Heavy Methyl SILAC Metabolic Labeling of Human Cell Lines for High-Confidence Identification of R/K-Methylated Peptides by High-Resolution Mass Spectrometry.
FDR
Heavy methyl SILAC
Liquid chromatography-tandem mass spectrometry (LC-MS/MS)
Metabolic labeling
Protein methylation
Protein methyltransferases
Proteomics
Journal
Methods in molecular biology (Clifton, N.J.)
ISSN: 1940-6029
Titre abrégé: Methods Mol Biol
Pays: United States
ID NLM: 9214969
Informations de publication
Date de publication:
2023
2023
Historique:
entrez:
12
11
2022
pubmed:
13
11
2022
medline:
16
11
2022
Statut:
ppublish
Résumé
Protein methylation is a widespread post-translational modification (PTM) involved in several important biological processes including, but not limited to, RNA splicing, signal transduction, translation, and DNA repair. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is considered today the most versatile and accurate technique to profile PTMs with high precision and proteome-wide depth; however, the identification of protein methylations by MS is still prone to high false discovery rates. In this chapter, we describe the heavy methyl SILAC metabolic labeling strategy that allows high-confidence identification of in vivo methyl-peptides by MS-based proteomics. We provide a general protocol that covers the steps of heavy methyl labeling of cultured cells, protein sample preparation, LC-MS/MS analysis, and downstream computational analysis of the acquired MS data.
Identifiants
pubmed: 36370279
doi: 10.1007/978-1-0716-2863-8_14
doi:
Substances chimiques
Peptides
0
Proteome
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
173-186Informations de copyright
© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.
Références
Murn J, Shi Y (2017) The winding path of protein methylation research: milestones and new frontiers. Nat Rev Mol Cell Biol 18(8):517–527
doi: 10.1038/nrm.2017.35
pubmed: 28512349
Greer EL, Shi Y (2012) Histone methylation: a dynamic mark in health, disease and inheritance. Nat Rev Genet 13(5):343–357
doi: 10.1038/nrg3173
pubmed: 22473383
pmcid: 4073795
Blanc RS, Richard S (2017) Arginine methylation: the coming of age. Mol Cell 65(1):8–24
doi: 10.1016/j.molcel.2016.11.003
pubmed: 28061334
Spadotto V, Giambruno R, Massignani E, Mihailovich M, Maniaci M, Patuzzo F et al (2020) PRMT1-mediated methylation of the microprocessor-associated proteins regulates microRNA biogenesis. Nucleic Acids Res 48(1):96–115
doi: 10.1093/nar/gkz1051
pubmed: 31777917
Qamar S, Wang G, Randle SJ, Ruggeri FS, Varela JA, Lin JQ et al (2018) FUS phase separation is modulated by a molecular chaperone and methylation of arginine cation-pi interactions. Cell 173(3):720–734. e15
doi: 10.1016/j.cell.2018.03.056
pubmed: 29677515
pmcid: 5927716
Wu Q, Schapira M, Arrowsmith CH, Barsyte-Lovejoy D (2021) Protein arginine methylation: from enigmatic functions to therapeutic targeting. Nat Rev Drug Discov 20:509–530
doi: 10.1038/s41573-021-00159-8
pubmed: 33742187
Cao XJ, Garcia BA (2016) Global proteomics analysis of protein lysine methylation. Curr Protoc Protein Sci 86:24.8.1–24.8.19
doi: 10.1002/cpps.16
Levy D (2019) Lysine methylation signaling of non-histone proteins in the nucleus. Cell Mol Life Sci 76(15):2873–2883
doi: 10.1007/s00018-019-03142-0
pubmed: 31123776
Bhat KP, Umit Kaniskan H, Jin J, Gozani O (2021) Epigenetics and beyond: targeting writers of protein lysine methylation to treat disease. Nat Rev Drug Discov 20(4):265–286
doi: 10.1038/s41573-020-00108-x
pubmed: 33469207
pmcid: 8035164
Guo A, Gu H, Zhou J, Mulhern D, Wang Y, Lee KA et al (2014) Immunoaffinity enrichment and mass spectrometry analysis of protein methylation. Mol Cell Proteomics 13(1):372–387
doi: 10.1074/mcp.O113.027870
pubmed: 24129315
Aslebagh R, Wormwood KL, Channaveerappa D, Wetie AGN, Woods AG, Darie CC (2019) Identification of posttranslational modifications (PTMs) of proteins by mass spectrometry. Adv Exp Med Biol 1140:199–224
doi: 10.1007/978-3-030-15950-4_11
pubmed: 31347049
Hart-Smith G, Yagoub D, Tay AP, Pickford R, Wilkins MR (2016) Large scale mass spectrometry-based identifications of enzyme-mediated protein methylation are subject to high false discovery rates. Mol Cell Proteomics 15(3):989–1006
doi: 10.1074/mcp.M115.055384
pubmed: 26699799
pmcid: 4813715
Ong SE, Mittler G, Mann M (2004) Identifying and quantifying in vivo methylation sites by heavy methyl SILAC. Nat Methods 1(2):119–126
doi: 10.1038/nmeth715
pubmed: 15782174
Geoghegan V, Guo A, Trudgian D, Thomas B, Acuto O (2005) Comprehensive identification of arginine methylation in primary T cells reveals regulatory roles in cell signalling. Nat Commun 6:6758
doi: 10.1038/ncomms7758
Ong SE, Mann M (2006) Identifying and quantifying sites of protein methylation by heavy methyl SILAC. Curr Protoc Protein Sci 14:Unit 14.9
pubmed: 18429299
Tay AP, Geoghegan V, Yagoub D, Wilkins MR, Hart-Smith G (2018) MethylQuant: a tool for sensitive validation of enzyme-mediated protein methylation sites from heavy-methyl SILAC data. J Proteome Res 17(1):359–373
doi: 10.1021/acs.jproteome.7b00601
pubmed: 29057651
Massignani E, Cuomo A, Musiani D, Jammula S, Pavesi G, Bonaldi T (2019) hmSEEKER: identification of hmSILAC doublets in MaxQuant output data. Proteomics 19(5):e1800300
doi: 10.1002/pmic.201800300
pubmed: 30656827
Yang F, Shen Y, Camp DG 2nd, Smith RD (2012) High-pH reversed-phase chromatography with fraction concatenation for 2D proteomic analysis. Expert Rev Proteomics 9(2):129–134
doi: 10.1586/epr.12.15
pubmed: 22462785
pmcid: 3337716
Batth TS, Olsen JV (2016) Offline high pH reversed-phase peptide fractionation for deep phosphoproteome coverage. Methods Mol Biol 1355:179–192
doi: 10.1007/978-1-4939-3049-4_12
pubmed: 26584926
Lim Y, Lee JY, Ha SJ, Yu S, Shin JK, Kim HC (2020) Proteome-wide identification of arginine methylation in colorectal cancer tissues from patients. Proteome Sci 18:6
doi: 10.1186/s12953-020-00162-8
pubmed: 32467672
pmcid: 7236946
Hartel NG, Chew B, Qin J, Xu J, Graham NA (2019) Deep protein methylation profiling by combined chemical and immunoaffinity approaches reveals novel PRMT1 targets. Mol Cell Proteomics 18(11):2149–2164
doi: 10.1074/mcp.RA119.001625
pubmed: 31451547
pmcid: 6823857
Musiani D, Massignani E, Cuomo A, Yadav A, Bonaldi T (2020) Biochemical and computational approaches for the large-scale analysis of protein arginine methylation by mass spectrometry. Curr Protein Pept Sci 21(7):725–739
doi: 10.2174/1389203721666200426232531
pubmed: 32338214
Cox J, Mann M (2008) MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol 26(12):1367–1372
doi: 10.1038/nbt.1511
pubmed: 19029910
Cox J, Neuhauser N, Michalski A, Scheltema RA, Olsen JV, Mann M (2011) Andromeda: a peptide search engine integrated into the MaxQuant environment. J Proteome Res 10(4):1794–1805
doi: 10.1021/pr101065j
pubmed: 21254760