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Suppression effects in enzymatic peptide ladder sequencing using ultraviolet - Matrix assisted laser desorption/ionization - Mass spectrometry
Abstract
The techniques of enzymatic and chemical peptide ladder sequencing, coupled with ultraviolet - matrix assisted laser desorption/ionization - mass spectrometry (UV-MALDI-MS) have been improving continuously in the last five years and have now become important tools for primary structure identification. In this work, signal suppression effects, appearing in UV-MALDI-MS (excitation 337 nm) of ladder peptides, were investigated using the 17-amino acid peptide dynorphin A. We show, with examples of simple "two-peptide" systems and more complex "multi-peptide" systems, that suppression effects do in fact exist. The magnitude of the observed suppression is strongly dependent upon both the nature and the amount of the suppressing peptide. Suppression behavior of individual ladder peptides was investigated on equimolar mixtures of ten ladder peptides. Significant signal suppression was recorded for all ladder peptides, with some of them being approximately 170 times lower in signal intensity than the pure, i.e., unsuppressed peptide at the same concentration. For the investigated system--dynorphin A, 4-hydroxy-alpha-cyanocinnamic acid (4-HCCA) matrix, UV excitation--a correlation between the extent of suppression and an intractable combination of peptide hydrophobicity and the presence of several basic amino acids can be seen.
... Sample preparation may be accompanied by incomplete trypsinolysis when, for example, the C-terminal part of the protein amino acid sequence contains proline residues connected to arginine or lysine [5], or spatial hindrances to proteolysis occur in the presence of disulfide bridges [2]. Another factor is the weak ionization of peptides under MALDI conditions, which is associated with the relatively low basicity of peptides in the gas phase [6][7][8][9] and their relatively low hydrophobicity [10,11]. The effect of basicity and hydrophobicity/hydrophilicity is manifested through the mechanism of suppressing the ionization of some components in the mixtures with other components [6,10,11]. ...
... Another factor is the weak ionization of peptides under MALDI conditions, which is associated with the relatively low basicity of peptides in the gas phase [6][7][8][9] and their relatively low hydrophobicity [10,11]. The effect of basicity and hydrophobicity/hydrophilicity is manifested through the mechanism of suppressing the ionization of some components in the mixtures with other components [6,10,11]. ...
... Note that, in publications, conclusions regarding the dominance of the hydrophobicity/hydrophilicity of peptides under the conditions of MALDI with the matrix under consideration are not unambiguous. The conclusion was opposite in [10,11]; we observe partial compliance with the study [21], in which a mixture of synaptic acid and CHCA was used as a matrix. Another conclusion is that hydrophobicity or hydrophilicity is not one of the main features taken into account in the multivariate prediction of the mass spectra of peptides under MALDI conditions [12,13]. ...
Characteristics of tryptic peptides that provide the detection and identification of these compounds and corresponding proteins by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry with α-cyano-4-hydroxycinnamic acid as a matrix are determined and discussed. Twenty-five proteins are identified; the features of reliably identified peptides are compared to those of the compounds that might be produced by trypsinolysis but were not detected. Two key factors enhancing a possibility of the detection of tryptic peptides are determined. One refers to the basicity of peptides in the gas phase, which is the highest for peptides with the C-terminal arginine residue; this reason for the emergence of significant analytical signals is well known. Another factor contributing to the ionization of peptides in study under MALDI conditions is their hydrophilicity. This result did not correlate with the most conclusions of earlier relevant studies discussed in the article.
... Despite a quantitative improvement in the average mass accuracy by employing the internal calibration, the standard deviations observed were in some cases larger than the ones observed with external calibration (see Figure 15B). This is due to the fact that the amount of the Fibrinopeptide B on a spot is low and the presence of peptides at higher concentrations and different composition on the same spot can induce peak suppression of the Fibrinopeptide B [90]. For this reason in some spots the Fibrinopeptide B peak may not be present or not labelled because of a low S/N. ...
... Most of the peptides released after enzymatic digestion, which are susceptible of detection by MALDI MS are in the mass range between 800-2500 Da. Because all peptides obtained by in-gel digest are ionized together for PMF analysis, peptide competition for the available protons can cause signal suppression [90]. Therefore peak suppression may play a role in detection of peptides originated from digestion of gel spots. ...
... [106], but it has also been reported that more hydrophobic peptides are preferentially ionized in CHCA matrix [90]. Previous studies have found that the MALDI technique is more prone to identified tryptic peptides ending with arginine, whereas ESI identifies rather peptides with a lysine at the C-terminus [105, 107]. ...
Large-scale proteome analysis of complex biological mixtures requires high resolving power separations, high-throughput mass spectrometry and accurate detection, and bioinformatics tools. In the present work LC-MALDI TOF/TOF mass spectrometry was combined for the first time with a two-dimensional separation based on reversed-phase chromatography at high and low pH for the analysis of the cytosolic proteome of Corynebacterium glutamicum. The proteome coverage achieved by this approach, 55%, is the highest reported up to day for this bacterium. A total of 1644 proteins including single-peptide based identifications were identified.
The sample was also analysed by other conventional methods in the proteome analysis. The classical 2D-PAGE approach presents more limitations and delivered 166 different proteins including enzymes of the main metabolic pathways. Complementary results were found at peptide level for the LC-MALDI and 2D-PAGE approaches. Fractions collected during the first dimension at high pH were analysed by LC-MALDI MS and compared with the analysis carried out by LC-ESI-IT MS, which identified 745 proteins [1]. Further comparison with results found in literature confirmed that the two-dimensional reversed phase combination with LC-MALDI MS/MS is a promising tool in the proteome analysis. The MALDI based approach showed higher sensitivity than other approaches and was able to identify proteins over a larger dynamic range, based on the codon adaptation indexes of the proteins.
... The data shown in Additional evidence for a lack of either complete retention during binding or low elution is observed from the low intensity of the antiestrogen peptide. -1000 counts were observed for 2.5 pmol/xL'^ starting concentration, in comparison to the PKC substrate-derived peptide, where 5500 counts were observed, with a starting concentration of 500 Although peptides ionisation efficiencies are known to vary in a sequence-dependent manner, (Janek, 2001;Karas, 1995;Kratzer, 1998), such a disparity indicates a problem with the IMAC process, since MALDI analysis of the peptide mixture prior to IMAC did not indicate a particular difference in ionisation efficiency. Further work to elucidate the cause of the lack of observation of certain peptides following IMAC is presented in Chapter 5. phosphoric acid respectively. ...
... In order to elucidate which of the possible adverse factors in phosphopeptide separation by IMAC has the most significant influence upon phosphopeptide detection, a quantitative investigation of the separation process during IMAC was required. As alluded to in Section 5.1, multiple factors influence mass spectrometric observation of phosphorylated analytes, largely based upon their individual properties and the conditions under which ionisation takes place (Beck, 2001;Carr, 1996;Cramer, 2000;Kratzer, 1998;Liao, 1994). Differential analyte ionisation means that the concentration of a target peptide may not give a linear response in the detected ion intensity from different sample compositions (Borchers, 1999;Zhou, 2000). ...
Reversible modification of proteins by phosphorylation of serine, threonine and tyrosine residues is the most common post-translational modification, which is estimated to occur in 30-90% of the cellular expressed protein component at any one time. Phosphorylation can alter proteins' subcellular distribution, enzymatic activity and specificity. Altered protein phosphorylation may correlate with disease states such as cellular transformation. The analysis of phosphorylated proteins is therefore of vital importance to the field of biology and in particular signal transduction. Protein phosphorylation sites are increasingly investigated using mass spectrometric methods, exploiting the inherent accuracy and sensitivity of these methods. However, the presence of unphosphorylated peptides in enzymatic digests of proteins causes ion suppression of phosphopeptides, reducing the effective sensitivity of detection; this sensitivity is further decreased by the relative lability of the phosphate moiety in the mass spectrometer and the occurrence of sub-stoichiometric modification, which together further reduce the achievable sensitivity. This study has examined techniques for the analysis of protein phosphorylation sites, with particular emphasis upon mass spectrometry. The technique of immobilised metal ion affinity chromatography (IMAC) was investigated in detail as a method suited to phosphorylation site analysis. IMAC exploits the relatively specific affinity of phosphorylated peptides for metal ions, particularly Fe(III), to isolate phosphopeptides upon a solid-phase affinity matrix, separating the suppressing non- phosphorylated component and allowing improved detection of phosphorylated peptides. Conditions for the application of IMAC to phosphopeptide segregation have been established and applied. Using IMAC, protein phosphorylation site identification of both standards and signal transduction mediators has been carried out. Apparent sequence-specific binding of phosphorylated and non-phosphorylated peptides to IMAC resins has been found and investigated. IMAC methodology has been further improved to optimise phosphopeptide analysis using mass spectrometry. The developed methods have clear utility for phosphorylation site analysis, which is vital to the understanding of signal transduction.
... The lability of the phosphate bond leads to prompt fragmentation of the peptide [11][12][13]. In matrix assisted laser desorption ionization-time of flight (MALDI-TOF) MS, ion suppression effects further reduce the intensity of the peak of a phosphorylated species when analyzed with non phosphorylated ones [14,15]. Moreover, the in vivo phosphorylation rates are usually low and lead to low abundant species. ...
... Alcanethiols have been extensively used [29,35,36,[38][39][40], as well as dithiol, leaving a free thiol extremity to label the derivatized peptide with an affinity purification tag [38,[40][41][42][43]. Another effective strategy is to use a thiol substituted with a moiety that enhances the MS signal, like a protonable group [36,[44][45][46] or a pre-charged moiety, for example thiocholine [36,37,47]. Finally, in MALDI-TOF MS, it was observed that aromatic moieties or residues in a peptide improved its detection [14,39,[48][49][50]. Nucleophiles substituted with aromatic moieties usually allow an intensity enhancement for the derivatized peptide [32,51,52]. ...
This study aims at improving the MALDI-TOF detection of a phosphorylated peptide containing a cysteine residue by β-elimination of H(3)PO(4) hardly enriched by classical methods. The experimental conditions were optimized on this phosphopeptide (biot-pAdd) and its nonphosphorylated counterpart (biot-Add). The major side-reactions were H(2)S elimination on the cysteine residues and H(2)O elimination on the non phosphorylated serine residue of biot-Add. The former dilutes the MALDI-TOF signal for the desired species. The latter gives a product similar to what is obtained by H(3)PO(4) elimination and should prompt to caution when working with a mixture between phosphorylated and non phosphorylated peptides. Modifications on the solvent, the reaction temperature and time, the nature, and concentration of the base were made. Major improvement of the selectivity of the reaction was observed in 30 % ACN, at room temperature for 4 h. However, these optimizations are specific to these sequences and should be performed anew for different peptides. The selectivity of the reaction towards H(3)PO(4) elimination is improved, but the persistence of side-reactions renders a previous sample fractionation necessary. In these optimized conditions, the ionization enhancement is 3-fold and the detection limits for biot-pAdd are similar to biot-Add (100 fmol).
... Different matrix molecules may crystallize in different shapes and dimensions; additionally, proteins are likely to accumulate at the droplet periphery, so the composition of the matrix solution and the rate of crystal growth strongly influence the spectral output [53,54]. Kratzer et al. [55] have described the ion-suppression effect which occurs when an ion suppresses the peak signal of other ions in the sample, and peptides with greater hydrophobicity show the greatest suppression effects. Furthermore, one of studies performed by Juhasz and Biemann [56] found that highly acidic compounds are responsible for producing weak signals in MALDI-TOF MS, but the mixing of them with a basic peptide and forming noncovalent complexes caused the signals to improve. ...
In recent years, matrix-assisted laser desorption/ionization (MALDI) has become the main tool for the study of biological macromolecules, such as protein nano-machines, especially in the determination of their molecular masses, structure, and post-translational modifications. A key role in the classical process of desorption and ionization of the sample is played by a matrix, usually a low-molecular weight weak organic acid. Unfortunately, the interpretation of mass spectra in the mass range of below m/z 500 is difficult, and hence the analysis of low molecular weight compounds in a matrix-assisted system is an analytical challenge. Replacing the classical matrix with nanomaterials, e.g., silver nanoparticles, allows improvement of the selectivity and sensitivity of spectrometric measurement of biologically important small molecules. Nowadays, the nanostructure-assisted laser desorption/ionization (NALDI) approach complements the classic MALDI in the field of modern bioanalytics. In particular, the aim of this work is to review the recent advances in MALDI and NALDI approaches.
... Still, many MS 2 spectra obtained from fractions showed high quality. This suggests that the fractionation could decrease problems with interfering analytes and ion suppression [41]. Differences in relative peak intensities between spectra of the desalted sample and the fractions yielded non-overlapping acquisition of MS 2 spectra (i.e. ...
A method for off-line CE-MALDI-TOF-MS and MS², and on-target digestion under a fluorocarbon lid was developed and applied for the analysis of proteins in the spermatophore of the butterfly Pieris napi. Fractionation revealed many peptides otherwise not detected or resolved. Automated fractionation was performed with an in-lab developed robotic system, and automated on-target tryptic digestion under a fluorocarbon lid was demonstrated with the same system. Fractionation onto a pre-structured MALDI-concentration plate facilitated aligned deposition of trypsin and MALDI-matrix with the deposited sample, also under the fluorocarbon lid. Some indications of indigenous proteolysis of spermatophore proteins were seen, and searching MS² spectra suggested three tentative sequence homologies to P. rapae. The study demonstrates the functionality of the lab-made robot. Detailed manufacturing instructions and code are provided. The feasibility of automated on-target digestion under a fluorocarbon lid, and the usefulness of a structured concentration plate in CE-MALDI fractionation was shown. Further, it constitutes a preliminary study of P. napi spermatophore proteins.
... Some of the most popular computational resources used in proteomics are listed in Table I.6. presence of other analytes or contaminants [248,276]. It was also reported that peptides with arginine at the C-terminus are preferentially ionized in MALDI than lysine C-terminal peptides, and therefore possess a higher intensity in the mass spectrum [277]. ...
... Nano-LC-ESI-QqTOF-MS/MS analysis was carried out on both tryptic digests of glycoconjugates with TF:protein ratio 2:1 and 8:1, and this was followed by de novo peptide sequencing using low-energy collision dissociation tandem mass spectrometry CID-MS/MS to identify the possible S-and N-glycated sites. [55,56] The obtained low-energy CID-MS/MS was submitted to the Mascot library to identify the glycopeptides and peptides released from BSA. The Mascot reports of MS/MS data of the tryptic digests of both glycoconjugates identified two serum albumin isoforms from Bos taurus species. ...
We present in this manuscript the characterization of the exact glycation sites of the Thomsen-Friedenreich antigen-BSA vaccine (TF antigen:BSA) prepared using a Michael addition reaction between the saccharide antigen as an electrophilic acceptor and the nucleophilic thiol and L-Lysine ε-amino groups of BSA using different ligation conditions. Matrix laser desorption ionization time-of-flight mass spectrometry of the neoglycoconjugates prepared with TF antigen:protein ratios of 2:1 and 8:1, allowed to observe, respectively, the protonated molecules for each neoglycoconjugates: [M + H](+) at m/z 67 599 and 70 905. The measurements of these molecular weights allowed us to confirm exactly the carbohydrate:protein ratios of these two synthetic vaccines. These were found to be closely formed by a TF antigen:BSA ratios of 2:1 and 8:1, respectively. Trypsin digestion and liquid chromatography coupled with electrospray ionization mass spectrometry allowed us to identify the series of released glycopeptide and peptide fragments. De novo sequencing affected by low-energy collision dissociation tandem mass spectrometry was then employed to unravel the precise glycation sites of these neoglycoconjugate vaccines. Finally, we identified, respectively, three diagnostic and characteristic glycated peptides for the synthetic glycoconjugate possessing a TF antigen:BSA ratio 2:1, whereas we have identified for the synthetic glycoconjugate having a TF:BSA ratio 8:1 a series of 14 glycated peptides. The net increase in the occupancy sites of these neoglycoconjugates was caused by the large number of glycoforms produced during the chemical ligation of the synthetic carbohydrate antigen onto the protein carrier. Copyright © 2014 John Wiley & Sons, Ltd.
Copyright © 2014 John Wiley & Sons, Ltd.
... Those analytes producing strong signals, especially those which exist in high concentration, can "drown" the signals of other analytes, which are less ameniable to ionisation. This feature has been noted by King et al. (2000) and Tang et al. (2004) for electronspray ionisation experiments and by Kratzer et al. (1998) where the Fourier coefficients are computed by ...
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... It is well established that chromatographic separation of protein digests increases data density, i.e. the number of peptides that can be identified in complex samples by mass spectrometry (32). In particular, peptides of low abundance or low ionization efficiency are more readily identified in separated digests, where suppression (41,42) of ionization by other peptides is minimized. To further increase peptide coverage, data sets were collected from two different combinations of ionization strategies and machine architectures: on-line LC-ESI on a Qq-TOF instrument versus off-line collection of LC fractions for subsequent analysis using the TOF/TOF instrument (LC-MALDI). ...
... The CID-MS/MS spectrum of each precursor ion was acquired using the Multi-Channel Analysis (MCA) mode, generating an average spectrum of different scans monitored at different collision energies. It is important to state that for all the CID-MS/MS sequencing the monoisotopic m/z values of the [M + H] + ions of the released peptides were used in the Mascot search engine (Matrix Science Inc., Boston, MA, USA).2627282930 Peptide mass fingerprint and MS/MS ions search Identification of the cryptocyanin protein from the peptide mass fingerprints and the MS/MS product ions search was carried out using the Mascot search engine on the National Center for Biotechnology Information Non-redundant protein database (NCBInr). ...
RATIONALEWe report the matrix-assisted laser desorption/ionization mass spectrometric (MALDI-MS) characterization of the cryptocyanin proteins of the juvenile Chionoecetes opilio crabs during their molting and non-molting phases. In order to assess the structural cryptocyanin protein differences between the molting and non-molting phases, the obtained peptides were sequenced by MALDI low-energy collision-induced dissociation tandem mass spectrometry (CID-MS/MS). METHODS
The cryptocyanin protein was isolated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by MALDI-TOF/TOF-MS. The purified cryptocyanin protein was sequenced, using the 'bottom-up' approach. After tryptic digestion, the peptide mixture was analyzed by MALDI-QqTOF-MS/MS and the data obtained were used for the peptide mass fingerprinting (PMF) identification by means of the Mascot database. RESULTSIt was demonstrated using MALDI-TOF/TOF-MS that the actual molecular weights of the non-molting and molting cryptocyanin proteins were different; these were, respectively, 67.6 kDa and 68.1 kDa. Using low-energy CID-MS/MS we have sequenced the trytic peptides to monitor the differences and similarities between the cryptocyanin molecular structures during the molting and non-molting stages. CONCLUSIONS
We have demonstrated for the first time that the actual molecular masses of the cryptocyanin protein during the molting and non-molting phases were different. The MALDI-CID-MS/MS analyses allowed the sequencing of the cryptocyanins after tryptic digestion, during the molting and non-molting stages, and showed some similarities and staggering differences between the identified cryptocyanin peptides. Copyright © 2013 John Wiley & Sons, Ltd.
... To further complicate matters the high negative charge introduced into a peptide by the incorporation of a phosphoamino acids makes the purification of phosphopeptides and particularly multiphosphorylated peptides difficult by traditional reversed phase chromatographic methods. Further, mass spectral analysis of phosphopeptides is challenging due to suppression effects and low sensitivity for phosphopeptides in the MS mode (Liao et al. 1994;Kratzer et al. 1998). ...
Protein phosphorylation is a critical mechanism in the regulation of cellular biochemical pathways and phosphopeptides can
play an important role in determining function. However, the use of phosphopeptides especially multiphosphorylated peptides
is hampered by their low abundance, difficulty in isolation from biological samples and in their chemical synthesis. Here
we describe methodologies for the Fmoc synthesis, purification and mass spectral analysis of the multiphosphorylated sequence
H-[Asp-(Ser(P))2]3-Asp-OH from phosphophoryn a protein involved in dentine mineralization. Critical steps in the synthesis of phosphophoryn
using Fmoc-Ser(PO3Bzl,H)-OH as the building block were double acylation steps for each residue, alternating HBTU and HATU as the acylating agents
and synthesis on a chlorotrityl resin which was essential for complete removal of the benzyl-side chain protecting groups.
The synthetic phosphophoryn was only effectively purified by anion exchange and size exclusion chromatography as both alkaline
and acid buffers failed to aid in purification by reversed phase HPLC. MALDI-TOF analysis of phosphophoryn was achieved with
good sensitivity (20 fmol/ml) and resolution using the DNA matrix 3-hydroxypicolinic acid, whereas typical protein/peptide
matrices failed to provide mass spectra. The synthetic phosphophoryn peptide was found to bind calcium, binding 6 mol of calcium
per mole of peptide. In conclusion the methodology described here can be easily adopted for the synthesis and analysis of
a wide variety of multiphosphorylated peptides.
... However, it must be noted that Fragmentation con®rmed a FindMod prediction that the peptide is of sequence YAMIGDPTGALTR, where the methionine has been oxidised to a methionine sulphoxide, M(so). SeeFigure 9 peptides may not be detected during mass spectrometry if they are not ef®ciently ionised (Kratzer et al., 1998). Finally, it is worth exploring the potential that the FindMod approach has for the large-scale discovery of protein post-translational modi®cations. ...
The availability of genome sequences, affordable mass spectrometers and high-resolution two-dimensional gels has made possible the identification of hundreds of proteins from many organisms by peptide mass fingerprinting. However, little attention has been paid to how information generated by these means can be utilised for detailed protein characterisation. Here we present an approach for the systematic characterisation of proteins using mass spectrometry and a software tool FindMod. This tool, available on the internet at http://www.expasy.ch/sprot/findmod.html, examines peptide mass fingerprinting data for mass differences between empirical and theoretical peptides. Where mass differences correspond to a post-translational modification, intelligent rules are applied to predict the amino acids in the peptide, if any, that might carry the modification. FindMod rules were constructed by examining 5153 incidences of post-translational modifications documented in the SWISS-PROT database, and for the 22 post-translational modifications currently considered (acetylation, amidation, biotinylation, C-mannosylation, deamidation, flavinylation, farnesylation, formylation, geranyl-geranylation, gamma-carboxyglutamic acids, hydroxylation, lipoylation, methylation, myristoylation, N-acyl diglyceride (tripalmitate), O-GlcNAc, palmitoylation, phosphorylation, pyridoxal phosphate, phospho-pantetheine, pyrrolidone carboxylic acid, sulphation) a total of 29 different rules were made. These consider which amino acids can carry a modification, whether the modification occurs on N-terminal, C-terminal or internal amino acids, and the type of organisms on which the modification can be found. We illustrate the utility of the approach with proteins from 2-D gels of Escherichia coli and sheep wool, where post-translational modifications predicted by FindMod were confirmed by MALDI post-source decay peptide fragmentation. As the approach is amenable to automation, it presents a potentially large-scale means of protein characterisation in proteome projects.
... We successfully isolated two tryptic plasma peptides using a TXP antibody specific for the short C-terminal EGYR sequence originating from coagulation factor IX and vitamin K-dependent protein S. The detection of these two proteins, which have been reported to be present at 4 and 25 g/ml in plasma, was not possible when only using C18 reversed phase ZipTip columns. This was to be expected, however, given the concentration range and breadth of the plasma proteome and the matrix and analyte ion suppression effects characteristic of the MALDI (along with the electrospray) ionization techniques (35,37). Nevertheless, the domination of nontargeted peptides derived from highly abundant proteins following anti-EGYR antibody immunoprecipitation was unexpected. ...
Blood plasma is a valuable source of potential biomarkers. However, its complexity and the huge dynamic concentration range of its constituents complicate its analysis. To tackle this problem, an immunoprecipitation strategy was employed using antibodies directed against short terminal epitope tags (triple X proteomics antibodies), which allow the enrichment of groups of signature peptides derived from trypsin-digested plasma. Isolated signature peptides are subsequently detected using MALDI-TOF/TOF mass spectrometry. Sensitivity of the immunoaffinity approach was, however, compromised by the presence of contaminant peaks derived from the peptides of nontargeted high abundant proteins. A closer analysis of the enrichment strategy revealed nonspecific peptide binding to the solid phase affinity matrix as the major source of the contaminating peptides. We therefore implemented a sucrose density gradient ultracentrifugation separation step into the procedure. This yielded a 99% depletion of contaminating peptides from a sucrose fraction containing 70% of the peptide-antibody complexes and enabled the detection of the previously undetected low abundance protein filamin-A. Assessment of this novel approach using 15 different triple X proteomics antibodies demonstrated a more consistent detection of a greater number of targeted peptides and a significant reduction in the intensity of nonspecific peptides. Ultracentrifugation coupled with immunoaffinity MS approaches presents a powerful tool for multiplexed plasma protein analysis without the requirement for demanding liquid chromatography separation techniques.
Erwinia amylovora is one of the major pathogens of apple. In order to prevent infections by this bacterium, various chemical products are commonly used in industrial agriculture. This, however, poses a heavy burden on the environment, so alternative methods of crop protection are being widely explored. In this regard, the induced formation of natural defense compounds (including phytoalexins) represents a promising alternative to conventional methods of plant protection. Phytoalexins are small antimicrobial secondary metabolites, synthesized "de novo" inplants in response to an infection or abiotic stress. The present work evaluates the impact of Bion® 50 WG, a plant resistance inducer (PRI) on the metabolomic profile of apple seedlings infected with E. amylovora. The chemical profile of samples were studied by two different methods : Laser desorptionionization mass spectrometry (LDI-MS) and liquid chromatography coupled to masss pectrometry, using electro spray ionization (LC-ESI-MS). While both methods revealed different chemical profiles, identical group separation by statistical analysis was observed for all analyzed samples. Moreover, marker signals, responsible for the statistical separation of differently treated plant groups, were identified. Based on database research, high-resolution (HR) MS as well as MS fragmentation patterns, specific immune markers responsible for acquired resistance against E. amylovora are proposed.
Biological imaging modalities are commonly used for characterization of health and disease, but each have trade-offs in the areas of spatial resolution, molecular coverage and specificity, and targeted or untargeted approaches. Stained tissue microscopy allows high spatial resolution to visualize tissue morphologies and cellular structure but provides little molecular information. Immunohistochemistry allows high spatial resolution similar to that of organic stains with the benefit of specific molecular information with the use of antibodies. However, molecular targets must be known in advance and antibodies may select for multiple molecular species. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) allows for the untargeted detection of hundreds to thousands of molecular species within a single experiment. Although it has somewhat lower spatial resolution than microscopy, IMS enables the detection of a wide range of biological species at increased molecular coverage with high spatial resolution and sensitivity using advanced data processing techniques. Herein, we present up-to-date examples of the current technologies of MALDI IMS.
This chapter presents an introduction to infective diseases and potential biological agents that could potentially be used for bioterrorism. Technical brief description of the possible treatments and preventions of these biothreats is presented with emphasis placed on the principles of immunological defenses, vaccination, and preparation of vaccines. In principle, various types of vaccines that are commercially used as “antibacterial or anticancer vaccines” can be produced using various types of antigenic carbohydrate haptens containing relevant epitopes. This review highlights the strategies used for the characterization of such synthetic neoglycoconjugate vaccines used as a means of protection against biothreats. A complete mass spectrometry-based strategy for validating the preparations of the neoglycoconjugate vaccine is presented.
Nowadays, the protection of environment and in particular the preservation of human health is one of the major concerns of policymakers over the world. Annaba region offers the most favorable conditions for economic development, through multiple possibilities, landscape diversity, basic infrastructure and an industrial potential. Facing the severity of environmental problems in Annaba due to the dense industrial activities, an environmental health survey was conducted in order to highlight the specific morbidity of air pollution in the region. The obtained results show that 30% of consultations and are for respiratory diseases, 40% of infant mortality (children under 1 year) is caused by respiratory diseases and 600000 asthmatics suffer permanently. A sustainable control, periodic monitoring and the establishment of a database connecting with air pollution, can play a key role to effective assessment and reducing the negative impacts of air pollution in the study area.
Keywords Air pollution · Regulation · Environmental protection · Health risks · Annaba .
في الوقت الحاضر ، تعد حماية البيئة وعلى وجه الخصوص الحفاظ على صحة الإنسان أحد
الاهتمامات الرئيسية لواضعي السياسات في جميع أنحاء العالم. توفر منطقة عنابة أفضل الظروف المواتية لتحقيق التنمية الاقتصادية ، من خلال الإمكانيات المتعددة والتنوع الطبيعي والبنية التحتية الأساسية والإمكانيات الصناعية.
لمواجهة خطورة المشاكل البيئية في عنابة بسبب الأنشطة الصناعية الكثيفة ، تم إجراء مسح صحي بيئي لتسليط الضوء على معدلات الإصابة بالأمراض الخاصة بتلوث الهواء في المنطقة.
النتائج المتحصل عليها تشير إلى أن 30٪ من الاستشارات تخص أمراض الجهاز التنفسي ، و 40٪ من وفيات الرضع (الأطفال دون السنة) سببها أمراض الجهاز التنفسي و 600.000 مصاب بالربو يعانون بشكل دائم .
يمكن أن تلعب المراقبة المستدامة والرصد الدوري وإنشاء قاعدة بيانات مرتبطة بتلوث الهواء دورًا رئيسيًا في التقييم الفعال وتقليل الآثار السلبية لتلوث الهواء في منطقة الدراسة.
Rationale:
The molecular complexity of tissue features several signal-suppression effects which reduce the ionization of analytes significantly and thereby weakens the quality of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) imaging (MALDI imaging). We report a novel approach in MALDI imaging by reducing signal-suppression effects for the analysis of beta-amyloid (Aβ) plaques, one pathological hallmark of Alzheimer's disease (AD).
Methods:
We analyzed Aβ proteoforms from postmortem AD brains and brains from transgenic mice (APPPS1-21) overexpressing familial AD mutations by combining two techniques: (1) laser capture microdissection (LCM) to accumulate Aβ plaques and (2) phosphoric acid (PA) as additive to the super-2,5-dihydroxybenzoic acid matrix.
Results:
LCM and MALDI-MS enabled tandem mass spectrometric fragmentation of stained Aβ plaques. PA improved the signal-to-noise (S/N) ratio, especially of the Aβ1-42 peptide, by three-fold compared with the standard matrix additive trifluoroacetic acid. The beneficial effect of the PA matrix additive in MALDI imaging was particularly important for AD brain tissue. We identified several significant differences in Aβ plaque composition from AD compared with APPPS1-21, underlining the value of reducing signal-suppressing effects in MALDI imaging.
Conclusions:
We present a novel strategy for overcoming signal-suppression effects in MALDI imaging of Aβ proteoforms.
In principle, various types of vaccines that are commercially used as “antibacterial or anticancer vaccines” can be produced using various types of antigenic carbohydrate haptens containing relevant epitopes. This review highlights the strategies used for the characterization of such glycoconjugate vaccines as well as a complete mass spectrometry-based strategy for validating their synthesis. The location of the covalently linked antigenic saccharide haptens on the protein carrier is usually termed as “glycation sites” and these are determined using tandem mass spectrometry. The ratio of carbohydrate hapten: BSA is determined by availing of the matrix-assisted laser desorption/ionization-TOF-MS analyses of the glycoconjugates. The outcome of our different studies was that all the glycated residues were located mainly near the outer surface of the carrier protein.
IntroductionFrom Protein Analysis to Proteome AnalysisMethods of Proteome AnalysisSummary and Outlook
Serum samples (33 healthy women, 34 ovarian cancer, 28 colorectal cancer, 34 syphilis patients and 136 patients with various benign gynecological diseases) were analyzed by MALDI-TOF MS peptide profiling and respective predictive models were generated by genetic and supervised neural network algorithms. Classification models for pathology versus healthy control showed up to 100% sensitivity and specificity for all target diseases. However, the specificity dropped to unsatisfactory 25–40% in case of target versus nontarget disease diagnostics. Expansion of the control group to an artificial “nominal control” group by adding profiles of benign gynecological diseases considerably improved specificity of the models distinguishing ovarian cancer from healthy control and benign gynecological diseases. The suggested version of MALDI-TOF MS profiling of sera could be applied to differentiate between cancers and benign neoplasms of the same localization which is a challenging task for classical methods. To increase the specificity of diagnostic methods based on peptidome analysis of blood samples, it is necessary to identify sets of concrete peptide structures which qualitatively or quantitatively differ among patients with different diseases.
For proteins, phosphorylation is the most important known post-translational modification that affects activity. Virtually any basic process of a eukaryotic cell is regulated at some point by the phosphorylation of one or more of its key protein components (Krebs 1994). The regulation of gene transcription, cell-cycle progression, cell division and proliferation, cell differentiation, cytoskeletal dynamics, energy storage and retrieval are all phosphorylation dependent. The functional relevance of protein phosphorylation is even more striking, considering its relatively small quantitative importance. Only a few percent of proteins are phosphorylated and, of these, only 0.03% are phosphorylated at a tyrosine residue.
The article describes the latest advances in the development of techniques and approaches in the field of proteomics in the Sigma-Aldrich company. It focuses on three major areas - on sample preparation for two-dimensional electrophoresis, on sample preparation for MS and on quantitative determinations in proteomic studies. A part of the article is devoted to new custom synthesis of labeled peptides for the determination of peptides using MS technique.
This report reviews the introductory aspects of electrospray ionization coupled with mass spectrometry and highlights those aspects of electrospray that play an important role in its application to analyzing real‐world samples. Following a discussion of matrix effects, factors that affect analyte response and sensitivity, and the flow rate factors of the spray solvent, the relative benefits of on‐line HPLC or UPLC are contrasted with an infusion electrospray sample introduction approach, which, of course, precludes any aspects of chromatographic separation. The introduction of samples via infusion bypasses the need for a chromatographic system, including its pumps, autosampler, separation column, and the compromise of a mobile phase, which must accomplish the chromatographic separation while also allowing the electrospray ionization process to occur. Thus, infusion sample introduction is in principle much easier to accomplish. However, there are clear benefits to employing HPLC for the analysis of complex samples. So, how can infusion sample introduction be worthwhile?
It can be worthwhile because nanoelectrospray, or electrospray at 20–500 nL/min solvent flow rates, provides some unique benefits over the higher flow rate regime that has been customarily used. Thus, nanoelectrospray provides improved sensitivity, reduced matrix suppression, and normalized response from differing analytes and conserves precious samples if this is an issue. This review overviews these principles and provides several successful examples of published work where infusion nanoelectrospray experiments have merit.
A protocol consisting of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), high performance liquid chromatography (HPLC), Edman degradation and cDNA cloning has been optimized for the rapid identification and structural characterization of peptides induced during the humoral immune response of Drosophila. MALDI-MS analysis led to the detection of at least 24 immune-induced molecules (DIMs) in the hemolymph (body fluid, 0.1 µl) of a single fruitfly in response to an experimental challenge. The use of micro-purification by HPLC, peptide sequencing and molecular biology techniques allowed the characterization of three out of the 24 DIMs from a hemolymph sample collected from less than 140 flies. This study clearly shows that MALDI-MS differential mapping constitutes a powerful tool to detect compounds involved in inducible physiological processes.
This review article describes the origins, advantages, and application of an indirect approach with which to study protein and other macromolecular complexes and identify the nature and site of interaction interfaces by means of conventional matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). First reported in 1999, it involves the detection of ion depletion or the absence of ions associated with a binding partner or domain in the MALDI mass spectrum of a mixture of interacting components compared to that for an untreated control. Later referred to as intensity-fading in some applications, the method offers numerous advantages over the direct detection of protein and other macromolecule complexes by MALDI-MS and even electrospray ionization (ESI) MS. The origins of this indirect method, its development for use with gel-separated components, validation using companion biochemical assays, and application to a range of protein-antibody and protein-drug complexes are reviewed together with software specifically developed to aid with data interpretation. The sensitivity of the approach for revealing how subtle differences in the structure of the binding partners can be detected by MALDI-MS is also demonstrated. © 2015 Wiley Periodicals, Inc. Mass Spec Rev 999:1-16, 2015.
© 2015 Wiley Periodicals, Inc.
In this review, we present the determination of the various glycation sites of synthetic neoglycoconjugates formed by conjugation of the antigenic saccharide hapten to BSA using tandem mass spectrometry. The ratio of hapten: BSA was determined by the matrix-assisted laser desorption/ionization-TOF/TOF-MS analyses of the glycoconjugates. We also tentatively propose that all glycated residues are located mainly near the outer surface of the protein.
GIP receptor knockout mice were shown to be protected from the development of obesity on a high fat
diet, suggesting a role of GIP in the development of obesity. In our study we aimed to test the hypothesis
if excess of GIP could accelerate development of obesity and to identify GIP gene targets in adipose
tissue. Therefore, mice were kept on a chow or a high fat diet and during the last 2 weeks D-Ala2-GIP
or PBS injections were performed. Afterwards, serum LPL activity and several biochemical parameters
(TG, FFA, cholesterol, glucose, insulin, resistin, IL-6, IL-1�, TNF�, GIP) were measured. Fat tissue was
isolated and QPCR was performed for a set of genes involved in energy metabolism and inflammation.
A DNA-microarray was used to identify GIP gene targets in adipose tissue of the chow diet group. We
found that the D-Ala2-GIP injections caused a significant decrease in both body weight and LPL activity
compared to controls. Serum biochemical parameters were not affected by D-Ala2-GIP, with an exception
for resistin and insulin. The set of inflammatory genes were significantly decreased in adipose tissue in the
D-Ala2-GIP injected animals on a chow diet. A DNA-microarray revealed that APO-genes and CYP-genes
were affected by D-Ala2-GIP treatment in adipose tissue. These results suggest that the body weight-reducing
effect of D-Ala2-GIP may be explained by lower LPL activity and insulin serum level. Moreover,
the identified GIP candidate gene targets in adipose tissue link GIP action to lipid metabolism exerted by
APO and CYP genes.
The early factors inducing insulin resistance are not known. Therefore, we are interested in
studying the secretome of the human visceral adipose tissue as a potential source of unknown
peptides and proteins inducing insulin resistance. Surface-enhanced laser desorption/ionization
time-of-flight (SELDI-TOF) mass spectrometry is a high-throughput proteomics technology to
generate peptide and protein profiles (MS spectra). To obtain good quality and reproducible
data from SELDI-TOF, many factors in the sample pretreatment and SELDI protocol should be
optimized. To identify the optimal combination of factors resulting in the best and the most
reproducible spectra, we designed an experiment where factors were varied systematically
according to a fractional factorial design. In this study, seven protein chip preparation protocol
factors were tested in 32 experiments. The main effects of these factors and their interactions
contributing to the best quality spectra were identified by ANOVA. To assess the reproducibility,
in a subsequent experiment the eight protocols generating the highest quality spectra were applied
to samples in quadruplicates on different chips. This approach resulted in the development of
an improved chip protocol, yielding higher quality peaks and more reproducible spectra.
Laser induced breakdown spectroscopy (LIBS) and endoscope observation were combined to design a remote probing device. We use this probing device to inspect a crack of the inner wall of the heat exchanger. Crack inspection requires speed at first, and then it requires accuracy. Once Eddy Current Testing (ECT) finds a crack with a certain signal level, another method should confirm it visually. We are proposing Magnetic particle Testing (MT) using specially fabricated the Magnetic Particle Micro Capsule (MPMC). For LIBS, a multichannel spectrometer and a Q-switch YAG laser were used. Irradiation area is 270 μm, and the pulse energy was 2 mJ. This pulse energy corresponds to 5-2.2 MW/cm2. A composite-type optical fiber was used to deliver both laser energy and optical image. Samples were prepared to heat a zirconium alloy plate by underwater arc welding in order to demonstrate severe accidents of nuclear power plants. A black oxide layer covered the weld surface and white particles floated on water surface. Laser induced breakdown plasma emission was taken into the spectroscope using this optical fiber combined with telescopic optics. As a result, we were able to simultaneously perform spectroscopic measurement and observation. For MT, the MPMC which gathered in the defective area is observed with this fiber. The MPMC emits light by the illumination of UV light from this optical fiber. The size of a defect is estimated with this amount of emission. Such technology will be useful for inspection repair of reactor pipe.
Milk is an excellent source of bioactive peptides. However, the composition of the native milk peptidome has only been partially elucidated. The present study applied matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) directly or after prefractionation of the milk peptides by reverse-phase high-performance liquid chromatography (RP-HPLC) or OFFGEL fractionation for the comprehensive analysis of the peptide profile of raw milk. The peptide sequences were determined by MALDI-TOF/TOF or nano-ultra-performance liquid chromatography-nano-electrospray ionization-LTQ-Orbitrap-MS. Direct MALDI-TOF-MS analysis led to the assignment of 57 peptides. Prefractionation by both complementary methods led to the assignment of another 191 peptides. Most peptides originate from αS1-casein, followed by β-casein, and αS2-casein. κ-Casein and whey proteins seem to play only a minor role as peptide precursors. The formation of many, but not all peptides could be explained by the activity of the endogenous peptidases, plasmin or cathepsin D, B and G. Database searches revealed the presence of 22 peptides with established physiological function, including those with ACE-inhibitory, immunomodulating or antimicrobial activity.
Based on a labeling regent1-(4-Cyanophenyl)-4-piperidinecarbohydrazide(CPH), a derivatization method was developed for the determination of oligosaccharides with high sensitivity by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The hydrazones were generated by the reaction of the reducing ends of oligosaccharides and the hydrazide of CPH. The derivatives were directly analyzed by MALDI-TOF-MS. Reaction conditions such as reaction temperature, concentration of acetic acid, and excess ratio of CPH regent were optimized. When the oligosaccharides reacted with 100-fold excess of CPH at 100 °C and in 0.125% (V/V) acetic acid, the yield was the highest. The results showed that the derivatization with CPH increased the sensitivity of MS detection, and this method was suitable for the analysis of oligosaccharides' compositions.
The molecular scanner combines protein separation using gel electrophoresis with peptide mass fingerprinting (PMF) techniques to identify proteins in a highly automated manner. Proteins separated in a 2-dimensional polyacrylamide gel (2D-PAGE) are digested ‘in parallel’ and transferred onto a membrane keeping their relative positions. The membrane is then sprayed with a matrix and inserted into a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer, which measures a peptide mass fingerprint at each site on the scanned grid. First, visualization of PMF data allows surveying all fingerprints at once and provides very useful information on the presence of chemical noise. Chemical noise is shown to be a potential source for erroneous identifications and is therefore purged from the mass fingerprints. Then, the correlation between neighboring spectra is used to recalibrate the peptide masses. Finally, a method that clusters peptide masses according to the similarity of the spatial distributions of their signal intensities is presented. This method allows discarding many of the false positives that usually go along with PMF identifications and allows identifying many weakly expressed proteins present in the gel.
Despite the progress in performance of MS-driven analytical methods during the last decade with regard to sensitivity and selectivity, the identification of phosphorylation sites is still not a trivial task. Thus, today no single method can reliably detect and characterize all modified residues in a phosphoprotein and far most successful analysis strategies for phosphoprotein and phosphoproteome, including multiple levels of enrichment and separation methods as well as biological follow-up analysis. However, recent improvements in MS have spawned improved and far more robust analytical strategies. An improved effiency of enrichment and separation techniqes on both peptide and protein level, the improved data quality by ECD or ETD peptide fragmentation and the improved confidence in phosphopeptide detection by MS 3 phosphopeptide sequencing using high mass accuracy FTICR-based mass spectrometers have enabled multiple comprehensive studies of protein phsophorylation. Last, but not least, multiple complementary MS-driven strategies for relative and absolute quantitation of protein phosphorylation will ease rapid investigations of signal transduction systems and, thus, provide the basis for great advances.
Zusammenfassung
Die SELDI-TOF-Massenspektrometrie (MS) ist eine der wichtigsten aktuell verfügbaren Technologien zur Hochdurchsatzanalyse von komplexen Proteinmischungen. In zahlreichen Untersuchungen der letzten Jahre konnte gezeigt werden, dass sich durch die Methode des vergleichenden Proteinprofilings mittels SELDI-TOF-MS neue Wege in der diagnostischen Proteinanalytik und insbesondere in der Identifizierung neuer Biomarker beschreiten lassen. Gleichzeitig haben diese Untersuchungen aber auch die Grenzen der Methode verdeutlicht und die besondere Bedeutung der Standardisierung und Qualitätskontrolle sowohl der präanalytischen und analytischen Einflussfaktoren, als auch der nachfolgenden bioinformatischen Auswertung belegt. So hat sich gezeigt, dass das Studiendesign, die Auswahl entsprechender Kontrollgruppen und die Verwendung besonderer, aus der Analyse von Microarraydaten bekannter, statistischer Methoden eine wesentliche Bedeutung für die Reduktion der Rate falsch-signifikanter Peaks haben. Obwohl eine essentielle Forderung zur Validierung der in Pilotstudien identifizierten potentiellen diagnostischen Peakmuster darin besteht, diese in entsprechend großen prospektiven und multizentrischen Studien zu validieren, mangelt es in einer Vielzahl von Studien gerade daran. Bevor diese Untersuchungen nicht in ausreichendem Maße durchgeführt und an einer größeren Anzahl von Fragestellungen gezeigt werden konnte, dass sich die unter standardisierten Bedingungen erhobenen Proteinprofile auch noch Monate/Jahre später unabhängig vom Untersucher und dem verwendeten Gerät reproduzierbar zur Unterscheidung distinkter Patientenpopulationen verwenden lassen, ist die Translation dieser Technologie in die klinische Routinediagnostik potentiell möglich, aber nicht konkret absehbar.
Biomedical application of spectroscopic methods is on the rise in the last few years. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and microscopic Fourier transform infrared spectroscopy (FTIR) were applied for the analysis of human blood samples in order to detect spectral peaks which might serve as biomarkers for monitoring and identification of specific diseases. In the present study, samples of serum were obtained from healthy and patient persons suffering from diarrhea and analyzed by MALDI-TOF and advanced FTIR microscopy. The results show consistent spectral peaks in all examined serums obtained from healthy persons when tested in MALDI-TOF on the one hand, and in FTIR microscopy on the other hand. In all tested patient samples, two unique interesting peaks appeared at m/z 11,553 and 11,710 in MALDI-TOF spectra. When these patients samples were examined by FTIR microscopy, a peak at area position near 1600cm−1 completely disappeared in FTIR spectra. These parameters could be used as a basis for developing a spectral method for the detection and identification of specific human diseases and probably other disorders.
A comparison of the mass spectral response for myoglobin, cytochrome c, and lysozyme is presented for laser electrospray mass spectrometry (LEMS) and electrospray ionization mass spectrometry (ESI-MS). Analysis of multi-component protein solutions using nonresonant femtosecond (fs) laser vaporization with electrospray post-ionization mass spectrometry exhibited significantly reduced ion suppression effects in comparison with conventional ESI analysis, enabling quantitative measurements over four orders of magnitude in concentration. No significant charge reduction was observed in the LEMS experiment while the ESI measurement revealed charge reduction for myoglobin and cytochrome c as a function of increasing protein concentration. Conventional ESI-MS of each analyte from a multi-component solution reveals that the ion signal detected for myoglobin and cytochrome c reaches a plateau and then begins to decrease with increasing protein concentration preventing quantitative analysis. The ESI mass spectral response for lysozyme from the mixture initially decreased, before increasing, with increasing multi-component solution concentration.
The fast and univocal identification of different species in mixtures of pollen grains is still a challenge. Apart from microscopic evaluation and Raman spectroscopy, no other techniques are available.
Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry was applied to the analysis of extracts of single pollen grains and pollen mixtures. Pollen grains were fixed, treated and covered with matrix directly on the MALDI target.
Clearly resolved MALDI ion intensity images could be obtained enabling the identification of single pollen grains in a mixture.
Our results demonstrate the potential and the suitability of MALDI imaging mass spectrometry as an additional method for the identification of pollen mixtures. Copyright
In order to elucidate the role of desorption/ionization efficiency of peptides in MALDI-MS, we focused on peptides with disulfide bonds, which form a rigid tertiary structure. We synthesized seven sets of peptides with one disulfide bond (oxytocin, somatostatin, [Arg(8)]-vasopressin, [Arg(8)]-vasotocin, cortistatin, melanin-concentrating hormone, urotensin II-related peptide) and five sets of peptides with two disulfide bonds (tertiapin, α-conotoxin GI, α-conotoxin ImI, α-conotoxin MI and α-conotoxin SI). Each peptide set consisted of three peptides: the oxidized form (S-S type), the reduced form (SH type), and an internal standard peptide in which all cysteine residues were substituted with alanine residues. In the case of urotensin II-related peptide, tertiapin, α-conotoxin ImI and α-conotoxin MI, the reduced form showed higher desorption/ionization efficiency than the oxidized form. In contrast, the other peptides revealed higher desorption/ionization efficiency in the oxidized form relative to the reduced form. These results imply that a rigid structure of peptides formed by disulfide bonds does not correlate with desorption/ionization efficiency in MALDI-MS.
Surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) using inorganic nanoparticles has been reported as an organic matrix-free approach. However, the correlation of desorption/ionization (DI) efficiency with analyte chemical structures in SALDI-MS is not clear. In this study, we investigated the DI efficiency of 20 common amino acids and several peptides in SALDI-MS with Pt nanoparticles with thin projections on the surface (termed with Pt nanoflowers, Pt Nfs) on silicon substrates. The fluorocarbon-based hydrophobic perfluorodecyltrichlorosilane (FDTS)-Pt Nf substrates enabled the simultaneous analysis of all 20 common amino acids in negative-ion mode, whereas MALDI-MS was able to detect only two amino acids, proline and glutamic acid, from the same mixture in negative-ion mode. The SALDI-MS produced high ion yields for arginine and proline in positive-ion mode as well as for glutamic acid and aspartic acid in negative-ion mode. A linear correlation was found between the ion yield and the gas-phase proton affinity or acidity of amino acids in SALDI-MS, consistent with the MALDI-MS analysis of amino acids, although the linear correlation in the SALDI-MS was poor in comparison with that of MALDI-MS. It was suggested that the ion yields of amino acids (i.e., the DI process) are mainly determined by the same factors regardless of the ionization method employed in both MALDI performed using organic matrix and organic matrix-free SALDI.
A piezoelectric device containing a polymeric monolithic column was used to dispense eluents of proteolyzed material onto a matrix-assisted laser desorption ionization time of flight (MALDI-TOF) MS target for PMF. The LC separation of the bovine serum albumin (BSA) digest peptides generated during gradient elution improved sequence coverage and protein identification. This microfluidic approach to protein digest sample separation facilitated the PMF of low-concentration proteins and offered a new instrument for proteomic analysis. It was demonstrated that The chromatographic separation of peptides by the inkjet system reduced ion suppression and improved the resolution of MALDI-TOF MS analysis for applications, such as peptide mass fingerprinting. The separation of tryptic digests of BSA resulted in a larger number of peptides identified in comparison to the control.
Recent developments in desorption/ionisation mass spectrometry techniques have made their application to biological analysis a realistic and successful proposition. Developments in primary ion source technology, mainly through the advent of polyatomic ion beams, have meant that the technique of secondary ion mass spectrometry (SIMS) can now access the depths of information required to allow biological imaging to be a viable option.Here the role of the primary ion C60+ is assessed with regard to molecular imaging of lipids and pharmaceuticals within tissue sections. High secondary ion yields and low surface damage accumulation are demonstrated on both model and real biological samples, indicating the high secondary ion efficiency afforded to the analyst by this primary ion when compared to other cluster ion beams used in imaging. The newly developed 40 keV C60+ ion source allows the beam to be focused such that high resolution imaging is demonstrated on a tissue sample, and the greater yields allow the molecular signal from the drug raclopride to be imaged within tissue section following in vivo dosing.The localisation shown for this drug alludes to issues regarding the chemical environment affecting the ionisation probability of the molecule; the importance of this effect is demonstrated with model systems and the possibility of using laser post-ionisation as a method for reducing this consequence of bio-sample complexity is demonstrated and discussed.
The identification of naturally processed peptides presented by molecules of the major histocompatibility complex (MHC) has progressed significantly over the past decade. The elution of peptides from immunoaffinity purified complexes of MHC class I or class II molecules has provided highly specific biochemical information regarding the nature of endogenous peptides capable of binding to and being presented by particular MHC alleles. Whilst Edman chemistry is sufficient for the identification of abundant or homogeneous immunodominant peptides contained in samples of fractionated peptides, mass spectrometry has proved more powerful for sequencing less abundant species present in the typically heterogeneous fractions of eluted peptides. This review focuses on the characterisation of T cell determinants by matrix-assisted laser desorption/ionisation (MALDI)–time-of-flight (TOF) mass spectrometry (MS). We demonstrate, with specific examples, the utility of post-source decay in MALDI–TOF MS for the characterisation of the amino acid sequences of both native and modified T cell determinants. The potential advantages and pitfalls of this technique relative to the more commonly used forms of tandem mass spectrometry in electrospray and ion spray modes of ionisation as well as hybrid quadrupole–quadrupole-TOF instruments are discussed. We highlight the complementarity between these techniques and discuss the advantages in the combined use of both MALDI- and electrospray-based instrumentation in epitope identification strategies.
We describe a novel approach to infrared matrix-assisted laser desorption-ionization mass spectrometry using a tunable, picosecond pulse laser to selectively excite specific modes of a solid, thereby creating a high local density of vibrational quanta. The concept is based on recent results from our experiments employing a free-electron laser to explore ‘matrix-less’ mass spectrometry in which an infrared chromophore intrinsic to the sample, rather than an exogenous matrix, is excited by the laser. Examples from both environmental mass spectrometry and a proteomics-driven research project are presented, showing how the principle of selective vibrational excitation can be used to make possible novel and useful ion generation protocols. We conclude with an analysis of possible mechanisms for the phenomena of infrared desorption, ablation and ionization using very short laser pulses. Prospects for achieving similar results with more conventional laser sources are discussed.
A new approach to protein sequencing is described. It consists of two steps: (i) ladder-generating chemistry, the controlled
generation from a polypeptide chain by wet chemistry of a family of sequence-defining peptide fragments, each differing from
the next by one amino acid; and (ii) data readout, a one-step readout of the resulting protein sequencing ladder by matrix-assisted
laser-desorption mass spectrometry. Each amino acid was identified from the mass difference between successive peaks, and
the position in the data set defined the sequence of the original peptide chain. This method was used to directly locate a
phosphoserine residue in a phosphopeptide. The protein ladder sequencing method lends itself to very high sample throughput
at very low per cycle cost.
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF) is becoming a standard tool in mass spectrometry in general and protein analysis in particular. Its advantages include high sensitivity, tolerance to buffers, fast data acquisition, and simple and robust instrumentation. Disadvantages have been relatively low resolution and mass accuracy and have mainly been caused by factors related to sample preparation. Here we describe a simple new sample preparation procedure in which matrix and sample handling are completely decoupled. Fast evaporation of a matrix solution based on, e.g,, acetone produces a thin layer, presumably consisting of microcrystals. In a second step a drop of analyte solution is deposited on the layer. Some of the features of this new procedure are as follows: (i) there is an improvement in sensitivity; peptides can routinely be analyzed in the attomole range; (ii) salts and impurities are more easily washed off the prepared samples; (iii) sample surfaces become much more homogeneous, allowing very fast data acquisition; (iv) spectra show a more linear mass scale and higher resolution, especially when a reflector is used. In examples, a sensitivity of 5 amol and a resolution of up to 5700 (fwhm) could be achieved for peptides.
The unimolecular decompositions of protonated heterodimers of native and derivatized amino acids to yield the protonated monomers
were studied as a guide to charge location in peptide ions. Analyses using a hybrid instrument of BEqQ geometry demonstrated
the advantages (with respect to mass resolution, sensitivityr reproducibility, and the elimination of extraneous signals)
of the detection of product ions formed in the radiofrequency-only quadrupole region (q) rather than in the field-free region
between Band E. Conversion of arginine to dimethylpyrimidylomithine (DMPO) reduced the proton affinity, as evidenced by the
decomposition of the protonated arginine/DMPO heterodimer. Conversion of cysteine to pyridylethylcysteine enhanced the proton
affinity. Application of these derivatization procedures to peptides resulted in changes in the observed fragmentations of
the protonated precursors consistent with the predicted modifications in charge location. Unimolecular decomposition of the
protonated dimer composed of glycine and N-acetylglycine yielded both protonated monomers with abundances differing by a factor of only 2; this suggests that in protonated
peptides, the amide bonds are competitive with the N-terminal amino group as sites of protonation. It is clear that the propensities
to proton’ or metal-cation location at particular sites in peptides are influenced by both short- and long-range intraionic
interactions. In peptides composed of amino acids of similar cation affinities, it may be postulated that the ion population
is heterogeneous with respect to the site of charge, with consequent promotion of multiple low-energy fragmentation routes.
The authors report on the inclusion of protein molecules in growing crystals of trans-3,5-dimethoxy-4-hydroxy-cinnamic acid (trans-sinapic acid). This material is important as the 'matrix' compound in matrix-assisted lasar desorption, a promising new ion source for heavy molecule mass spectrometry. The crystal structure of trans-sinapic acid was determined. It belongs to the space group P21/n and has a structure in which the planar trans-sinapic acid molecules are hydrogen bonded into extended sheets. Dyes were used to follow protein incorporation into growing trans-sinapic crystals. The staining pattern obtained demonstrated that protein molecules were included only on crystal faces parallel to these extended sheets. This result is the first demonstration that proteins can be incorporated into a growing crystal by a selective interaction with one of the crystal faces. The structure of the crystal plane that interacts with the protein was consistent with a 'hydrophobic' bonding of the protein to the crystal face.
Matrix-assisted laser desorption/ionization (MALDI) mass spectra and methods to improve their quality are reported for three hydrophobic, membrane-bound proteins: porin from Escherichia coli, bacteriorhodopsin from Halobacterium salinarium and cholesterolesterase from Pseudomonas fluorescens. Several commonly used UV and IR matrices have been tested. In addition, the susceptibility of MALDI mass spectrometry to various neutral and ionic detergents, known usually to degrade the quality of MALDI mass spectra, has been tested systematically. For porin, consisting of three identical non-covalently bound subunits, a new sample preparation is reported, resulting in the desorption of the intact quaternary protein structure. This leads to a better understanding of the way a given analyte is embedded into the host matrix crystals.
Suppression of low-mass ion peaks in matrix-assisted ultraviolet laser desorption has been found to occur at low matrix-to-analyte molar ratios when using nicotinic acid as matrix, independent of the angle of illumination. Microscopic Raman scattering spectroscopy has been employed to investigate the matrix–analyte solid-state composition. The matrix-to-analyte molar ratios employed in preparing the solutions are reliable guides to the relative amounts of matrix and analyte molecules in the solid crystals, given the method of sample preparation employed involving drying under a stream of nitrogen. A qualitative model based on the proton supply-and-demand argument is tentatively proposed to explain the suppression phenomenon.
In matrix-assisted laser desorption/ionization (MALDI) the analyte signal produced depends strongly on the analyte and on the sample preparation procedure. Comparing the results obtained with the dried-drop method with the results obtained with a homogeneous sample preparation procedure, discrimination effects, as well as sensitivity variations, are shown to be dependent on the molecular weight of the analysed compounds. The signal obtained in MALDI is shown to be dependent on the hydrophobicity and basicity of the analyte. With the dried-drop method, in particular, evidence of peripheral sample deposition of hydrophilic compounds is shown. These last effects are due to mass transfer caused by differences in surface activities during solvent evaporation, and are known as Marangoni effects. © 1997 John Wiley & Sons, Ltd.
Examination, by optical microscopy, of the matrix/analyte morphology of a sample propared from a droplet in matrix-assisted UV laser-desorption mass spectrometry (MUVLDMS) reveals that the matrix crystallizes to a large extent, thus altering the concentration of the material in regions on the surface from that of the bulk applied solution. Microscopy reveals that vanillic acid and to a lesser extent nicotinic acid matrices form prominent crystalline rings around the dried sample spot while sinapic acid forms a more uniform dispersion of the crystallized matrix. Insulin analyte investigated in this report tendss to form a thin isotropic phase in the center of the drops. The first reported use of a KrF excimer laser (249 nm) in MUVLDMS is also discussed.
This study encompasses a collection of experiences with regard to numerous matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) sample preparation techniques in terms of their suitability for different peptide and protein analytes. Variants of both established and new sample preparation techniques for the MALDI-MS analysis of peptides and proteins are described. The importance of matrix selection, matrix and analyte concentration, pH adjustment, crystallization conditions and the use of additives is evaluated. The tolerance of the different sample preparations towards salts, buffers, synthetic polymers, detergents, denaturants and other contaminants, and also the influence of the preparation methods on undesired amino acid side-chain oxidation, are investigated. Moreover, the performance of on-target tryptic digestion and on-target disulfide reduction is shown and a microscale purification procedure is described. According to this study, there is no universally applicable sample preparation for a broad variety of analytes. Rather, it is necessary to specifically adapt the sample preparation to the analyte properties. © 1997 John Wiley & Sons, Ltd.
The ionization in ultraviolet laser desorption was investigated for a large number of small polar organic molecules which have a strong resonance absorption at the laser wavelength. In many cases, both positive- and/or negative-ion mass spectra show strong signals of ion species which deviate from the simple scheme of even-electron quasimolecular and fragment ion formation commonly expected for desorption techniques. These are radical cations and ion species formed by single and multiple hydrogen cleavage or addition. A model is proposed and discussed which explains these features assuming photoionization as the common initial ionization step followed by ion-molecule reactions to the final product ions. The mass spectra of all compounds proved to function well in matrix-assisted ultraviolet laser desorption/ionization show characteristic features indicative of their photochemical reactivity. This observation substantiates the hypothesis of tbe essential role of the matrix in analyte ionization.
Ion yields were investigated in matrix-assisted laser desorption/ionization (MALDI) as a function of amino acid composition using a variable wavelength ion source. In the case of nitrogen laser excitation (337 nm), [M+H]+ ions were abundant for short peptides containing basic or polar amino acid residues. The lack of basic residues led to diminishing ion formation at 337 nm. Increasing the chain length led to enhanced ionization even for peptides with non-polar side chains. In contrast to the liquid phase basicities the basic residues showed Arg > His > Lys order in their affinity to protons. Variations in the average ion yield showed decreasing trend with increasing basicity of the residues pointing to potential differences in the matrix incorporation of different guest molecules. Dye laser excitation at 280 nm resulted in extensive fragmentation and enhanced ion formation from peptides containing aromatic side chains indicating the possible role of analyte excited states in ion formation. Semi-empirical quantum chemical calculations were used to explore the sites of protonation. Heat of formation diagrams of the matrix-analyte complexes were examined as a function of their configuration. Investigation of the optimized geometries allowed the location of bifurcated and linear hydrogen bonds between the non-polar analyte (e.g., Gly-Gly) and matrix molecules. In addition to these intermolecular bonds, in the case of His-His an intramolecular hydrogen bond was also formed within the analyte molecule. Proton affinity values were calculated for every dipeptide while the site of protonation was varied. The results seemed to indicate that for every dipeptide the amino terminus was more susceptible to protonation than the peptide bond. Due to increased stabilization effects in homo-oligomers (Glyn), increasing the number of residues led to an increase in proton affinity. For basic diopeptides (e.g., His-His), the side chains had the highest proton affinity, underlining their role in MALDI of proteins.
A new chemical method for carboxy-terminal (C-terminal) protein sequencing has been developed. This approach has been successfully used to sequence 5 residues of standard proteins and 5 to 10 residues of synthetic peptides at low nanomole levels. The sequencing procedure consists of converting the C-terminal amino acid into a thiohydantoin (TH) derivative, followed by transformation of the TH into a good leaving group by alkylation. Next, the alkylated TH is cleaved mildly and efficiently with (N = C V S)- anion, which simultaneously forms a TH on the newly truncated protein or peptide. Thus, after the initial TH derivatization, there is no return to a free carboxyl group at the C-terminus. An additional benefit of this method is that the alkylating moiety can be chosen with a variety of properties allowing for variation in the detection method. This chemistry has been adapted to automated protein sequencers with a cycle time of about 1 h.
A novel method of peptide sequencing by mass spectrometry is described. Metastable decay of laser-desorbed ions, taking place in the first field-free drift region of a reflection time-of-flight mass spectrometer, has been monitored to get structural information from larger peptides. Fragment ions from metastable decay are mass analysed by adjusting the potentials of the ion reflectron according to the kinetic energies of the ions. The features of the technique and its significance for future applications are outlined.
Peptides or proteins were hydrolyzed by vapors of 90% pentafluoropropionic acid or heptafluorobutyric acid at 90 degrees C for various time periods. The hydrolyzate mixtures analyzed by both fast-atom-bombardment and electrospray ionization mass spectrometry showed a series of C-terminal successive degradation molecular ions. The degradation reaction may be due to the selective formation of an oxazolone ring at the C-terminal amino acid, followed by hydrolytic removal of the C-terminal amino acid. The major side reactions were cleavages of the peptide bonds at the C side of the internal aspartic acid residue and the N side of serine residue.
The protein sequenator is an instrument for the automatic determination of amino acid sequences in proteins and peptides. It operates on the principle of the phenylisothiocyanate degradation scheme. The automated process embraces the formation of the phenylthiocarbamyl derivative of the protein and the splitting off of the N-terminal amino acid as thiazolinone. The degradation proceeds at a rate of 15.4 cycles in 24 hours and with a yield in the individual cycle in excess of 98%. The material requirements are approximately 0.25 μmoles of protein. The thiazolinones are converted to the corresponding phenylthiohydantoins in a separate operation, and the latter identified by thin layer chromatography. The process has been applied to the whole molecule of apomyoglobin from the humpback whale, and it has been possible to establish the sequence of the first 60 amino acids from the N-terminal end.
A conceptually novel approach to protein sequencing involves the generation of ragged-end polypeptide chains followed by mass spectroscopic analysis of the resulting nested set of fragments. We report here on the synthesis and development of a volatile isothiocyanate (trifluoroethylisothiocyanate) that allows the identification of several consecutive residues starting with a few picomoles of peptide. The nested set of peptides is generated simply by adding equal aliquots of starting peptide each cycle and driving both the coupling and cleavage reactions to completion. No additional reagents are required to act as chain terminators and retention of the peptide terminal amine allows for subsequent modification with quaternary ammonium alkyl NHS esters to improve sensitivity. Complex washing procedures are not required each cycle, as reagents and by-products are efficiently removed under vacuum, eliminating extractive loss. Multiple peptide samples can be processed simultaneously, with each degradation cycle completed in 35-40 min. The inherent simplicity of the process should allow for easy automation and permit rapid processing of samples in parallel.
Matrix-assisted laser desorption/ionization (MALDI) was used for several small proteins (such as insulin) and for peptides. It was found that the detection efficiencies of MALDI for the insulin B chain and the insulin A chain are drastically different. Similar phenomena were also observed for various types of peptides. The positive-ion signal of MALDI in detecting proteins or peptides was found to be greatly enhanced by the presence of a basic amino acid in their chains. The experimental results indicate that this enhancement may arise from proton transfer in solution by an acid-base reaction between the protein/peptide and matrix molecule. This pre-protonated mechanism provides a low energy barrier for the ionization of peptides in a MALDI process, and greatly reduces the energy threshold of MALDI. Matrix effects on the ionization mechanism are discussed.
The utility of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry for the analysis of C-terminal peptide ladders from carboxypeptidase Y (CPY) digestions is discussed. MALDI analysis of aliquots of an optimized time-dependent CPY digestion of ACTH 7-38 fragment allowed for the sequence of the first 19 amino acids from the C-terminus to be determined in 25 min of digestion time. A strategy for performing parallel concentration-dependent digestions on the MAL-DI plate is proven to be superior to the time-dependent approach as the method development time and practical amounts of both peptide and enzyme consumed are reduced significantly. The on-plate approach offered the same sequence information from the ACTH 7-38 fragment and was used to digest 22 peptides of various amino acid composition, size, charge, and polarity. Of the 22 peptides digested on-plate, sequence information was derived from 19 of them. A statistical analysis strategy for ladder sequencing utilizing t-statistics is offered as a method for placing confidence intervals on residue assignments.
An overview is provided of methods for the study of complex biological processes by using micro-column liquid chromatography-electrospray ionization tandem mass spectrometry. Procedures discussed include electrospray ionization, micro-column liquid chromatography, tandem mass spectrometry, tandem mass spectra data interpretation for peptides, and database searching with mass spectral data. Several problems in immunology are discussed to illustrate this approach.
By the incorporation of delayed extraction (DE) into matrix-assisted laser desorption/ionization time-of-flight mass spectrometry a dramatic improvement of performance with respect to sensitivity, mass resolution and mass accuracy of precursor ions up to approximately 10 kDa has been achieved. Since DE reduces collisional in-source activation to a large extent, the rate of subsequent metastable decay is considerably reduced. Results are presented which demonstrate that under DE the loss of total post-source decay (PSD) fragment ion yield can be as large as one order of magnitude but that, in terms of sensitivity, part of this loss is balanced by a better S/N ratio which results from a significantly improved mass resolution of the PSD fragment ions (M/delta M up to 1800 compared with M/delta M = 200-500 under prompt extraction). While this compensatory effect is true for the middle to high mass range of PSD fragment ions, it gradually vanishes towards the low mass end of the PSD mass scale where, in the case of linear peptides some important information (immonium ions) is lost. It appears, however, that in the majority of practical PSD work, DE improves the qualty of the PSD spectra and that high energy collisional post-source activation can compensate for the occasional loss of analytical information.
Sample-matrix preparation procedures are shown to greatly influence the quality of the matrix-assisted laser desorption/ionization (MALDI) mass spectra of peptides and proteins. In particular, dramatic mass discrimination effects are observed when the matrix 4-hydroxy-alpha-cyanocinnamic acid is used for analyzing complex mixtures of peptides and proteins. The discrimination effects are found to be strongly dependent on the sample-matrix solution composition, pH, and the rates at which the sample-matrix cocrystals are grown. These findings demonstrate the need to exercise great care in performing and interpreting the MALDI analysis of biological samples. The results also indicate that there is a reverse-phase chromatographic-like dimension in the sample-matrix preparation procedures that can be exploited to optimize the analysis. The present work describes the conditions under which the majority of components of a complex mixture of peptides and proteins can be successfully measured.
A chemical procedure for the degradation of peptides and analysis by matrix-assisted-laser-desorption ionization mass spectrometry (MALDI-MS) was used for C-terminal sequence determination. The method allowed us to determine up to eight amino acid residues in the lower picomole range by mass analysis without any repetitions of the degradation nor any extraction or purification of the truncated peptide chains. The C-terminal degradation of all 20 common amino acid residues was proved by applying this method. Extended C-terminal sequence information from enzymatic digests using carboxypeptidase P was obtained by combining the enzymatic with the chemical mass spectrometric approach. Furthermore the amino acids lysine and glutamine, with the same masses, were distinguishable due to the formation of acetyl-lysine in the chemical process.