Evaluation of mass spectrometry MS/MS spectra for the presence of isopeptide crosslinked peptides

Isopeptide crosslinked proteins can be the product of transglutaminase or of exposure to organophosphorus toxicants (OP). Transglutaminase links glutamine to lysine with loss of ammonia. OP toxicants induce a link between glutamic acid and lysine with loss of water. Our goal was to establish criteria to distinguish real from false isopeptide crosslinks reported by software searches of mass spectrometry data. We used fragmentation spectra of tryptic peptides from MAP-rich tubulin Sus scrofa as a test system for detection of naturally-occurring isopeptide crosslinks. Data were analyzed with Protein Prospector. Criteria for the assignments included the presence of at least 1 crosslink specific product ion, fragment ions from both peptides, Protein Prospector scores ≥20, and best fit of the MS/MS data to the crosslinked peptide as opposed to a linear peptide. Out of 301,364 spectra, 15 potential transglutaminase-type crosslinked peptide candidates were identified. Manual evaluation of these MS/MS spectra reduced the number to 1 valid crosslink between Q112 of NFH and K368 of Tau. Immunopurification with anti-isopeptide 81D1C2 confirmed that MAP-rich tubulin contained only one isopeptide. Support for this isopeptide bond was obtained by showing that transglutaminase was capable of incorporating dansyl-aminohexyl -QQIV into K368. A model of the KIETHK-QLEAHNR isopeptide was synthesized with the aid of transglutaminase. MS/MS spectra of the model validated our interpretation of the native isopeptide. An OP-induced isopeptide bond between K163 of tubulin alpha-1A and E158 of tubulin beta-4B was induced by treating MAP-rich tubulin with 100 μM chlorpyrifos oxon. This crosslink was supported by the criteria described above and by the presence of diethoxyphospho-lysine 163 in the tubulin alpha-1A peptide. The information obtained in this work is valuable for future studies that aim to understand why exposure to OP is associated with increased risk of neurodegenerative disease.

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Mass Spectrometry Data Repository Enhances Novel Metabolite Discoveries with Advances in Computational Metabolomics

Metabolites ◽

10.3390/metabo9060119 ◽

2019 ◽

Vol 9 (6) ◽

pp. 119 ◽

Cited By ~ 8

Author(s):

Hiroshi Tsugawa ◽

Aya Satoh ◽

Haruki Uchino ◽

Tomas Cajka ◽

Makoto Arita ◽

...

Keyword(s):

Mass Spectrometry ◽

Acyl Chain ◽

Algal Species ◽

Mass Spectrometry Data ◽

Data Repository ◽

Data Repositories ◽

Specific Product ◽

Untargeted Analysis ◽

Targeted Analysis ◽

Chlorella Variabilis

Mass spectrometry raw data repositories, including Metabolomics Workbench and MetaboLights, have contributed to increased transparency in metabolomics studies and the discovery of novel insights in biology by reanalysis with updated computational metabolomics tools. Herein, we reanalyzed the previously published lipidomics data from nine algal species, resulting in the annotation of 1437 lipids achieving a 40% increase in annotation compared to the previous results. Specifically, diacylglyceryl-carboxyhydroxy-methylcholine (DGCC) in Pavlova lutheri and Pleurochrysis carterae, glucuronosyldiacylglycerol (GlcADG) in Euglena gracilis, and P. carterae, phosphatidylmethanol (PMeOH) in E. gracilis, and several oxidized phospholipids (oxidized phosphatidylcholine, OxPC; phosphatidylethanolamine, OxPE; phosphatidylglycerol, OxPG; phosphatidylinositol, OxPI) in Chlorella variabilis were newly characterized with the enriched lipid spectral databases. Moreover, we integrated the data from untargeted and targeted analyses from data independent tandem mass spectrometry (DIA-MS/MS) acquisition, specifically the sequential window acquisition of all theoretical fragment-ion MS/MS (SWATH-MS/MS) spectra, to increase the lipidomic annotation coverage. After the creation of a global library of precursor and diagnostic ions of lipids by the MS-DIAL untargeted analysis, the co-eluted DIA-MS/MS spectra were resolved in MRMPROBS targeted analysis by tracing the specific product ions involved in acyl chain compositions. Our results indicated that the metabolite quantifications based on DIA-MS/MS chromatograms were somewhat inferior to the MS1-centric quantifications, while the annotation coverage outperformed those of the untargeted analysis of the data dependent and DIA-MS/MS data. Consequently, integrated analyses of untargeted and targeted approaches are necessary to extract the maximum amount of metabolome information, and our results showcase the value of data repositories for the discovery of novel insights in lipid biology.

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Natural isotope correction improves analysis of protein modification dynamics

10.1101/2020.10.31.361725 ◽

2020 ◽

Author(s):

Jörn Dietze ◽

Alienke van Pijkeren ◽

Mathias Ziegler ◽

Marcel Kwiatkowski ◽

Ines Heiland

Keyword(s):

Mass Spectrometry ◽

Protein Modification ◽

High Resolution Mass Spectrometry ◽

Mass Spectrometry Data ◽

Stable Isotope Labelling ◽

Isotope Labelling ◽

Naturally Occurring ◽

Natural Isotope ◽

Experimental Approaches ◽

Correct Estimation

AbstractStable isotope labelling in combination with high resolution mass spectrometry approaches are increasingly used to analyse both metabolite and protein modification dynamics. To enable correct estimation of the resulting dynamics it is critical to correct the measured values for naturally occurring stable isotopes, a process commonly called isotopologue correction or deconvolution. While the importance of isotopologue correction is well recognized in metabolomics, it has received far less attention in proteomics approaches. Although several tools exist that enable isotopologue correction of mass spectrometry data, none of them is universally applicable for all potential experimental approaches. We here present PICor which has been streamlined for multiple isotope labelling isotopologue correction in proteomics or metabolomics approaches. We demonstrate the importance for accurate measurement of the dynamics of protein modifications, such as histone acetylation.

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Natural isotope correction improves analysis of protein modification dynamics

Analytical and Bioanalytical Chemistry ◽

10.1007/s00216-021-03732-7 ◽

2021 ◽

Author(s):

Jörn Dietze ◽

Alienke van Pijkeren ◽

Anna-Sophia Egger ◽

Mathias Ziegler ◽

Marcel Kwiatkowski ◽

...

Keyword(s):

Mass Spectrometry ◽

Protein Modification ◽

High Resolution Mass Spectrometry ◽

Accurate Determination ◽

Mass Spectrometry Data ◽

Stable Isotope Labelling ◽

Isotope Labelling ◽

Naturally Occurring ◽

Correct Estimation

AbstractStable isotope labelling in combination with high-resolution mass spectrometry approaches are increasingly used to analyze both metabolite and protein modification dynamics. To enable correct estimation of the resulting dynamics, it is critical to correct the measured values for naturally occurring stable isotopes, a process commonly called isotopologue correction or deconvolution. While the importance of isotopologue correction is well recognized in metabolomics, it has received far less attention in proteomics approaches. Although several tools exist that enable isotopologue correction of mass spectrometry data, the majority is tailored for the analysis of low molecular weight metabolites. We here present PICor which has been developed for isotopologue correction of complex isotope labelling experiments in proteomics or metabolomics and demonstrate the importance of appropriate correction for accurate determination of protein modifications dynamics, using histone acetylation as an example.

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Recognizing Contamination Fragment Ions in Liquid Chromatography–Tandem Mass Spectrometry Data

Journal of the American Society for Mass Spectrometry ◽

10.1021/jasms.0c00478 ◽

2021 ◽

Author(s):

Shipei Xing ◽

Huaxu Yu ◽

Min Liu ◽

Qingquan Jia ◽

Zhi Sun ◽

...

Keyword(s):

Mass Spectrometry ◽

Liquid Chromatography ◽

Tandem Mass Spectrometry ◽

Mass Spectrometry Data ◽

Tandem Mass ◽

Fragment Ions ◽

Chromatography Tandem Mass Spectrometry ◽

Liquid Chromatography Tandem Mass ◽

Tandem Mass Spectrometry Data

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High Throughput Analysis of Tandem Mass Spectrometry Data for Peptides

Laboratory Automation News ◽

10.1177/221106829700200206 ◽

1997 ◽

Vol 2 (2) ◽

pp. 28-31

Author(s):

John R. Yates ◽

Edwin Carmack ◽

Lara Hays ◽

Jimmy Eng

Keyword(s):

Mass Spectrometry ◽

Tandem Mass Spectrometry ◽

High Throughput ◽

Mass Spectrometry Data ◽

Mass Analyzer ◽

Tandem Mass ◽

Peptide Ions ◽

High Throughput Analysis ◽

Substantial Impact ◽

Fragment Ions

In recent years tandem mass spectrometry has made a substantial impact on the sequence analysis of peptides ( 1 ). In this process peptide ions are dissociated in a collision cell to produce a collection of fragment ions. The m/z values of the fragment ions are determined in the second mass analyzer. Fortuitously, peptide ions fragment primarily around the amide linkages or peptide bonds in a manner that produces a ladder of sequence ions. This method of analysis for peptides has several advantages; high throughput and sensitivity, the ability to analyze peptides contained in mixtures, and lastly the ability to observe covalent modifications to the structure.

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