Evaluation Of Mass Spectrometry Ms/Ms Spectra For The Presence Of Isopeptide Crosslinked Peptides
| Autor: | Seda Onder, Oksana Lockridge, Lawrence M. Schopfer |
|---|---|
| Rok vydání: | 2021 |
| Předmět: |
0301 basic medicine
Tissue transglutaminase Glutamine Lysine Peptide Biochemistry Physical Chemistry Database and Informatics Methods Tandem Mass Spectrometry Cross-Linking Amino Acids Database Searching Gel Electrophoresis chemistry.chemical_classification Gel electrophoresis Isopeptide bond Multidisciplinary biology Spectrometers Organic Compounds Acidic Amino Acids Neurodegenerative Diseases Proteases Enzymes Chemistry Physical Sciences Engineering and Technology Medicine Basic Amino Acids Mass Spectrometers Research Article Science Equipment macromolecular substances Mass spectrometry Research and Analysis Methods 03 medical and health sciences Electrophoretic Techniques Tubulins Measurement Equipment 030102 biochemistry & molecular biology Chemical Bonding Organic Chemistry Chemical Compounds Biology and Life Sciences Proteins Glutamic acid Cytoskeletal Proteins 030104 developmental biology Tubulin chemistry biology.protein Enzymology Serine Proteases Peptides |
| Zdroj: | PLoS ONE, Vol 16, Iss 7, p e0254450 (2021) PLoS ONE |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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