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Similarity of tandem mass spectra shown as a “heat map” for all combinations of collision energies determined on (A) Waters QTof and Waters QQQ, (B) Waters QQQ and Bruker ion trap, and (C) Thermo Orbitrap and Bruker QTof instruments. Figures taken from references Bazsó et al. (2016) and Szabó et al. (2021) with permission
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![CID spectrum of (A) QQQHLFGSN[+1]VTDCSGNFCLFR³⁺ peptide and (B) that of...](publication/356756468/figure/fig2/AS:11431281176234272@1690075130415/CID-spectrum-of-A-QQQHLFGSN-1VTDCSGNFCLFR-peptide-and-B-that-of-its-glycosylated_Q320.jpg)
Mass‐spectrometry coupled to liquid chromatography is an indispensable tool in the field of proteomics. In the last decades, more and more complex and diverse biochemical and biomedical questions have arisen. Problems to be solved involve protein identification, quantitative analysis, screening of low abundance modifications, handling matrix effect...
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Proteomics aims to characterise system-wide protein expression and typically relies on mass-spectrometry and peptide fragmentation, followed by a database search for protein identification. It has wide ranging applications from clinical to environmental settings and virtually impacts on every area of biology. In that context, de novo peptide sequen...
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... Kolejnym ważnym czynnikiem w przypadku analizy DDA jest energia kolizji jonów użyta do ich fragmentacji. Przy zbyt małej energii jon albo nie ulegnie rozbiciu, albo jego dysocjacja będzie tylko częściowa, co spowoduje niewystarczającą intensywność sygnału jonów fragmentacyjnych [12]. Odwrotnie, przy zbyt wysokiej energii kolizyjnej, obserwuje się efekt nadmiernej fragmentacji, co z kolei prowadzi do otrzymania widm z niewspółmiernie reprezentowanymi jonami o niskich wartościach m/z. ...
Mass spectrometry is an important tool in proteomic, metabolomic and lipidomic analysis. To fully use its potential, it is crucial to select and configure the appropriate analytical approach. For untargeted research, there are two main strategies available: data-dependent analysis (DDA) and data-independent analysis (DIA). Both methods differ in the way the analysis is carried out and in the degree of coverage of the obtained data, which is why each of them can be used in various types of research. The DDA method is based on continuous scanning of the analyzed ions, as a result of which the precursors with the highest intensity are fragmented in the MS2 mode. On the other hand, DIA, due to the use of combined ranges of precursor ion isolation, allows for a deeper analysis of the analyzed compounds. Both approaches also have modifications that improve their operation and enable obtaining more valuable data. Methods combining both techniques are also appearing on the horizon, such as DDIA, which uses the advantages of both methods, opening new analytical possibilities.
... One important piece of metadata required by some models is the normalized collision energy (NCE), which affects fragmentation patterns. NCE is an important parameter to tune based on the type of peptides being fragmented 23,24 . Indeed, other computational workflows have also noted the importance of calibrating NCE to maximize the similarity between experimental spectra and model predictions 1,12 . ...
Recent developments in machine-learning (ML) and deep-learning (DL) have immense potential for applications in proteomics, such as generating spectral libraries, improving peptide identification, and optimizing targeted acquisition modes. Although new ML/DL models for various applications and peptide properties are frequently published, the rate at which these models are adopted by the community is slow, which is mostly due to technical challenges. We believe that, for the community to make better use of state-of-the-art models, more attention should be spent on making models easy to use and accessible by the community. To facilitate this, we developed Koina, an open-source containerized, decentralized and online-accessible high-performance prediction service that enables ML/DL model usage in any pipeline. Using the widely used FragPipe computational platform as example, we show how Koina can be easily integrated with existing proteomics software tools and how these integrations improve data analysis.
... For example, Zhang and Ge (2011) utilized topdown MS/MS with ECD to identify two potential monophosphorylation sites on the positional isoform of monophosphorylated human cardiac troponin I (cTnI): the well-documented Protein Kinase A (PKA) site at Serine 22 (Ser22) and a newly discovered site at either Serine 76 (Ser76) or Threonine 77 (Thr77). While some technical challenges remain, developments in top-down protein analysis over the past few years have improved its ability to unambiguously identify, characterize and quantify thousands of protein forms at high throughput (Révész et al., 2023). ...
This review focuses on changes in nutrition and functional properties of protein-rich foods, primarily attributed to alterations in protein structures. We provide a comprehensive overview and comparison of commonly used laboratory methods for protein structure identification, aiming to offer readers a convenient understanding of these techniques. The review covers a range of detection technologies employed in food protein analysis and conducts an extensive comparison to identify the most suitable method for various proteins. While these techniques offer distinct advantages for protein structure determination, the inherent complexity of food matrices presents ongoing challenges. Further research is necessary to develop and enhance more robust detection methods to improve accuracy in protein conformation and structure analysis.
... The results highlight that CID under conventional conditions is the result of a high number of collisions, even as many as 20-50 collision events. In proteomics, it has become fashionable to use 'composite spectra', which are a combination of low-and high-energy CID spectra [18,20,21,47]. The result is qualitatively similar to the low-pressure CID spectra studied here. ...
We have performed CID experiments on a triple quadrupole instrument, lowering the collision gas pressure by 50 times compared to its conventional value. The results show that at very low-collision gas pressure, single collisions dominate the spectra. Indirectly, these results suggest that under conventional conditions, 20–50 collisions may be typical in CID experiments. The results show a marked difference between low- and high-pressure CID spectra, the latter being characterized in terms of ‘slow heating’ and predominance of consecutive reactions. The results indicate that under single collision conditions, the collisional energy transfer efficiency is very high: nearly 100% of the center of mass kinetic energy is converted to internal energy.
... In addition, Skyline automatically calculates ranges of CE sets based on the analyst's setting and sums the peak area observed over replicates to enhance the accuracy of the optimal CE [16]. In proteomics, Skyline suggests the primary CE value for doubly-or triply-charged peptides using instrument-specific and built-in linear equations from the precursor mass-to-charge ratio (m/z) [16,17]. However, a linear equation for CE prediction during small-molecule analysis is not supported so far. ...
As the number of prohibited drugs has been progressively increasing and analytical methods for detecting such substances are renewed continuously for doping control, the need for more sensitive and accurate doping analysis has increased. To address the urgent need for high throughput and accurate analysis, liquid chromatography with tandem mass spectrometry is actively utilized in case of most of the newly designated prohibited substances. However, because all mass spectrometer vendors provide data processing software that is incapable of handling other instrumental data, it is difficult to cover all doping analysis procedures, from method development to result reporting, on one platform. Skyline is an open-source and vendor-neutral software program invented for the method development and data processing of targeted proteomics. Recently, the utilization of Skyline has been expanding for the quantitative analysis of small molecules and lipids. Herein, we demonstrated Skyline as a simple platform for unifying overall doping control, including the optimization of analytical methods, monitoring of data quality, discovery of suspected doping samples, and validation of analytical methods for detecting newly prohibited substances. For method optimization, we selected the optimal collision energies for 339 prohibited substances. Notably, 195 substances exhibited a signal intensity increase of >110% compared with the signal intensity of the original collision energy. All data related to method validation and quantitative analysis were efficiently visualized, extracted, or calculated using Skyline. Moreover, a comparison of the time consumed and the number of suspicious samples screened in the initial test procedure highlighted the advantages of using Skyline over the commercially available software TraceFinder in doping control.
... These techniques have greatly benefitted from the development of bioorthogonal chemistries, 169 the commercial availability of noncanonical biological buliding blocks, 170 and advances in mass spectrometry techniques. [171][172][173][174] Nevertheless, more efforts are still needed to resolve remaining questions. First, most reported studies represent a snapshot of host-pathogen interactions at certain infection time points but the interplay between host and pathogen is more complex and dynamic and may vary across different stages of infection. ...
With the rapid emergence and the dissemination of microbial resistance to conventional chemotherapy, the shortage of novel antimicrobial drugs has raised a global health threat. As molecular interactions between microbial pathogens and their mammalian hosts are crucial to establish virulence, pathogenicity, and infectivity, a detailed understanding of these interactions has the potential to reveal novel therapeutic targets and treatment strategies. Bidirectional molecular communication between microbes and eukaryotes is essential for both pathogenic and commensal organisms to colonise their host. In particular, several devastating pathogens exploit host signalling to adjust the expression of energetically costly virulent behaviours. Chemical proteomics has emerged as a powerful tool to interrogate the protein interaction partners of small molecules and has been successfully applied to advance host–pathogen communication studies. Here, we present recent significant progress made by this approach and provide a perspective for future studies.
... 21 Higher charge-state ions require higher collision energy to produce similar abundant fragmentation ions as in lower charge-state ions, resulting in insufficient fragmentation spectra for database search. 22 Although previous studies have demonstrated that multiple-CV FAIMS returns more identifications than single-CV FAIMS in DDA proteomics, 5,23 our results showed that multiple-CV FAIMS moderately reduces proteome coverage compared with single-CV FAIMS when using DIA and partially validated previous observations. 4 Importantly, a significant portion of the peptides were identified exclusively in CV = À35 V (Figures 3I and S3I). ...
Here, we present a standardized, “off-the-shelf” proteomics pipeline working in a single 96-well plate to achieve deep coverage of cellular proteomes with high throughput and scalability. This integrated pipeline streamlines a fully automated sample preparation platform, a data-independent acquisition (DIA) coupled with high-field asymmetric waveform ion mobility spectrometer (FAIMS) interface, and an optimized library-free DIA database search strategy. Our systematic evaluation of FAIMS-DIA showing single compensation voltage (CV) at −35 V not only yields the deepest proteome coverage but also best correlates with DIA without FAIMS. Our in-depth comparison of direct-DIA database search engines shows that Spectronaut outperforms others, providing the highest quantifiable proteins. Next, we apply three common DIA strategies in characterizing human induced pluripotent stem cell (iPSC)-derived neurons and show single-shot mass spectrometry (MS) using single-CV (−35 V)-FAIMS-DIA results in >9,000 quantifiable proteins with <10% missing values, as well as superior reproducibility and accuracy compared with other existing DIA methods.
... Following the establishment of the concept of the proteome [53] and mass-spectrometry-based proteomics as a highly useful screening tool in the modern biosciences [54], there has been a steady improvement of sample preparation, mass spectrometric instrumentation and data analysis pipelines using both bottom-up [55][56][57] and top-down proteomic techniques [58][59][60]. Importantly, modern biochemical analyses focus on the unifying concept of dynamic proteoforms being the basic units of protein activity [61][62][63]. ...
The progressive loss of skeletal muscle mass and concomitant reduction in contractile strength plays a central role in frailty syndrome. Age-related neuronal impairments are closely associated with sarcopenia in the elderly, which is characterized by severe muscular atrophy that can considerably lessen the overall quality of life at old age. Mass-spectrometry-based proteomic surveys of senescent human skeletal muscles, as well as animal models of sarcopenia, have decisively improved our understanding of the molecular and cellular consequences of muscular atrophy and associated fiber-type shifting during aging. This review outlines the mass spectrometric identification of proteome-wide changes in atrophying skeletal muscles, with a focus on contractile proteins as potential markers of changes in fiber-type distribution patterns. The observed trend of fast-to-slow transitions in individual human skeletal muscles during the aging process is most likely linked to a preferential susceptibility of fast-twitching muscle fibers to muscular atrophy. Studies with senescent animal models, including mostly aged rodent skeletal muscles, have confirmed fiber-type shifting. The proteomic analysis of fast versus slow isoforms of key contractile proteins, such as myosin heavy chains, myosin light chains, actins, troponins and tropomyosins, suggests them as suitable bioanalytical tools of fiber-type transitions during aging.
... To further confirm the emerging PST analogues' assignments, and in the absence of commercially available certified reference materials (CRMs), their MS 2 spectra obtained by collision-induced dissociation (CID) and higher-energy collisional dissociation (HCD) (see Supplementary Figures S1-S34) were compared with those reported in previous studies [10,13,14,22]. HCD is known to originate spectra similar to those obtained using triple quadrupole fragmentation [29]. The unequivocal identification of classical PSTs in samples was performed by comparing the observed retention times, exact masses, isotope distributions, and fragmentation spectra obtained by CID and HCD with the same parameters obtained after injection of the authentic standards (CRMs). ...
Saxitoxin and its more than 50 analogues are a group of naturally occurring neurotoxins collectively designated as paralytic shellfish toxins (PSTs). PSTs are toxic to humans and maximum legal limits in seafood have been implemented by regulatory authorities worldwide. In the European Union, monitoring of PSTs is performed using the AOAC Official Method 2005.06, based on liquid chromatography coupled with fluorescence detection (LC- FLD). However, this method has been suggested to not effectively detect the emerging C-11 hydroxyl (M-toxins) and benzoate (GC-toxins) analogues, with these analogues currently not being surveyed in monitoring programs. In this study, a liquid chromatography-high resolution mass spectrometry (LC-HRMS) method was used to search for these emerging PSTs in mussels, Mytilus galloprovincialis, contaminated following an intense Gymnodinium catenatum bloom in the Tagus estuary (Lisbon, Portugal). Five M-toxins (M1, M2, M6, dcM6, and dcM10), but no GC-toxins, were detected in the mussels’ whole-soft body tissue. Moreover, the classical PSTs (C1 to C4, GTX 4 to GTX6, dcGTX1 to dcGTX4, dcSTX, dcNEO, and STX) were also found and comprised the largest fraction of the PSTs’ profile. The presence of unregulated PSTs in edible mussel samples suggests potential seafood safety risks and urges further research to determine the frequency of these analogues in seafood and their contribution to toxicity.
