Abstract
The study of the N-terminome and the precise identification of proteolytic processing events are key in biology. Dedicated methodologies have been developed as the comprehensive characterization of the N-terminome can hardly be achieved by standard proteomics methods. In this context, we have set up a trimethoxyphenyl phosphonium (TMPP) labeling approach that allows the characterization of both N-terminal and internal digestion peptides in a single experiment. This latter point is a major advantage of our strategy as most N-terminomics methods rely on the enrichment of N-terminal peptides and thus exclude internal peptides.
We have implemented a double heavy/light TMPP labeling and an automated data validation workflow that make our doublet N-terminal oriented proteomics (dN-TOP) strategy efficient for high-throughput N-terminome analysis.
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References
Hartmann EM, Armengaud J (2014) N-terminomics and proteogenomics, getting off to a good start. Proteomics 14(23–24):2637–2646
Krug K, Nahnsen S et al (2011) Mass spectrometry at the interface of proteomics and genomics. Mol Biosyst 7(2):284–291
Marino G, Eckhard U et al (2015) Protein termini and their modifications revealed by positional proteomics. ACS Chem Biol 10(8):1754–1764
Gevaert K, Goethals M et al (2003) Exploring proteomes and analyzing protein processing by mass spectrometric identification of sorted N-terminal peptides. Nat Biotechnol 21(5):566–569
Prudova A, auf dem Keller U et al (2010) Multiplex N-terminome analysis of MMP-2 and MMP-9 substrate degradomes by iTRAQ-TAILS quantitative proteomics. Mol Cell Proteomics 9(5):894–911
Gallien S, Perrodou E et al (2009) Ortho-proteogenomics: multiple proteomes investigation through orthology and a new MS-based protocol. Genome Res 19(1):128–135
Helsens K, Van Damme P et al (2011) Bioinformatics analysis of a Saccharomyces cerevisiae N-terminal proteome provides evidence of alternative translation initiation and post-translational N-terminal acetylation. J Proteome Res 10(8):3578–3589
Lange PF, Huesgen PF et al (2014) Annotating N termini for the human proteome project: N termini and Nalpha-acetylation status differentiate stable cleaved protein species from degradation remnants in the human erythrocyte proteome. J Proteome Res 13(4):2028–2044
Vaca Jacome AS, Rabilloud T et al (2015) N-terminome analysis of the human mitochondrial proteome. Proteomics 15(14):2519–2524
Venne AS, Solari FA et al (2015) An improved workflow for quantitative N-terminal charge-based fractional diagonal chromatography (ChaFRADIC) to study proteolytic events in Arabidopsis thaliana. Proteomics 15(14):2458–2469
Vogtle FN, Wortelkamp S et al (2009) Global analysis of the mitochondrial N-proteome identifies a processing peptidase critical for protein stability. Cell 139(2):428–439
Huang ZH, Wu J et al (1997) A picomole-scale method for charge derivatization of peptides for sequence analysis by mass spectrometry. Anal Chem 69(2):137–144
Bertaccini D, Vaca S et al (2013) An improved stable isotope N-terminal labeling approach with light/heavy TMPP to automate proteogenomics data validation: dN-TOP. J Proteome Res 12(6):3063–3070
Ayoub D, Bertaccini D et al (2015) Characterization of the N-terminal heterogeneities of monoclonal antibodies using in-gel charge derivatization of alpha-amines and LC-MS /MS. Anal Chem 87(7):3784–3790
Geer LY, Markey SP et al (2004) Open mass spectrometry search algorithm. J Proteome Res 3(5):958–964
Carapito C, Burel A et al (2014) MSDA, a proteomics software suite for in-depth mass spectrometry data analysis using grid computing. Proteomics 14(9):1014–1019
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Westermann, B., Jacome, A.S.V., Rompais, M., Carapito, C., Schaeffer-Reiss, C. (2017). Doublet N-Terminal Oriented Proteomics for N-Terminomics and Proteolytic Processing Identification. In: Schilling, O. (eds) Protein Terminal Profiling. Methods in Molecular Biology, vol 1574. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6850-3_6
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DOI: https://doi.org/10.1007/978-1-4939-6850-3_6
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