyric acid type A (GABAA) receptors are the primary inhibitory neurotransmitter-gated ion channels in the mammalian central nervous system. Maintenance of GABAA receptor protein homeostasis (proteostasis) in cells utilizing its interacting proteins is essential for the function of GABAA receptors. However, how the proteostasis network orchestrates GABAA receptor biogenesis in the endoplasmic reticulum (ER) is not well understood. Here, we employed a proteomics-based approach to systematically identify the interactomes of GABAA receptors. We carried out a quantitative immunoprecipitation-tandem mass spectrometry (IP-MS/MS) analysis utilizing stable isotope labeling by amino acids in cell culture (SILAC). Further, we performed comparative proteomics by using both wild type α1 subunit and a misfolding-prone α1 subunit carrying the A322D variant as the bait proteins. We identified 125 interactors for wild type α1-containing receptors, 105 proteins for α1(A322D)-containing receptors, and 54 overlapping proteins within these two interactomes. Our bioinformatics analysis identified potential GABAA receptor proteostasis network components, including chaperones, folding enzymes, trafficking factors, and degradation factors, and we assembled a model of their potential involvement in the cellular folding, degradation, and trafficking pathways for GABAA receptors. Additionally, we verified endogenous interactions between α1 subunits and selected interactors by using co-immunoprecipitation in mouse brain homogenates. Moreover, we showed that TRIM21, an E3 ubiquitin ligase, positively regulated the degradation of misfolding-prone α1(A322D) subunits selectively. This study paves the way for understanding the molecular mechanisms as well as fine-tuning of GABAA receptor proteostasis to ameliorate related neurological diseases such as epilepsy.
... Affinity-based enrichments, subcellular fractionations, and targeted proteomics were also used in a few focused investigations. Discussion of these techniques was outside the scope of this review; interested readers should consult reviews and protocols broadly available in the scientific literature (e.g., Carrette et al., 2006;Dupree et al., 2020;Gonzalez-Galarza et al., 2012;Hamdan & Righetti, 2003;Harsha et al., 2008;Kulyyassov et al., 2021;Wilson et al., 2015) regarding their principles and implementations. The focus of this review is on MS's specific contributions to advance mammalian and avian retina proteomics by cataloging and quantifying retinal protein expression, determining their PTMs, as well as their applications to study the retinal proteome in the context of biology, health, diseases, and therapy developments. ...
A subfield of neuroproteomics, retina proteomics has experienced a transformative growth since its inception due to methodological advances in enabling chemical, biochemical, and molecular biology techniques. This review focuses on mass spectrometry's contributions to facilitate mammalian and avian retina proteomics to catalog and quantify retinal protein expressions, determine their posttranslational modifications, as well as its applications to study the proteome of the retina in the context of biology, health and diseases, and therapy developments.
... This set of techniques involves quantitation using popular isotopic species, which comprises techniques such as tandem mass tags (TMT), stable isotope labeling by amino acids (SILAC), and isotopically labeled standards. [7][8][9][10][11] This suite of quantitation methods has been developed to the point that robust and reproducible workflows exist, and quantitation using these methods is generally considered to be highly accurate when performed correctly. There are, however, also limitations to the labeling techniques: they are expensive, can suffer from incomplete labeling efficiencies, and are limited in the number of samples that can be analyzed in parallel within a single experiment. ...
RATIONALE
Analyte quantitation by mass spectrometry underpins a diverse range of scientific endeavors. The fast growing field of mass spectrometer development has resulted in several targeted and untargeted acquisition modes suitable for these applications. By characterizing the acquisition methods available on an ion mobility (IM) enabled orthogonal acceleration time-of-flight (oa-ToF) instrument, the optimum modes for analyte semi-quantitation can be deduced.
METHODS
Serial dilutions of commercial metabolite, peptide, or crosslinked peptide analytes were prepared in matrices of human urine or E. coli digest. Each analyte dilution was introduced into an IM separation enabled oa-ToF mass spectrometer by reversed phase liquid chromatography and electrospray ionization. Data were acquired for each sample in duplicate using nine different acquisition modes, including four IM enabled acquisitions modes, available on the mass spectrometer.
RESULTS
Five (metabolite) or seven (peptide/crosslinked peptide) point calibration curves were prepared for analytes across each of the acquisition modes. A non-linear response was observed at high concentrations for some modes, attributed to saturation effects. Two correction methods, one MS1 isotope-correction and one MS2 ion intensity-correction, were applied to address this observation, resulting in an up to two-fold increase in dynamic range. By averaging the semi-quantitative results across analyte classes, two parameters, linear dynamic range (LDR) and lower limit of quantitation (LLOQ), were determined to evaluate each mode.
CONCLUSION
Comparison of the acquisition modes revealed that data independent acquisition and parallel reaction monitoring methods are most robust for semi-quantitation when considering achievable LDR and LLOQ. IM enabled modes exhibited sensitivity increases, but a simultaneous reduction in dynamic range which required correction methods to recover. These findings will assist users in identifying the optimum acquisition mode for their analyte quantitation needs, supporting a diverse range of applications and providing guidance for future acquisition mode developments.
... SILAC is an in vivo labeling strategy for mass spectrometry-based quantitative proteomics (32,68). HEK293T cells stably expressing either WT α1β2γ2 or α1(A322D)β2γ2 GABA A receptors were labeled with heavy media (SILAC DMEM media (Pierce, #88420) plus 10% dialyzed FBS (Sigma, #F0392), 1% Penicillin Streptomycin, 0.01% 13 C 6 L-Lysine-2HCL (Pierce, #89988), 0.01% 13 C 6 L-Arginine-HCL (Pierce, #88210), and 0.002% L-Proline (Pierce, #88430)), whereas HEK293T cells that were transfected with empty vector (EV) plasmids were cultured in normal light media (SILAC DMEM media plus 10% dialyzed FBS, 1% Penicillin Streptomycin, 0.01% L-Lysine-2HCL (Pierce, #88429), 0.01% L-Arginine-HCL (Pierce, #88427) and 0.002% L-Proline) for 14 days to ensure complete labeling. ...
Gamma-aminobutyric acid type A (GABA A ) receptors, the primary inhibitory neurotransmitter-gated ion channels in the mammalian central nervous system, inhibit neuronal firing to preserve balanced neuronal activity. Maintenance of GABA A receptor protein homeostasis (proteostasis) in the cell utilizing its interacting proteins is essential for the function of GABA A receptors. However, how the proteostasis network orchestrates GABA A receptor biogenesis in the endoplasmic reticulum (ER) is not well understood. To address this question systematically, we employed a proteomics-based approach to identify the interactomes of GABA A receptors by carrying out a quantitative immunoprecipitation-tandem mass spectrometry (IP-MS/MS) analysis utilizing stable isotope labeling by amino acids in cell culture (SILAC). To enhance the coverage and reliability of the identified proteins, we performed comparative proteomics by using both wild type α1 subunit and a misfolding-prone α1 subunit carrying the A322D variant as the bait proteins. The wild type α1 interactome contains 125 proteins, the α1(A322D) interactome contains 105 proteins, and 54 proteins overlap within two interactomes. Bioinformatics analysis identified potential GABA A receptor proteostasis network components, including chaperones, folding enzymes, trafficking factors, and degradation factors. Further, their potential involvement is modelled in the cellular folding, degradation and trafficking pathways for GABA A receptors. In addition, we verified endogenous interactions between α1 subunit and their selected interactors by carrying out co-immunoprecipitation assay in mouse brain homogenates. This study paves the way for understanding the molecular mechanisms as well as fine-tuning of GABA A receptor proteostasis to ameliorate related neurological diseases such as epilepsy.
... In-solution digestion of protein lysates were carried out as previously described [22]. Briefly, the protein lysate from each condition (~1 mg) was subjected to reduction and alkylation using 10 mM dithiothreitol (DTT) (I6125, Sigma Aldrich, Bangalore, India) and 20 mM iodoacetamide (IAA) (D9779, Sigma Aldrich, Bangalore, India) respectively. ...
Interleukin-33 (IL-33), a member of the IL-1 superfamily cytokines, is an endogenous danger signal and a nuclear-associated cytokine. It is one of the essential mediators of both innate and adaptive immune responses. Aberrant IL-33 signaling has been demonstrated to play a defensive role against various infectious and inflammatory diseases. Although the signaling responses mediated by IL-33 have been previously reported, the temporal signaling dynamics are yet to be explored. To this end, we applied quantitative temporal phosphoproteomics analysis to elucidate pathways and proteins induced by IL-33 in THP-1 monocytes. Employing a TMT labeling-based quantitation and titanium dioxide (TiO2)-based phosphopeptide enrichment strategy followed by mass spectrometry analysis, we identified and quantified 9448 unique phosphopeptides corresponding to 3392 proteins that showed differential regulation. Of these, 171 protein kinases, 60 phosphatases and 178 transcription factors were regulated at different phases of IL-33 signaling. In addition to the confirmed activation of canonical signaling modules including MAPK, NFκB, PI3K/AKT modules, pathway analysis of the time-dependent phosphorylation dynamics revealed enrichment of several cellular processes, including leukocyte adhesion, response to reactive oxygen species, cell cycle checkpoints, DNA damage and repair pathways. The detailed quantitative phosphoproteomic map of IL-33 signaling will serve as a potentially useful resource to study its function in the context of inflammatory and pathological conditions.
... In the present study, we employed BioID coupled to BioSITe to map the interacting proteins of TNK2 in triple negative breast cancer cells. By using SILAC labeling-based quantitative proteomics strategy (23,24) to differentially label full-length TNK2 and truncated version of TNK2, we were able to more clearly identify real proteins associated with full-length TNK2 from false positive interactors and potential background contaminants. Collectively, our global study on the TNK2 interactome highlight a more specific approach to provide insights in oncogenic TNK2 signaling and identifying potential targets which may be used in combination therapy to apply multiple approaches of inhibiting TNK2 deregulated pathways or any compensatory/refractory mechanisms. ...
... Stable isotope labeling with amino acids in cell culture (SILAC) is a powerful tool to quantify changes in global protein levels under different conditions. 291, 292 We therefore used this approach to identify which proteins are differentially expressed after inhibition of Hsp70 with JG-98 in MCF-7 cells. Parallel cultures were grown in media spiked with either 12 C 6 ("light") or 13 It is important to note that this analysis did not identify either of the "control" proteins (Hsp90 clients Raf-1 and AKT1), likely because of the low expression levels of those proteins. ...
Networks of protein-protein interactions (PPIs) are essential in all aspects of cellular biology. At the nodes of these networks are multi-protein complexes that are often composed of dynamic, exchangeable modules assembled around a central enzyme. In this thesis, I have used the molecular chaperone heat shock protein 70 (Hsp70) as a model to develop ways of creating inhibitors of PPIs that tune the assembly and function of multi-protein complexes. Hsp70 is an ATPase and master regulator of protein homeostasis that interacts with co-chaperones, including nucleotide exchange factors (NEFs) and J-proteins. There is interest in creating chemical inhibitors that selectively interrupt PPIs between Hsp70 and its co-chaperones, as these molecules would be powerful chemical probes for validating Hsp70 as a target in cancer and other diseases. In this dissertation, I first review how advances in chemical screening methodologies, structural and computational biology, and proteomics have paved the way for the discovery of potent PPI inhibitors, even for difficult targets such as Hsp70 complexes. In Chapter 2, I develop a new high throughput screening (HTS) method in which Hsp70 is combined with co-chaperones and the ATPase activity of the combination is measured. I use this method to identify new inhibitors of Hsp70, characterizing their binding sites and molecular mechanism by NMR, mutagenesis and biochemical approaches. Importantly, I found that this HTS method reveals inhibitors of multiple PPIs within the Hsp70 system, including the interactions with NEFs and J proteins. This approach allowed me to find that Hsp70-NEF complexes control the stability of inhibitor of apoptosis (IAP) protein family members. In Chapter 3, I characterize IAPs as new “clients” of the Hsp70 system and explore the physical interaction between these proteins. That work establishes IAPs as the first sensitive, selective biomarkers suitable for use in pre-clinical studies of Hsp70 inhibitors. Finally, I show how inhibiting the Hsp70-NEF interaction has effects throughout the broader PPI network in Chapter 4. Together, these findings not only have important implications for Hsp70 drug discovery, but they also illustrate, more broadly, how small molecules can be used to re-shape multi-protein complexes and propagate changes throughout PPI networks.
... Furthermore, knockdown of Hbo1 in CGR8 ES cells also resulted in a marked reduction of Oct4 and Sox2 proteins ( Figure 3G, right panel) and significantly diminished mES stemness activity ( Figure 3H), consistent with a role of Hbo1 in maintaining pluripotency and self-renewal of embryonic stem cells (65). Thus, these results demonstrate that HBO1 is required for bulk histone acetylation, propionylation, butyrylation and crotonylation in various cell lines including mES cells To more precisely define the role of HBO1 in bulk histone acylations, we carried out SILAC-based proteomic analysis (66) to quantitatively compare the relative levels of histone acylations in control HeLa and HeLa cells with transient knockdown of HBO1 by shHBO1. We used transient knockdown cells for analysis to minimize the potential secondary effect on histone acylations by long-term loss of HBO1. ...
Recent studies demonstrate that histones are subjected to a series of short-chain fatty acid modifications that is known as histone acylations. However, the enzymes responsible for histone acylations in vivo are not well characterized. Here, we report that HBO1 is a versatile histone acyltransferase that catalyzes not only histone acetylation but also propionylation, butyrylation and crotonylation both in vivo and in vitro and does so in a JADE or BRPF family scaffold protein-dependent manner. We show that the minimal HBO1/BRPF2 complex can accommodate acetyl-CoA, propionyl-CoA, butyryl-CoA and crotonyl-CoA. Comparison of CBP and HBO1 reveals that they catalyze histone acylations at overlapping as well as distinct sites, with HBO1 being the key enzyme for H3K14 acylations. Genome-wide chromatin immunoprecipitation assay demonstrates that HBO1 is highly enriched at and contributes to bulk histone acylations on the transcriptional start sites of active transcribed genes. HBO1 promoter intensity highly correlates with the level of promoter histone acylation, but has no significant correlation with level of transcription. We also show that HBO1 is associated with a subset of DNA replication origins. Collectively our study establishes HBO1 as a versatile histone acyltransferase that links histone acylations to promoter acylations and selection of DNA replication origins.
... In the present study, we employed BioID coupled to BioSITe to map the interacting proteins of TNK2 in triple negative breast cancer cells. By using SILAC labeling-based quantitative proteomics strategy (23,24) to differentially label full-length TNK2 and truncated version of TNK2, we were was not certified by peer review) is the author/funder. All rights reserved. ...
Non-receptor tyrosine kinases represent an important class of signaling molecules which are involved in driving diverse cellular pathways. Although, the large majority have been well-studied in terms of their protein-binding partners, the interactomes of some of the key non-receptor tyrosine kinases such as TNK2 (also known as activated Cdc42-associated kinase 1 or ACK1) have not been systematically investigated. Aberrant expression and hyperphosphorylation of TNK2 has been implicated in a number of cancers. However, the exact proteins and cellular events that mediate phenotypic changes downstream of TNK2 are unclear. Biological systems that employ proximity-dependent biotinylation methods, such as BioID, are being increasingly used to map protein-protein interactions as they provide increased sensitivity in discovering interaction partners. In this study, we employed BioID coupled to the biotinylation site identification technology (BioSITe) method that we recently developed to perform molecular mapping of intracellular proteins associated with TNK2. We also employed stable isotope labeling with amino acids in cell culture (SILAC) to quantitatively explore the interactome of TNK2. By performing a controlled comparative analysis between full-length TNK2 and its truncated counterpart, we were not only able to confidently identify site-level biotinylation of previously well-established TNK2 binders and substrates such as NCK1, NCK2, CTTN, STAT3, but also discover several novel TNK2 interacting partners. We validated TNK2 interaction with one of the novel TNK2 interacting protein, clathrin interactor 1 (CLINT1), using immunoblot analysis. Overall, this work reveals the power of the BioSITe method coupled to BioID and highlights several molecules that warrant further exploration to assess their functional significance in TNK2-mediated signaling.
... Importantly, also iTRAQ and TMT can be used for any kind of sample because, with these approaches, the isotope labels are introduced at the peptide level as well. Only with SILAC, the isotopic labels are incorporated during cell culture (Harsha et al., 2008). Thus, this method cannot be applied to human tissues, for instance, and is highly challenging for non-proliferating primary cells. ...
The application of quantitative proteomics provides a new and promising tool for standardized toxicological research. However, choosing a suitable quantitative method still puzzles many researchers because the optimal method needs to be determined.
In this study, we investigated the advantages and limitations of two of the most commonly used global quantitative proteomics methods, namely label-free quantitation (LFQ) and tandem mass tags (TMT). As a case study, we exposed hepatocytes (HepG2) to the environmental contaminant benzo[a]pyrene (BaP) using a concentration of 2 µM. Our results revealed that both methods yield a similar proteome coverage, in which for LFQ a wider range of fold changes was observed but with less significant p-values compared to TMT. We detected 37 and 47 significantly enriched pathways by LFQ and TMT, respectively, with 17 overlapping pathways.
To define the minimally required effort in proteomics as a benchmark, we artificially reduced the LFQ, and TMT data sets stepwise and compared the pathway enrichment. Thereby, we found that fewer proteins are necessary for detecting significant enrichment of pathways in TMT compared to LFQ, which might be explained by the higher reproducibility of the TMT data that was observed.
In summary, we showed that the TMT approach is the preferable one when investigating toxicological questions because it offers a high reproducibility and sufficient proteome coverage in a comparably short time.
... One advantage of the SILAC method is that ± treatment samples are mixed together prior to any processing (Fig 4.4), thus minimizing sample-to-sample variability. Additional SILAC reagents have been generated, allowing multiplexing up to five samples [60,61]. ...
Introduction:
Viruses induce profound changes in the cells they infect. Understanding these perturbations will assist in designing better therapeutics to combat viral infection. System-based proteomic assays now provide unprecedented opportunity to monitor large numbers of cellular proteins.
Areas covered:
This review will describe various quantitative and functional mass spectrometry-based methods, and complementary non-mass spectrometry-based methods, such as aptamer profiling and proximity extension assays, and examples of how each are used to delineate how viruses affect host cells, identify which viral proteins interact with which cellular proteins, and how these change during the course of a viral infection. PubMed was searched multiple times prior to manuscript submissions and revisions, using virus, viral, proteomics; in combination with each keyword. The most recent examples of published works from each search were then analyzed.
Expert opinion:
There has been exponential growth in numbers and types of proteomic analyses in recent years. Continued development of reagents that allow increased multi-plexing and deeper proteomic probing of the cell, at quantitative and functional levels, enhancements that target more important protein modifications, and improved bioinformatics software tools and pathway prediction algorithms will accelerate this growth and usher in a new era of host proteome understanding.
... In addition to label-free approaches, metabolic (stable isotope labeling by amino acids, SILAC) [60,61] and chemical labeling (dimethyl labeling, iTRAQ) [62,63] were also applied for quantitative CRC secretome analysis (Table 2). In the SILAC-based quantitative proteomics strategy, CRC cell lines are grown on heavy-( 13 C6-Lys/ 13 C6-Arg) and light ( 12 C6-Lys/ 12 C6-Arg)-labeled medium under standard conditions until the confluency is reached. ...
Searching for new cancer-related biomarkers is a key priority for the early detection of solid tumors, such as colorectal cancer (CRC), in clinically relevant biological fluids. The cell line and/or tumor tissue secretome represents a valuable resource for discovering novel protein markers secreted by cancer cells. The advantage of a secretome analysis is the reduction of the large dynamic range characterizing human plasma/serum, and the simultaneous enrichment of low abundance cancer-secreted proteins, thereby overcoming the technical limitations underlying the direct search in blood samples. In this review, we provided a comprehensive overview of recent studies on the CRC secretome for biomarker discovery, focusing both on methodological and technical aspects of secretome proteomic approaches and on biomarker-independent validation in CRC patient samples (blood and tissues). Secretome proteomics are mainly based on LC-MS/MS analyses for which secretome samples are either in-gel or in-solution trypsin-digested. Adequate numbers of biological and technical replicates are required to ensure high reproducibility and robustness of the secretome studies. Moreover, another major challenge is the accuracy of proteomic quantitative analysis performed by label-free or labeling methods. The analysis of differentially expressed proteins in the CRC secretome by using bioinformatic tools allowed the identification of potential biomarkers for early CRC detection. In this scenario, this review may help to follow-up the recent secretome studies in order to select promising circulating biomarkers to be validated in larger screenings, thereby contributing toward a complete translation in clinical practice.
... In-solution digestion was carried out as previously described. 23 Briefly, 50 μg of protein was reduced with 10 mM dithiothreitol (DTT) and alkylated using 20 mM iodoacetamide (IAA) followed by acetone precipitation. The proteins were then digested with trypsin (1:20) (TPCKtrypsin, Worthington Biochemical Corporation) at 37°C for 16 h. ...
Bleomycin (BLM)-induced pulmonary fibrosis is characterized by the inflammation in the alveoli and subsequent deposition of extracellular matrix (ECM), myofibroblasts and impaired fibrinolytic system. Here we describe major hematological changes, IL-17A mediated p53-fibrinolytic pathway and the high throughput hits of LC-MS analysis during the progression of pulmonary fibrosis and the therapeutic potential of curcumin against the disease progression. C57BL/6 mice were exposed to BLM, followed by the curcumin intervention after 24 and 48 h. Mice were sacrificed after 7 days to validate the hematology parameters, molecular pathways and proteomics. Various techniques such as western blot, immunofluorescence, RT-PCR, hematoxylin and eosin, Masson’s trichrome staining, and immunohistochemistry were used to validate the proposed theory. LC-MS analysis was done using Q-Orbitrap mass spectrometer. Schrödinger was used to perform the in silico molecular docking studies. BLM exposed mice exhibited the gradual weight loss and altered lung morphology, howevr, it was reversed by the curcumin treatment. The significant changes in the hematology parameters were confirmed the severity of BLM-exposure in the mice and IL-17A mediated p53-fibrinolytic system components expression and alveolar epithelial cells (AECs) apoptosis further confirmed the pathophysiology of pulmonary fibrosis. Differentially expressed proteins were characterized and mapped using proteomics approach. A strong interaction of curcumin is obsereved with p53, uPA, and PAI-I proteins. Key role of IL-17A mediated inflammation in the impairment of the p53-fibrinolytic system, and AECs apoptosis was confirmed during BLM-induced pulmonary fibrosis. Therapeutic efficacy of curcumin exhibited a protective role against the progression of pulmonary fibrosis, which promises the potent therapeutic modality to target IL-17A mediated p53-fibrinolytic system during pulmonary fibrosis.
... We used our previously established and validated cell culture models of DUX4 expression (Jagannathan et al., 2016) to conduct RNA-seq and Stable Isotope Labeling with Amino acids in Cell culture (SILAC) coupled with quantitative mass spectrometry (Harsha et al., 2008). The resulting data enabled us to measure DUX4-induced alterations in the transcriptome as well as protein levels for~4000 genes with high confidence. ...
DUX4 is a transcription factor whose misexpression in skeletal muscle causes facioscapulohumeral muscular dystrophy (FSHD). DUX4’s transcriptional activity has been extensively characterized, but the DUX4-induced proteome remains undescribed. Here, we report concurrent measurement of RNA and protein levels in DUX4-expressing cells via RNA-seq and quantitative mass spectrometry. DUX4 transcriptional targets were robustly translated, confirming the likely clinical relevance of proposed FSHD biomarkers. However, a multitude of mRNAs and proteins exhibited discordant expression changes upon DUX4 expression. Our dataset revealed unexpected proteomic, but not transcriptomic, dysregulation of diverse molecular pathways, including Golgi apparatus fragmentation, as well as extensive post-transcriptional buffering of stress-response genes. Key components of RNA degradation machineries, including UPF1, UPF3B, and XRN1, exhibited suppressed protein, but not mRNA, levels, explaining the build-up of aberrant RNAs that characterizes DUX4-expressing cells. Our results provide a resource for the FSHD community and illustrate the importance of post-transcriptional processes in DUX4-induced pathology.
... We used our previously established and validated cell culture models of DUX4 expression (Jagannathan et al., 2016) to conduct RNA-seq and Stable Isotope Labeling with Amino acids in Cell culture (SILAC) coupled with quantitative mass spectrometry (Harsha et al., 2008). The resulting data enabled us to measure DUX4-induced alterations in the transcriptome as well as protein levels for~4000 genes with high confidence. ...
DUX4 is a transcription factor whose misexpression in skeletal muscle causes facioscapulohumeral muscular dystrophy (FSHD). DUX4’s transcriptional activity has been extensively characterized, but the DUX4-induced proteome remains undescribed. Here, we report concurrent measurement of RNA and protein levels in DUX4-expressing cells via RNA-seq and quantitative mass spectrometry. DUX4 transcriptional targets were robustly translated, confirming the likely clinical relevance of proposed FSHD biomarkers. However, a multitude of mRNAs and proteins exhibited discordant expression changes upon DUX4 expression. Our dataset revealed unexpected proteomic, but not transcriptomic, dysregulation of diverse molecular pathways, including Golgi apparatus fragmentation, as well as extensive post-transcriptional buffering of stress-response genes. Key components of RNA degradation machineries, including UPF1, UPF3B, and XRN1, exhibited suppressed protein, but not mRNA, levels, explaining the build-up of aberrant RNAs that characterizes DUX4-expressing cells. Our results provide a resource for the FSHD community and illustrate the importance of post-transcriptional processes in DUX4-induced pathology.
... Approximately 250 μg of protein from vehicle treated CM and AICAR treated CM mixed secretome were subjected to in solution digestion as described previously (Harsha et al., 2008). The proteins in solution were reduced with 5 mM dithiothreitol followed by alkylation with 10 mM iodoacetamide. ...
Exercise has profound benefits for brain function in animals and humans. In rodents, voluntary wheel running increases the production of new neurons and upregulates neurotrophin levels in the hippocampus, as well as improving synaptic plasticity, memory function and mood. The underlying cellular mechanisms, however, remain unresolved. Recent research indicates that peripheral organs such as skeletal muscle, liver and adipose tissue secrete factors during physical activity that may influence neuronal function. Here we used an in vitro cell assay and proteomic analysis to investigate the effects of proteins secreted from skeletal muscle cells on adult hippocampal neural progenitor cell (aNPC) differentiation. We also sought to identify the relevant molecules driving these effects. Specifically, we treated rat L6 skeletal muscle cells with the AMP-kinase (AMPK) agonist 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) or vehicle (distilled water). We then collected the conditioned media (CM) and fractionated it using high-performance liquid chromatography (HPLC). Treatment of aNPCs with a specific fraction of the AICAR-CM upregulated expression of doublecortin (DCX) and Tuj1, markers of immature neurons. Proteomic analysis of this fraction identified proteins known to be involved in energy metabolism, cell migration, adhesion and neurogenesis. Culturing differentiating aNPCs in the presence of one of the factors, glycolytic enzyme glucose-6-phosphate isomerase (GPI), or AICAR-CM, increased the proportion of neuronal (Tuj1+) and astrocytic, glial fibrillary acidic protein (GFAP+) cells. Our study provides further evidence that proteins secreted from skeletal muscle cells may serve as a critical communication link to the brain through factors that enhance neural differentiation.
... [10] In addition, the use of labeled CO 2 gas ( 11 CO 2 , 13 CO 2 ,a nd 14 CO 2 )w ould provide an easy method to access carboxylic and amino acids with traceability in in vivo and in vitro systems. [11] Herein, we report the successful direct carboxylation of aromatic aldehydes,b yt aking advantage of an in situ generated umpoled aldehyde under 1atm of aCO 2 atmosphere,asan"artificial" CO 2 fixation process. [12] We commenced our investigation by attempting to form Breslow intermediates of the aldehyde 1 with N-heterocyclic carbenes (NHCs) and thiamine derivatives,t op romote an ucleophilic attack onto the CO 2 . ...
Carbon dioxide is an intrinsically stable molecule. Therefore, its activation requires extra energy input in the form of reactive reagents and/or activated catalysts and, often, harsh reaction conditions. We report here a direct carboxylation reaction of aromatic aldehydes with carbon dioxide, to afford α‐keto acids as added‐value products. In situ generation of a reactive cyanohydrin was key to the successful carboxylation reaction under operationally mild reaction conditions (25‐40 oC, 1 atm CO2). The resulting α‐keto acids served as a platform for alpha‐amino acid synthesis via reductive amination reactions, illustrating the chemical synthesis of essential bioactive molecules from carbon dioxide.
... A lot of mass spectrometry (MS)-based quantitative proteomic methods are commonly used today. Among them, stable isotope labeling with amino acids in cell culture (SILAC) is a simple and accurate method for relative quantitation of protein abundance [33][34][35]. ...
Hypoxia is associated with poor prognosis in most solid tumors due to its multiple effects on therapy resistance, angiogenesis, apoptotic resistance, and tumor invasion/metastasis. Here we used a comprehensive omics profiling to investigate hypoxia-regulated gene expression in HCT116 colon cancer cells. Quantitative analyses of proteome and secretome were performed in HCT116 cells cultured under hypoxic or normoxic conditions. A total of 5700 proteins were quantified in proteome analysis and 722 proteins were quantified in secretome analysis. Both datasets were combined with the transcriptome and translatome datasets for further analysis. Verification of candidate proteins/genes in this integrated omics analysis was performed using immunoblotting and quantitative real-time RT-PCR analyses. We also performed polysome profiling to assess changes in translational efficiency of hypoxia-induced genes. Notably, several genes were differently regulated at the transcriptional and translational levels in HCT116 cells during hypoxia. Bioinformatics analysis suggested that hypoxia regulates translation of genes involved in extracellular matrix organization, extracellular exosomes, and protein processing in endoplasmic reticulum. Aberrations in these metabolic pathways appear to be correlated with an increased risk of tumor invasion/metastasis.
Biological significance
This study integrates transcriptome/translatome and proteome/secretome to analyze gene expression changes in human colon cancer cells under hypoxic conditions. Candidate proteins/genes in this integrated omics analysis were further validated by immunoblotting, quantitative real-time RT-PCR, and polysome profiling. The datasets would be useful to uncover the molecular mechanisms of hypoxia-induced gene regulation in colorectal cancer.
... The cells treated with CSC were called H358-Smoke. Stable isotope labeling with amino acids in cell culture (SILAC) was employed to metabolically label the H358-Parental cells [28]. Stable Isotope Labeling with Amino acids in Cell culture (SILAC) is a technique of quantitative proteomics using mass spectrometry wherein proteins are labelled via metabolic incorporation of isotopic amino acid [29]. ...
Background:
Dysregulation of miRNAs is associated with the development of non-small cell lung cancer (NSCLC). It is imperative to study the dysregulation of miRNAs by cigarette smoke which will affect their targets, either leading to the overexpression of oncoproteins or downregulation of tumor suppressor proteins.
Objective and methods:
In this study, we carried out miRNA sequencing and SILAC-based proteomic analysis of H358 cells chronically exposed to cigarette smoke condensate. Using bioinformatics analysis, we mapped the dysregulated miRNAs to differentially expressed target proteins identified in our data. Gene ontology-based enrichment and pathway analysis was performed using the deregulated targets to study the role of cigarette smoke-mediated miRNA dysregulation in NSCLC cell line.
Results:
miRNA sequencing resulted in the identification of 208 miRNAs, of which 6 miRNAs were found to be significantly dysregulated (2 fold, Log Base 2; p-value ≤ 0.05) in H358-Smoke cells. Proteomic analysis of the smoke exposed cells compared to the untreated parental cells resulted in the quantification of 2,610 proteins, of which 690 proteins were found to be differentially expressed (fold change ≥ 2). Gene ontology based analysis of target proteins revealed enrichment of proteins driving metabolism and a decrease in expression of proteins associated with immune response in the cells exposed to cigarette smoke. Pathway study using Ingenuity Pathway Analysis (IPA) revealed activation of NRF2-mediated oxidative stress response and actin-cytoskeleton signaling, and repression of protein kinase A signaling in H358-Smoke cells. We also identified 5 novel miRNAs in H358-Smoke cells using unassigned reads of small RNA-Seq dataset.
Conclusion:
In summary, this study indicates that chronic exposure to cigarette smoke leads to widespread dysregulation of miRNAs and their targets, resulting in signaling aberrations in NSCLC cell line. The miRNAs and their targets identified in the study need to be further investigated to explore their role as potential therapeutic targets and/or molecular markers in NSCLC especially in smokers.
... Cells were maintained at 37°C in a humidified atmosphere with 5% CO 2 for some cell doublings and then transferred in SILAC medium. 8505C cell line was adapted to the SILAC media as described earlier [40]. SILAC labeling and culturing was performed using PIERCE SILAC Protein Quantitation KIT (Thermo Scientific, MA, USA) according to manufacturer' instructions. ...
BAG3 protein is an apoptosis inhibitor and is highly expressed in Anaplastic Thyroid Cancer. We investigated the entire set of proteins modulated by BAG3 silencing in the human anaplastic thyroid 8505C cancer cells by using the Stable- Isotope Labeling by Amino acids in Cell culture strategy combined with mass spectrometry analysis. By this approach we identified 37 up-regulated and 54 downregulated proteins in BAG3-silenced cells. Many of these proteins are reportedly involved in tumor progression, invasiveness and resistance to therapies. We focused our attention on an oncogenic protein, CAV1, and a tumor suppressor protein, SERPINB2, that had not previously been reported to be modulated by BAG3. Their expression levels in BAG3-silenced cells were confirmed by qRT-PCR and western blot analyses, disclosing two novel targets of BAG3 pro-tumor activity. We also examined the dataset of proteins obtained by the quantitative proteomics analysis using two tools, Downstream Effect Analysis and Upstream Regulator Analysis of the Ingenuity Pathways Analysis software. Our analyses confirm the association of the proteome profile observed in BAG3-silenced cells with an increase in cell survival and a decrease in cell proliferation and invasion, and highlight the possible involvement of four tumor suppressor miRNAs and TP53/63 proteins in BAG3 activity.
... enzyme substrates, membrane proteins, and temporal dynamics [55][56][57]. Pinto et al. demonstrated the utility of SILAC for exploring sheddase activity in cultured cells to identify two novel substrates for snake venom metalloproteinases [58,59]. A limitation of using SILAC is that the cells must be 270 cultured in protein-free medium to allow analysis of secreted proteins in the supernatant; thus, using this approach with post-mitotic cells such as cardiomyocytes is limiting. ...
Introduction: The cardiac extracellular matrix (ECM) provides anatomical, biochemical, and physiological support to the left ventricle. ECM proteins are difficult to detect using unbiased proteomic approaches due to solubility issues and a relatively low abundance compared to cytoplasmic and mitochondrial proteins present in highly prevalent cardiomyocytes.
Areas covered: Proteomic capabilities have dramatically improved over the past 20 years, due to enhanced sample preparation protocols and increased capabilities in mass spectrometry (MS), database searching, and bioinformatics analysis. This review summarizes technological advancements made in proteomic applications that make ECM proteomics highly feasible.
Expert commentary: Proteomic analysis of the ECM provides an important contribution to our understanding of the molecular and cellular processes associated with cardiovascular disease. Using results generated from proteomics approaches in basic science applications and integrating proteomics templates into clinical research protocols will aid in efforts to personalize medicine.
... While stable-isotope label technology helps to resolve the quantification problems [33][34][35] , those label strategies would increase the complexity of MS spectra. In addition, when quantification at the cellular level is required, SILAC (stable isotope labeling with amine acid in cell culture) would need 5 or 6 generation passages before cell detection by MS 36 . Compared to label strategies, label free methods recently developed are mainly depending on statistical calculation [37][38][39][40] . ...
This study proposed an easy-to-use method for cell identification and quantitation by ratiometric matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Two pairs of MS peaks in the molecular fingerprint of cells were selected as intracellular dual-biomarkers due to the stability and specificity of their ratio values in different types of hepatocellular cancer (HCC) cell lines. Five types of HCC cells can be thereafter differentiated based on these two pairs of intracellular peptides/proteins. Two types of HCC cells, Huh7 and LM3 were co-cultured as a model to test whether the method is feasible for cell quantitation. The results indicated that the ratiometric peak intensity of the two pair biomarkers exhibits linear relationship with the proportion of Huh7 cells. Furthermore, tumor heterogeneity was simulated by subcutaneously injecting the co-cultured cells into nude mice. The cell type and proportion in the section of grown tumor tissue can be discriminated using the ratiometric MALDI imaging approach. LC-MS/MS detection revealed that one of the biomarker pairs belongs to thymosin family, β4 and β10. The ratiometric MS spectral approach using intracellular dual-biomarkers might become a pervasive strategy for high-throughput cell identification and quantitation, which is vital in tumor heterogeneity study, clinical diagnosis and drug screening.
... Isotopically labeled samples are quantified by calculating the ratios of integrated signal intensities for labeled peptides to the signal intensities of the corresponding unlabeled peptides in a chromatogram by mass spectrometer. SILAC data could be analyzed effectively by several software packages like MaxQuant, Mascot Distiller, Xpress, and ASAPratio Mann, 2006, 2007;Boumediene et al., 2010;Amanchy et al., 2005;Harsha et al., 2008). ...
Being one of the impressing groups of macromolecules, proteins and their application
are crucial to understand the molecular logic of living cells. Being composed of amino
acids despite their similar structure, proteins are surrounded by several other molecules of
different chemical structures creating an overcrowded environment that makes them different
in their functions. Unraveling the importance of protein folding, protein�protein
interaction is critical to understand cellular processes and rational drug design. A major
breakthrough in protein purification technologies has greatly changed the landscape of
protein study leading to a deeper understanding of their role in disease biochemical pathway
and also in drug metabolism. Since the first protein sequencing in 1953 by Frederic
Sanger, proteomics, which is the large-scale study of proteins, became an essential tool in
understanding the protein structures and functions. Furthermore, the application of physicochemical
measures for protein content has lit up comparative and functional biology of
proteins. This chapter is intended to give a rudimentary understanding of the methods
and technologies behind proteins and their application.
Keywords: Proteins; Protein Identification; Protein Structural Analysis; protein purification;
Chromatography; ELISA; Protein Quantitation; Western blotting
... In fact, the expression pattern of miRNA regulation is not necessarily correlated with the integrity of the transcripts [28]. To measure changes in protein abundance, stable isotope labeling of amino acids in cell culture (SILAC) has been performed by utilizing mass spectrometry, wherein nonradioactive isotopes ( 13 C or 15 N) are used to discriminate mass spectrometry results from different miRNA expression profiles [111]. Although only 12 putative miR-1 targets were initially identified because of the limited coverage of detection (only ~500 proteins), the results monitored in miR-1 versus control transfected HeLa cells have been found to correlate with those obtained from the microarray profiling and computational prediction of seed sites [112]. ...
RNA interference (RNAi) has been widely adopted to repress specific gene expression and is easily achieved by designing small interfering RNAs (siRNAs) with perfect sequence complementarity to the intended target mRNAs. Although siRNAs direct Argonaute (Ago), a core component of the RNA-induced silencing complex (RISC), to recognize and silence target mRNAs, they also inevitably function as microRNAs (miRNAs) and suppress hundreds of off-targets. Such miRNA-like off-target repression is potentially detrimental, resulting in unwanted toxicity and phenotypes. Despite early recognition of the severity of miRNA-like off-target repression, this effect has often been overlooked because of difficulties in recognizing and avoiding off-targets. However, recent advances in genome-wide methods and knowledge of Ago–miRNA target interactions have set the stage for properly evaluating and controlling miRNA-like off-target repression. Here, we describe the intrinsic problems of miRNA-like off-target effects caused by canonical and noncanonical interactions. We particularly focus on various genome-wide approaches and chemical modifications for the evaluation and prevention of off-target repression to facilitate the use of RNAi with secured specificity.
We had reported the isotopic envelopes in differential IMS (FAIMS) separations depending on the ion structure. However, this new approach to distinguish isomers was constrained by the unit-mass resolution commingling all nominally isobaric isotopologues. Here, we directly couple high-definition FAIMS to ultrahigh-resolution (Orbitrap) MS and employ the resulting platform to explore the FAIMS spectra for isotopic fine structure. The peak shifts therein for isotopologues of halogenated anilines with 15N and 13C (split by 6 mDa) in N2/CO2 buffers dramatically differ, more than for the 13C, 37Cl, or 81Br species apart by 1 or 2 Da. The shifts in FAIMS space upon different elemental isotopic substitutions are orthogonal mutually and to the underlying separations, forming fingerprint multidimensional matrices and 3-D trajectories across gas compositions that redundantly delineate all isomers considered. The interlocking instrumental and methodological upgrades in this work take the structural isotopic shift approach to the next level.
Abnormal protein kinetics could be a cause of several diseases associated with essential life processes. An accurate understanding of protein dynamics and turnover is essential for developing diagnostic or therapeutic tools to monitor these changes. Raman Spectroscopy in combination with stable isotope probes such as carbon‐13, and deuterium has been a breakthrough in the qualitative and quantitative study of various metabolites. In this work, we are reporting the utility of Raman stable isotope probing (Raman‐SIP) for monitoring dynamic changes in the proteome at the community level. We have used 13C‐ labelled glucose as the only carbon source in the medium and verified its incorporation in the microbial biomass in a time‐dependent manner. A visible redshift in the Raman spectral vibrations of major biomolecules such as nucleic acids, phenylalanine, tyrosine, amide I and amide III were observed. Temporal changes in the intensity of these bands demonstrating the feasibility of protein turnover monitoring were also verified. Kanamycin, a protein synthesis inhibitor was used to assess the feasibility of identifying effects on protein turnover in the cells. Successful application of this work can provide an alternate/adjunct tool for monitoring proteome level changes in an objective and non‐destructive manner.
Tyrosine phosphorylation on proteins is an important posttranslational modification that regulates various processes in cells. Mass spectrometry-based phosphotyrosine profiling can reveal tyrosine kinase signaling activity in cells. Using quantitative proteomics strategies such as stable isotope labeling with amino acids in cell culture (SILAC) allows comparison of tyrosine kinase signaling activity across two to –three different conditions. In this book chapter, we discuss the reagents required and a step-by-step protocol to carry out phosphotyrosine profiling using SILAC.Key wordsSILACTyrosineProteomicsMass spectrometryPhosphorylationKinasesPhosphatasesPhosphopeptides
Background & Aims
There is an unmet need to develop novel, effective medical therapies for cholangiocarcinoma (CCA). The Hippo pathway effector, YAP, is oncogenic in CCA, but has been historically difficult to target therapeutically. Recently, we described a novel role for the Src-family kinase LCK in activating YAP through tyrosine phosphorylation. This led to the hypothesis that LCK is a viable therapeutic target in CCA via regulation of YAP activity.
Methods
A novel tyrosine kinase inhibitor with relative selectivity for LCK, NTRC 0652-0, was pharmacodynamically profiled in vitro and in CCA cells. A panel of eight CCA patient-derived organoids (PDO) were characterized and tested for sensitivity to NTRC 0652-0. Two patient-derived xenograft (PDX) models bearing FGFR2-rearrangements were utilized for in vivo assessment of pharmacokinetics, toxicity, and efficacy.
Results
NTRC 0652-0 demonstrated selectivity for LCK inhibition in vitro and in CCA cells. LCK inhibition with NTRC 0652-0 led to decreased tyrosine phosphorylation, nuclear localization, and co-transcriptional activity of YAP, and resulted in apoptotic cell death in CCA cell lines. A subset of patient-derived organoids tested demonstrated sensitivity to NTRC 0652-0. CCA with FGFR2 fusions were identified as a potentially susceptible and clinically relevant genetic subset. In PDX models of FGFR2 fusion-positive CCA, daily oral treatment with NTRC 0652-0 resulted in stable plasma and tumor drug levels, acceptable toxicity, decreased YAP tyrosine phosphorylation, and significantly decreased tumor growth.
Conclusions
A novel LCK inhibitor, NTRC 0652-0, inhibited YAP signaling and demonstrated preclinical efficacy in CCA cell-lines, and patient derived organoid and xenograft models.
Lay summary
Cholangiocarcinoma is an aggressive form of liver cancer arising in the biliary tract, which cannot be controlled by current chemotherapy or targeted therapy options. This paper describes a new targeted therapy approach, using an oral inhibitor of LCK. The data suggest this approach may be effective in cholangiocarcinoma with YAP dependence or FGFR2 fusions.
Numerous applications in clinical, pharmaceutical, and biotechnological analyses use absorption‐based measurements, as the method is universal and has a wide range of applications. In addition to absorption methods, fluorescence‐based methods are increasingly being used in detection. In clinical diagnostics, fluorescence detection is often used for the rapid and specific diagnosis of diseases. Absorption or fluorescence‐based methods enable samples to be quickly screened for specific parameters. The parameters to be determined must be known, and interference with signals from the matrix or other substances must be ruled out. A major disadvantage of the optical method is that no element and structure‐specific information can be obtained. A method that can provide both element‐specific and structure‐specific information is mass spectrometry, which determines the mass of atoms or molecules.
Bleomycin (BLM) injury is associated with the severity of acute lung injury (ALI) leading to fibrosis, a high‐morbidity, and high‐mortality respiratory disease of unknown etiology. BLM‐induced ALI is marked by the activation of a potent fibrogenic cytokine transcription growth factor beta‐1 (TGFβ‐1), which is considered a critical cytokine in the progression of alveolar injury. Previously, our work demonstrated that a diet‐derived compound curcumin (diferuloylmethane), represents its antioxidative and antifibrotic application in TGF‐β1‐mediated BLM‐induced alveolar basal epithelial cells. However, curcumin‐specific protein targets, as well as its mechanism using mass spectrometry‐based proteomic approach, remain elusive. To elucidate the underlying mechanism, a quantitative proteomics approach and bioinformatics analysis were employed to identify the protein targets of curcumin in BLM or TGF‐β1‐treated cells. With subsequent in vitro experiments, curcumin‐related pathways and cellular processes were predicted and validated. The current study discusses two separate proteomics experiments using BLM and TGF‐β1‐treated cells with the proteomics approach, various unique target proteins were identified, and proteomic analysis revealed that curcumin reversed the expressions of unique proteins like DNA topoisomerase 2‐alpha (TOP2A), kinesin‐like protein (KIF11), centromere protein F (CENPF), and so on BLM or TGF‐β1 injury. For the first time, the current study reveals that curcumin restores TGF‐β1 induced peroxisomes like PEX‐13, PEX‐14, PEX‐19, and ACOX1. This was verified by subsequent in vitro assays. This study generated molecular evidence to deepen our understanding of the therapeutic role of curcumin at the proteomic level and may be useful to identify molecular targets for future drug discovery.
Secretome analysis is crucial to unravel extracellular processes. However, secreted proteins are difficult to detect in mass-spectrometry-based analysis due to contamination of serum proteins deriving from cell culture media and to high glycosylation, which hampers tryptic digestion. Secret3D workflow is an optimized protocol for the global analysis of secretome from in vitro cultured cells. It allows efficient and robust protein identification and quantitation and provides information on putative N-glycosylation sites of the secreted proteins.
For complete details on the use and execution of this protocol, please refer to Matafora et al. (2020).
Interactions between RNA-binding proteins (RBPs) and RNAs are critical to cell biology. However, methods to comprehensively and quantitatively assess these interactions within cells were lacking. RNA interactome capture (RIC) uses in vivo UV crosslinking, oligo(dT) capture, and proteomics to identify RNA-binding proteomes. Recent advances have empowered RIC to quantify RBP responses to biological cues such as metabolic imbalance or virus infection. Enhanced RIC exploits the stronger binding of locked nucleic acid (LNA)-containing oligo(dT) probes to poly(A) tails to maximize RNA capture selectivity and efficiency, profoundly improving signal-to-noise ratios. The subsequent analytical use of SILAC and TMT proteomic approaches, together with high-sensitivity sample preparation and tailored statistical data analysis, substantially improves RIC’s quantitative accuracy and reproducibility. This optimized approach is an extension of the original RIC protocol. It takes 3 d plus 2 weeks for proteomics and data analysis and will enable the study of RBP dynamics under different physiological and pathological conditions. This protocol extension describes an improved method for global profiling of poly(A) RNA-binding proteins (RBPs) and quantitative analysis of RBP dynamics in response to biological and pharmacological cues that uses UV crosslinking, capture with LNA-modified oligo-dT probes, and proteomics.
Biomarkers are playing an increasingly important role in drug discovery and development and can be applied for many purposes, including disease mechanism study, diagnosis, prognosis, staging, and treatment selection. Advances in high-throughput “omics” technologies, including genomics, transcriptomics, proteomics and metabolomics, significantly accelerate the pace of biomarker discovery. Comprehensive molecular profiling using these “omics” technology has become a field of intensive research aiming at identifying biomarkers relevant for improved diagnostics and therapeutics. Although each “omics” technology plays important roles in biomarker research, different “omics” platforms have different strengths and limitations. This chapter aims to give an overview of these “omics” technologies and their current application in the biomarker discovery.
Determining variations in protein abundance and/or posttranslational modification as a function of time or upon induction by a signal in a particular cell type is central to quantitative proteomics. Isobaric labeling methodologies now allow for parallel quantification of proteins at various conditions concurrently or multiplexing in relatively quantitative proteomics workflows. Hence, mapping the protein expression profiles of various developmental stages of Leishmania parasites is possible with high-resolution mass spectrometry. To analyze global changes in protein expression and cellular signaling pathways during Leishmania differentiation and development is possible with a quantitative proteomics approach. The tandem mass tags (TMT) approach provides a chemical labeling method based on the principle of amine reactive tags; the maximum number of conditions that can be multiplexed is 10-plex. We describe herein a detailed method for sample preparation, TMT-labeling, mass spectrometry and data analysis of different developmental stages of Leishmania donovani parasites. This quantitative proteomic approach is useful to study dynamic changes in protein expression levels during L. donovani differentiation, and also allows in-depth analysis of signaling pathways via phosphoproteomics.
Quantitative proteomic platforms based on precursor intensity in mass spectrometry (MS1-level) uniquely support in vivo metabolic labeling with superior quantification accuracy but suffer from limited multiplexity (≤3-plex) and frequent missing quantities. Here we present a new MS1-level quantification platform that allows maximal multiplexing with high quantification accuracy and precision for the given labeling scheme. The platform currently comprises 6-plex in vivo SILAC or in vitro di-ethylation labeling with a dedicated algorithm, and is also expandable to higher multiplexity (e.g., nine-plex for SILAC). For complex samples with broad dynamic ranges such as total cell lysates, our platform performs highly accurately and free of missing quantities. Furthermore, we successfully applied our method to measure protein synthesis rate under heat shock response in human cells by 6-plex pulsed SILAC experiments, demonstrating the unique biological merits of our in vivo platform to disclose translational regulations for cellular response to stress.
Phosphorylation is one of the most extensively studied posttranslational modifications (PTM), which regulates cellular functions like cell growth, differentiation, apoptosis, and cell signaling. Kinase families cover a wide number of oncoproteins and are strongly associated with cancer. Identification of driver kinases is an intense area of cancer research. Thus, kinases serve as the potential target to improve the efficacy of targeted therapies. Mass spectrometry-based phosphoproteomic approach has paved the way to the identification of a large number of altered phosphorylation events in proteins and signaling cascades that may lead to oncogenic processes in a cell. Alterations in signaling pathways result in the activation of oncogenic processes predominantly regulated by kinases and phosphatases. Therefore, drugs such as kinase inhibitors, which target dysregulated pathways, represent a promising area for cancer therapy.
DUX4 is a transcription factor whose misexpression in skeletal muscle causes facioscapulohumeral muscular dystrophy (FSHD). While DUX4's transcriptional activity has been extensively characterized, the DUX4-induced proteome remains undescribed. Here, we report concurrent measurement of RNA and protein levels in DUX4-expressing cells via RNA-seq and quantitative mass spectrometry. DUX4 transcriptional targets were robustly translated, confirming the likely clinical relevance of proposed FSHD biomarkers. However, a multitude of mRNAs and proteins exhibited discordant expression changes upon DUX4 expression. Our dataset revealed unexpected proteomic, but not transcriptomic, dysregulation of diverse molecular pathways, including Golgi apparatus fragmentation, as well as extensive post-transcriptional buffering of stress response genes. Key components of RNA degradation machineries, including UPF1, UPF3B, and XRN1, exhibited suppressed protein, but not mRNA, levels, explaining the build-up of aberrant RNAs that characterizes DUX4-expressing cells. Our results provide a resource for the FSHD community and illustrate the importance of post-transcriptional processes to DUX4-induced pathology.
Carbon dioxide is an intrinsically stable molecule. Therefore, its activation requires extra energy input in the form of reactive reagents and/or activated catalysts and, often, harsh reaction conditions. We report here a direct carboxylation reaction of aromatic aldehydes with carbon dioxide, to afford α‐keto acids as added‐value products. In situ generation of a reactive cyanohydrin was key to the successful carboxylation reaction under operationally mild reaction conditions (25‐40 oC, 1 atm CO2). The resulting α‐keto acids served as a platform for alpha‐amino acid synthesis via reductive amination reactions, illustrating the chemical synthesis of essential bioactive molecules from carbon dioxide.
Proteomics allows the simultaneous detection and identification of thousands of proteins within a sample. Here, we describe a quantitative method to compare protein expression and subcellular localization of different cell lines representative of different stages of colorectal cancer using stable isotope labeling with amino acids in culture, or SILAC. We also describe a biochemical fractionation approach to separate different cellular compartments and the necessary steps to obtain a specific proteomic profile of each cell line. This technique enables a comprehensive proteomic analysis of cancer cell lines and the identification of pathways that are deregulated in different cancer cell lines.
We describe a protocol for multiplexed proteomic analysis using neutron-encoded (NeuCode) stable isotope labeling of amino acids in cells (SILAC) or mice (SILAM). This method currently enables simultaneous comparison of up to nine treatment and control proteomes. Another important advantage over traditional SILAC/SILAM is that shorter labeling times are required. Exploiting the small mass differences that correspond to subtle differences in the neutron-binding energies of different isotopes, the amino acids used in NeuCode SILAC/SILAM differ in mass by just a few milliDaltons. Isotopologs of lysine are introduced into cells or mammals, via the culture medium or diet, respectively, to metabolically label the proteome. Labeling time is ∼2 weeks for cultured cells and 3–4 weeks for mammals. The proteins are then extracted, relevant samples are combined, and these are enzymatically digested with lysyl endopeptidase (Lys-C). The resultant peptides are chromatographically separated and then mass analyzed. During mass spectrometry (MS) data acquisition, high-resolution MS¹ spectra (≥240,000 resolving power at m/z = 400) reveal the embedded isotopic signatures, enabling relative quantification, while tandem mass spectra, collected at lower resolutions, provide peptide identities. Both types of spectra are processed using NeuCode-enabled MaxQuant software. In total, the approximate completion time for the protocol is 3–5 weeks.
We describe an approach for the accurate quantification and concurrent sequence identification of the individual proteins within complex mixtures. The method is based on a class of new chemical reagents termed isotope-coded affinity tags (ICATs) and tandem mass spectrometry. Using this strategy, we compared protein expression in the yeast Saccharomyces cerevisiae, using either ethanol or galactose as a carbon source. The measured differences in protein expression correlated with known yeast metabolic function under glucose-repressed conditions. The method is redundant if multiple cysteinyl residues are present, and the relative quantification is highly accurate because it is based on stable isotope dilution techniques. The ICAT approach should provide a widely applicable means to compare quantitatively global protein expression in cells and tissues.
Quantitative proteomics has traditionally been performed by two-dimensional gel electrophoresis, but recently, mass spectrometric methods based on stable isotope quantitation have shown great promise for the simultaneous and automated identification and quantitation of complex protein mixtures. Here we describe a method, termed SILAC, for stable isotope labeling by amino acids in cell culture, for the in vivo incorporation of specific amino acids into all mammalian proteins. Mammalian cell lines are grown in media lacking a standard essential amino acid but supplemented with a non-radioactive, isotopically labeled form of that amino acid, in this case deuterated leucine (Leu-d3). We find that growth of cells maintained in these media is no different from growth in normal media as evidenced by cell morphology, doubling time, and ability to differentiate. Complete incorporation of Leu-d3 occurred after five doublings in the cell lines and proteins studied. Protein populations from experimental and control samples are mixed directly after harvesting, and mass spectrometric identification is straightforward as every leucine-containing peptide incorporates either all normal leucine or all Leu-d3. We have applied this technique to the relative quantitation of changes in protein expression during the process of muscle cell differentiation. Proteins that were found to be up-regulated during this process include glyceraldehyde-3-phosphate dehydrogenase, fibronectin, and pyruvate kinase M2. SILAC is a simple, inexpensive, and accurate procedure that can be used as a quantitative proteomic approach in any cell culture system.
To study the global dynamics of phosphotyrosine-based signaling events in early growth factor stimulation, we developed a mass spectrometric method that converts temporal changes to differences in peptide isotopic abundance. The proteomes of three cell populations were metabolically encoded with different stable isotopic forms of arginine. Each population was stimulated by epidermal growth factor for a different length of time, and tyrosine-phosphorylated proteins and closely associated binders were affinity purified. Arginine-containing peptides occurred in three forms, which were quantified; we then combined two experiments to generate five-point dynamic profiles. We identified 81 signaling proteins, including virtually all known epidermal growth factor receptor substrates, 31 novel effectors and the time course of their activation upon epidermal growth factor stimulation. Global activation profiles provide an informative perspective on cell signaling and will be crucial to modeling signaling networks in a systems biology approach.
Tyrosine kinases play a prominent role in human cancer, yet the oncogenic signaling pathways driving cell proliferation and survival have been difficult to identify, in part because of the complexity of the pathways and in part because of low cellular levels of tyrosine phosphorylation. In general, global phosphoproteomic approaches reveal small numbers of peptides containing phosphotyrosine. We have developed a strategy that emphasizes the phosphotyrosine component of the phosphoproteome and identifies large numbers of tyrosine phosphorylation sites. Peptides containing phosphotyrosine are isolated directly from protease-digested cellular protein extracts with a phosphotyrosine-specific antibody and are identified by tandem mass spectrometry. Applying this approach to several cell systems, including cancer cell lines, shows it can be used to identify activated protein kinases and their phosphorylated substrates without prior knowledge of the signaling networks that are activated, a first step in profiling normal and oncogenic signaling networks.
Reversible phosphorylation of proteins regulates the majority of all cellular processes, e.g. proliferation, differentiation, and apoptosis. A fundamental understanding of these biological processes at the molecular level requires characterization of the phosphorylated proteins. Phosphorylation is often substoichiometric, and an enrichment procedure of phosphorylated peptides derived from phosphorylated proteins is a necessary prerequisite for the characterization of such peptides by modern mass spectrometric methods. We report a highly selective enrichment procedure for phosphorylated peptides based on TiO2microcolumns and peptide loading in 2,5-dihydroxybenzoic acid (DHB). The effect of DHB was a very efficient reduction in the binding of nonphosphorylated peptides to TiO2 while retaining its high binding affinity for phosphorylated peptides. Thus, inclusion of DHB dramatically increased the selectivity of the enrichment of phosphorylated peptides by TiO2. We demonstrated that this new procedure was more selective for binding phosphorylated peptides than IMAC using MALDI mass spectrometry. In addition, we showed that LC-ESI-MSMS was biased toward monophosphorylated peptides, whereas MALDI MS was not. Other substituted aromatic carboxylic acids were also capable of specifically reducing binding of nonphosphorylated peptides, whereas phosphoric acid reduced binding of both phosphorylated and nonphosphorylated peptides. A putative mechanism for this intriguing effect is presented.
Quantitative proteomics can be used as a screening tool for identification of differentially expressed proteins as potential biomarkers for cancers. Candidate biomarkers from such studies can subsequently be tested using other techniques for use in early detection of cancers. Here we demonstrate the use of stable isotope labeling with amino acids in cell culture (SILAC) method to compare the secreted proteins (secretome) from pancreatic cancer-derived cells with that from non-neoplastic pancreatic ductal cells. We identified 145 differentially secreted proteins (>1.5-fold change), several of which were previously reported as either up-regulated (e.g. cathepsin D, macrophage colony stimulation factor, and fibronectin receptor) or down-regulated (e.g. profilin 1 and IGFBP-7) proteins in pancreatic cancer, confirming the validity of our approach. In addition, we identified several proteins that have not been correlated previously with pancreatic cancer including perlecan (HSPG2), CD9 antigen, fibronectin receptor (integrin beta1), and a novel cytokine designated as predicted osteoblast protein (FAM3C). The differential expression of a subset of these novel proteins was validated by Western blot analysis. In addition, overexpression of several proteins not described previously to be elevated in human pancreatic cancer (CD9, perlecan, SDF4, apoE, and fibronectin receptor) was confirmed by immunohistochemical labeling using pancreatic cancer tissue microarrays suggesting that these could be further pursued as potential biomarkers. Lastly the protein expression data from SILAC were compared with mRNA expression data obtained using gene expression microarrays for the two cell lines (Panc1 and human pancreatic duct epithelial), and a correlation coefficient (r) of 0.28 was obtained, confirming previously reported poor associations between RNA and protein expression studies.
We developed an integrated proteomic approach to decipher in vivo protein-protein interactions and applied this strategy to globally map the 26 S proteasome interaction network in yeast. We termed this approach QTAX for quantitative analysis of tandem affinity purified in vivo cross-linked (X) protein complexes. For this work, in vivo formaldehyde cross-linking was used to freeze both stable and transient interactions occurring in intact cells prior to lysis. To isolate cross-linked protein complexes with high purification efficiency under fully denaturing conditions, a new tandem affinity tag consisting of a hexahistidine sequence and an in vivo biotinylation signal was adopted for affinity-based purification. Tandem affinity purification after in vivo cross-linking was combined with tandem mass spectrometry coupled with a quantitative SILAC (stable isotope labeling of amino acids in cell culture) strategy to carry out unambiguous protein identification and quantification of specific protein interactions. Using this method, we captured and identified the full composition of yeast 26 S proteasome complex as well as the two known ubiquitin receptors, Rad23 and Dsk2. Quantitative mass spectrometry analysis allowed us to distinguish specific proteasome-interacting proteins (PIPs) from background proteins and led to the identification of a total of 64 potential PIPs of which 42 are novel interactions. Among the 64 putative specific PIPs, there are ubiquitin pathway components, ubiquitinated substrates, chaperones, and transcription and translation regulators, demonstrating the efficacy of the developed approach in capturing in vivo protein interactions. The method offers an advanced technical approach to elucidate the dynamic protein interaction networks of the proteasome and can find a wide range of applications in the studies of other macromolecular protein complex interaction networks.
Protein phosphorylation is a complex network of signaling and regulatory events that affects virtually every cellular process. Our understanding of the nature of this network as a whole remains limited, largely because of an array of technical challenges in the isolation and high-throughput sequencing of phosphorylated species. In the present work, we demonstrate that a combination of tandem phosphopeptide enrichment methods, high performance MS, and optimized database search/data filtering strategies is a powerful tool for surveying the phosphoproteome. Using our integrated analytical platform, we report the identification of 5,635 nonredundant phosphorylation sites from 2,328 proteins from mouse liver. From this list of sites, we extracted both novel and known motifs for specific Ser/Thr kinases including a “dipolar” motif. We also found that C-terminal phosphorylation was more frequent than at any other location and that the distribution of potential kinases for these sites was unique. Finally, we identified double phosphorylation motifs that may be involved in ordered phosphorylation.
• mass spectrometry
• proteomics
Detailed characterization of phosphoproteins as well as other post-translationally modified proteins is required to fully understand protein function and regulatory events in cells and organisms. Here we present a mass spectrometry (MS) based experimental strategy for the identification and mapping of phosphorylation site(s) using only low-picomole amounts of phosphoprotein starting material. Miniaturized sample preparation methods for MS facilitated localization of phosphorylation sites in phosphoproteins isolated by polyacrylamide gel electrophoresis. Custom made, nanoscale immobilized Fe(III) affinity chromatography (Fe(III)-IMAC) columns were employed for enrichment of phosphorylated peptides from crude peptide mixtures prior to off-line analysis by matrix-assisted laser desorption/ionization (MALDI) MS or nanoelectrospray tandem mass spectrometry (MS/MS). An optimized and sensitive procedure for alkaline phosphatase treatment of peptide mixtures was implemented, which in combination with nano-scale Fe(III)-IMAC and MALDI-MS allowed unambiguous identification of phosphopeptides by observation of 80 Da mass shifts. Nanoelectrospray MS/MS was used for phosphopeptide sequencing for exact determination of phosphorylation sites. The advantages and limitations of the experimental strategy was demonstrated by enrichment, identification and sequencing of phosphopeptides from the model proteins ovalbumin and bovine β-casein isolated by gel electrophoresis. Furthermore, an autophosphorylation site at Ser-3 in recombinant human casein kinase-2 β subunit was determined. The potential of miniaturized Fe(III)-IMAC and MALDI-MS for characterization of in vivo phosphorylated proteins was demonstrated by identification of tryptic phosphopeptides derived from the human p47/phox phosphoprotein isolated by two-dimensional gel electrophoresis.
We have recently described a method, stable isotope labeling by amino acids in cell culture (SILAC) for the accurate quantitation of relative protein abundances. Cells were metabolically labeled with deuterated leucine, leading to complete incorporation within about five cell doublings. Here, we investigate fully substituted C-13-labeled arginine in the SILAC method. After tryptic digestion, there is a single label at the C-terminal position in half of the peptides. Labeled and unlabeled peptides coelute in liquid chromatography-mass spectrometric analysis, eliminating quantitation error due to unequal sampling of ion profiles. Tandem mass spectrum interpretation and database identification are aided by the predictable shift of the y-ions in the labeled form. The quantitation of mixtures of total cell lysates in known ratios resolved on a one-dimensional SDS-PAGE gel produced consistent and reproducible results with relative standard deviations better than five percent under optimal conditions.
Detailed characterization of phosphoproteins as well as other post-translationally modified proteins is required to fully understand protein function and regulatory events in cells and organisms. Here we present a mass spectrometry (MS) based experimental strategy for the identification and mapping of phosphorylation site(s) using only low-picomole amounts of phosphoprotein starting material. Miniaturized sample preparation methods for MS facilitated localization of phosphorylation sites in phosphoproteins isolated by polyacrylamide gel electrophoresis. Custom made, nanoscale immobilized Fe(III) affinity chromatography (Fe(III)-IMAC) columns were employed for enrichment of phosphorylated peptides from crude peptide mixtures prior to off-line analysis by matrix-assisted laser desorption/ionization (MALDI) MS or nanoelectrospray tandem mass spectrometry (MS/MS). An optimized and sensitive procedure for alkaline phosphatase treatment of peptide mixtures was implemented, which in combination with nano-scale Fe(III)-IMAC and MALDI-MS allowed unambiguous identification of phosphopeptides by observation of 80 Da mass shifts. Nanoelectrospray MS/MS was used for phosphopeptide sequencing for exact determination of phosphorylation sites. The advantages and limitations of the experimental strategy was demonstrated by enrichment, identification and sequencing of phosphopeptides from the model proteins ovalbumin and bovine β-casein isolated by gel electrophoresis. Furthermore, an autophosphorylation site at Ser-3 in recombinant human casein kinase-2 β subunit was determined. The potential of miniaturized Fe(III)-IMAC and MALDI-MS for characterization of in vivo phosphorylated proteins was demonstrated by identification of tryptic phosphopeptides derived from the human p47/phox phosphoprotein isolated by two-dimensional gel electrophoresis.
Phosphoproteins and phosphoamino acids bind to ferric ions immobilized on iminodiacetate-agarose gel and can be eluted by increasing pH or by introducing phosphate ions to the eluant. Some other metals were found to resemble iron with regard to the interaction with protein-bound phosphate and phosphoamino acids. These observations were utilized to develop purification procedures for phosphoproteins. Hen egg albumin (ovalbumin) was fractionated into three components of varying phosphate contents. Porcine pepsin was purified in a similar manner.
Proteins were digested in normal and highly 18O-enriched water using proteases commonly employed for protein sequencing. The extent of 18O incorporation into the resulting peptide fragments was characterized by electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). The endoproteinases trypsin, Lys-C and Glu-C incorporate two atoms of 18O, resulting in a mass shift of +4 D for the peptide fragments. This indicates that, following proteolytic cleavage, peptide products continue to interact with these proteases and undergo repeated binding/hydrolysis cycles, resulting in complete equilibration of both oxygens in the carboxy terminus of the fragments with oxygen from solvent water. In contrast, chymotrypsin and Asp-N incorporate only one atom of 18O, resulting in a mass shift of +2 D, indicating that after the cleavage step these proteases do not accept the peptides as substrates. In addition, it was found that the proteases trypsin, Glu-C, and Lys-C exhibit minor or nontypical sequence specificities, resulting in unexpected peptide fragments. These fragments incorporate only one 18O atom, indicating that they do not undergo further binding/hydrolysis cycles with the enzyme. Thus, it is possible to discriminate between enzyme-typical peptide fragments with mass shifts of +4 D and nontypical fragments with mass shifts of only +2 D. Based on these observations, protein digest strategies are described for the generation of 1:1 ion doublets spaced either by 2 or 4 D. In addition, the C-terminus of a protein can be identified by the absence of an ion doublet in the corresponding peptide fragment. In protein sequencing by mass spectrometry, digest protocols generating ion doublets provide the most clear-cut analytical results for the recognition of ion series in ESI-MS/MS and MALDI post-source decay (PSD) product ion spectra. Only the mass spectrometric fragment ions of a C-terminal series show ion doublets spaced either by 2 or 4 D, whereas the fragment ions belonging to an N-terminal series remain unshifted. This assignment unequivocally reveals the direction of the identified sequence.
This paper focuses on identifying structural features responsible for resolution of heavy isotope coded peptides during reversed-phase chromatography. This was achieved by using labeled coding agents that varied in structure, number of deuterium atoms, placement of deuterium in the coding agent, and the functional group targeted by the reagent. Six coding agents were examined. Deuterated versions of the coding agents studied included succinic anhydride-2H4, acetic acid 2,5-dioxopyrrolidin-1-yl ester-2H3, propionic acid 2,5-dioxopyrrolidin-1-yl ester-2H5, pentanoic acid 2,5-dioxopyrrolidin-1-yl ester-2H9, [3-(2,5-dioxopyrrolidin-1-yloxycarbonyl)-propyl]-trimethylammonium chloride-2H9, and the commercial ICAT-2H8 reagent. It was found that these labeling agents vary widely in both their absolute and relative contribution to the chromatographic isotope effect. Relative effects were evaluated by normalizing resolution for the number of deuterium atoms in the derivatized peptide. The single, most dominant effect was the placement of deuterium atoms relative to hydrophilic functional groups in the coding agent. It was concluded that the probability of a deuterium atom interacting with the stationary phase of a reversed-phase chromatography (RPC) column and impacting resolution is greatly diminished by placing it adjacent to a hydrophilic group, as explained by solvophobic theory. But peptide size and coding agent size were also seen to correlate inversely with the magnitude of the isotope effect. This effect was explained as being due to the relative size of the coding agent versus that of the coding agent-peptide conjugate.
We have recently described a method, stable isotope labeling by amino acids in cell culture (SILAC) for the accurate quantitation of relative protein abundances. Cells were metabolically labeled with deuterated leucine, leading to complete incorporation within about five cell doublings. Here, we investigate fully substituted 13C-labeled arginine in the SILAC method. After tryptic digestion, there is a single label at the C-terminal position in half of the peptides. Labeled and unlabeled peptides coelute in liquid chromatography-mass spectrometric analysis, eliminating quantitation error due to unequal sampling of ion profiles. Tandem mass spectrum interpretation and database identification are aided by the predictable shift of the y-ions in the labeled form. The quantitation of mixtures of total cell lysates in known ratios resolved on a one-dimensional SDS-PAGE gel produced consistent and reproducible results with relative standard deviations better than five percent under optimal conditions.
ClpXP, a bacterial AAA+ protease, controls intracellular levels of many stress-response proteins. To investigate substrate profile changes caused by a specific environmental stress, quantitative mass spectrometry (SILAC) was used to analyze proteins trapped by ClpXP(trap) before and after DNA damage. The abundance of half of the trapped proteins changed more than 3-fold after damage. Overrepresented substrates included the DNA-repair proteins RecN and UvrA. Among SOS-response proteins, 25% were ClpXP substrates and, importantly, nearly all of the highly induced regulon members were rapidly degraded. Other proteins, including the stress regulator sigma(S), were underrepresented in ClpXP(trap) after DNA damage; overproduction experiments suggest that simple substrate competition does not account for this reduced recognition. We conclude that damage-response proteins are an unusually rapidly degraded family and that ClpXP has substantial capacity to process the influx of newly synthesized substrates while maintaining the ability to degrade its other substrates in an environmentally responsive manner.
Cell signaling mechanisms often transmit information via posttranslational protein modifications, most importantly reversible protein phosphorylation. Here we develop and apply a general mass spectrometric technology for identification and quantitation of phosphorylation sites as a function of stimulus, time, and subcellular location. We have detected 6,600 phosphorylation sites on 2,244 proteins and have determined their temporal dynamics after stimulating HeLa cells with epidermal growth factor (EGF) and recorded them in the Phosida database. Fourteen percent of phosphorylation sites are modulated at least 2-fold by EGF, and these were classified by their temporal profiles. Surprisingly, a majority of proteins contain multiple phosphorylation sites showing different kinetics, suggesting that they serve as platforms for integrating signals. In addition to protein kinase cascades, the targets of reversible phosphorylation include ubiquitin ligases, guanine nucleotide exchange factors, and at least 46 different transcriptional regulators. The dynamic phosphoproteome provides a missing link in a global, integrative view of cellular regulation.
Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns p u o r G g n i h s i l b u P e r u t a N 8 0 0 2 © natureprotocols / m o c . e r u t a n . w w w / / : p t t h 516 | VOL
- M R Larsen
- T E Thingholm
- O N Jensen
- P Roepstorff
- T Jorgensen
Larsen, M.R., Thingholm, T.E., Jensen, O.N., Roepstorff, P. & Jorgensen, T. Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns. Mol. Cell Proteomics 4, 873–886 (2005). p u o r G g n i h s i l b u P e r u t a N 8 0 0 2 © natureprotocols / m o c . e r u t a n . w w w / / : p t t h 516 | VOL.3 NO.3 | 2008 | NATURE PROTOCOLS PROTOCOL
Large-scale phosphorylation analysis of mouse liver
- J Villen
- S A Beausoleil
- S A Gerber
- S P Gygi
Villen, J., Beausoleil, S.A., Gerber, S.A. & Gygi, S.P. Large-scale
phosphorylation analysis of mouse liver. Proc. Natl. Acad. Sci. USA 104,
1488–1493 (2007).
Isolation of phosphoproteins by immobilized
- L Anderson
- J Porath
Anderson, L. & Porath, J. Isolation of phosphoproteins by immobilized. Metal
HCl ( 15 N 4, D7) 88.2 mg l À1 (0.398 mM) 254
- Arg
Arg: HCl ( 15 N 4, D7)
88.2 mg l À1 (0.398 mM)
254.8 mg l À1 (1.15 mM)
11
Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents
- P L Ross
- PL Ross
Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns
- M R Larsen
- T E Thingholm
- O N Jensen
- P Roepstorff
- T Jorgensen
- MR Larsen