Mass spectrometric analysis of protein deamidation – A focus on top-down and middle-down mass spectrometry

Jin, Yutong; Yi, Yunyu; Yeung, Bernice

doi:10.1016/j.ymeth.2020.08.002

Cited by 29 publications

(23 citation statements)

References 62 publications

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“…The detection of deamidated proteoforms in complex samples is challenging due to, for example, coisolation with nondeamidated forms. Therefore, protein deamidation analyses in the literature are usually performed in targeted approaches with a single protein or a simple protein mixture and are not further discussed here …”

Section: Resultsmentioning

confidence: 99%

“…Therefore, protein deamidation analyses in the literature are usually performed in targeted approaches with a single protein or a simple protein mixture and are not further discussed here. 23 The IBH (Figure 2B) is based on the summed intensity of mass features (derived from deconvoluted data) in the assigned bins. This procedure reduces noise and amplifies the influence of higher intensity mass features, which is helpful because deconvolution algorithms often report invalid mass features with low abundance.…”

Section: Data Representationmentioning

confidence: 99%

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MSTopDiff: A Tool for the Visualization of Mass Shifts in Deconvoluted Top-Down Proteomics Data for the Database-Independent Detection of Protein Modifications

et al. 2021

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Top-down proteomics analyzes intact proteoforms with all of their post-translational modifications and genetic and RNA splice variants. In addition, modifications introduced either deliberately or inadvertently during sample preparation, that is, via oxidation, alkylation, or labeling reagents, or through the formation of noncovalent adducts (e.g., detergents) further increase the sample complexity. To facilitate the recognition of protein modifications introduced during top-down analysis, we developed MSTopDiff, a software tool with a graphical user interface written in Python, which allows one to detect protein modifications by calculating and visualizing mass differences in top-down data without the prerequisite of a database search. We demonstrate the successful application of MSTopDiff for the detection of artifacts originating from oxidation, formylation, overlabeling during isobaric labeling, and adduct formation with cations or sodium dodecyl sulfate. MSTopDiff offers several modes of data representation using deconvoluted MS1 or MS2 spectra. In addition to artificial modifications, the tool enables the visualization of biological modifications such as phosphorylation and acetylation. MSTopDiff provides an overview of the artificial and biological modifications in top-down proteomics samples, which makes it a valuable tool in quality control of standard workflows and for parameter evaluation during method development.

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Section: Resultsmentioning

confidence: 99%

Section: Data Representationmentioning

confidence: 99%

MSTopDiff: A Tool for the Visualization of Mass Shifts in Deconvoluted Top-Down Proteomics Data for the Database-Independent Detection of Protein Modifications

et al. 2021

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“…Deamidation is a chemical reaction in which an amide functional group on the side chain of amino acids Asn or Gln is removed, typically converting Asn to aspartic acid (Asp) or isoaspartic acid (isoAsp) and Gln to glutamic acid (Glu) or isopyroglutamic acid (γ-Glu) [45]. There are two types of deamidation: enzymatic deamidation, which uses PNGase F to remove N-glycans from N-linked glycoproteins or glycopeptides [46], and nonenzymatic deamidation (also called chemical deamidation), which occurs spontaneously on proteins and peptides both in vivo and in vitro.…”

Section: Discussionmentioning

confidence: 99%

“…Deamidation on Gln can occur via a mechanism similar to Asn residue by direct hydrolysis at acidic pH or via a glutarimide intermediate at neutral or alkaline pH to yield α-Glu and γ-Glu [50]. In general, the deamidation rate of Gln is much slower than the rate of Asn [45].…”

Section: Discussionmentioning

confidence: 99%

Comprehensive Evaluation of Different TiO2-Based Phosphopeptide Enrichment and Fractionation Methods for Phosphoproteomics

Wang

Yan

et al. 2022

Cells

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Protein phosphorylation is an essential post-translational modification that regulates multiple cellular processes. Due to their low stoichiometry and ionization efficiency, it is critical to efficiently enrich phosphopeptides for phosphoproteomics. Several phosphopeptide enrichment methods have been reported; however, few studies have comprehensively compared different TiO2-based phosphopeptide enrichment methods using complex proteomic samples. Here, we compared four TiO2-based phosphopeptide enrichment methods that used four non-phosphopeptide excluders (glutamic acid, lactic acid, glycolic acid, and DHB). We found that these four TiO2-based phosphopeptide enrichment methods had different enrichment specificities and that phosphopeptides enriched by the four methods had different physicochemical characteristics. More importantly, we discovered that phosphopeptides had a higher deamidation ratio than peptides from cell lysate and that phosphopeptides enriched using the glutamic acid method had a higher deamidation ratio than the other three methods. We then compared two phosphopeptide fractionation methods: ammonia- or TEA-based high pH reversed-phase (HpH-RP). We found that fewer phosphopeptides, especially multi-phosphorylated peptides, were identified using the ammonia-based method than using the TEA-based method. Therefore, the TEA-based HpH-RP fractionation method performed better than the ammonia method. In conclusion, we comprehensively evaluated different TiO2-based phosphopeptide enrichment and fractionation methods, providing a basis for selecting the proper protocols for comprehensive phosphoproteomics.

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“…Deamidation has been reported to show promise in being a molecular marker of age in ancient protein studies and is frequently used to separate contaminant proteins from endogenous data (Ramsøe, et al, 2020, Chowdhury and Buckley, 2021). As proteins degrade, the amide bonds in N and Q amino acids undergo a hydrolysis reaction wherein a mass shift of 0.984 Da is observed per deamidation site (where the -NH2 group on the amino acid sidechain is replaced with an -OH) (Jin, et al, 2022). Analysis of the degree of deamidation identified in the collagen peptides showed that the peptides were not heavily deamidated with N residues averaging approximately 40 – 60% non-deamidated and Q residues averaging approximately 60 – 80% non-deamidated across the bone handles (Figure 6).…”

Section: Discussionmentioning

confidence: 99%

Species identification of early colonial bone artefacts excavated from Pyrmont, Australia, by mass spectrometric identification of collagen peptides

Multari¹,

Sullivan²,

Hartley

et al. 2022

Preprint

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Zooarchaeology by Mass Spectrometry (ZooMS) is a rapidly developing and increasingly utilised peptide mass fingerprinting (PMF) technique that analyses Collagen 1A1 and 1A2 marker peptides for the genus- or species-level identification of fragmentary bones in the archaeological record. Traditionally, this analysis is performed using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-ToF-MS) to identify characteristic m/z values of known marker peptides. Here we present data on the application of a modified ZooMS approach, using nanoflow liquid chromatography - tandem mass spectrometry proteomics, to the analysis of a collection of six early colonial Australian (early to mid-19th Century CE) worked bone artefacts, believed to be mostly knife handles, excavated from a site in Pyrmont, Sydney, Australia in 2017. We were successfully able to identify characteristic marker peptides for bovine COL1A1 and COL1A2 in all six bone artefacts.

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Mass spectrometric analysis of protein deamidation – A focus on top-down and middle-down mass spectrometry

Cited by 29 publications

References 62 publications

MSTopDiff: A Tool for the Visualization of Mass Shifts in Deconvoluted Top-Down Proteomics Data for the Database-Independent Detection of Protein Modifications

MSTopDiff: A Tool for the Visualization of Mass Shifts in Deconvoluted Top-Down Proteomics Data for the Database-Independent Detection of Protein Modifications

Comprehensive Evaluation of Different TiO2-Based Phosphopeptide Enrichment and Fractionation Methods for Phosphoproteomics

Species identification of early colonial bone artefacts excavated from Pyrmont, Australia, by mass spectrometric identification of collagen peptides

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