A strategy for distinguishing modified peptides based on post-digestion 18O labeling and mass spectrometry.
The simultaneous identification of multiple different protein modifications, with or without known mass changes, is a challenging application of mass spectrometry. In this contribution, a strategy for distinguishing modified peptides within a large background of unmodified peptides was demonstrated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) analysis of cytochrome c (Cyt-c) modified with 4-hydroxy-2-nonenal (HNE), based on post-digestion 18O labeling. Labeling of control Cyt-c peptides obtained from in-solution or in-gel digestion with 18O, prior to mixing in the ratio of 1:1 with peptides derived from a modified sample, identified more HNE modifications than a method based on a known mass increment search (Isom AL, Barnes S, Wilson L, Kirk M, Coward L, Darley-Usmar V. J. Am. Soc. Mass Spectrom. 2004; 15: 1136), demonstrating the potential of this strategy to enhance the detection of modified peptides by mass spectrometry. A virtue of the strategy is that it obviates the need for isotopic labeling of the modifier, making the method applicable to the detection of modifications occurring in vivo. Additionally, this technique identified protease auto-cleavage peptides by their altered mass isotopomer distribution due to incomplete 18O exchange, and modified peptides containing 'protein carbonyls' by partial 18O exchange, allowing these peptides to be differentiated during data analysis.
Sun
Gang
G
Department of Biochemistry, School of Medicine, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106, USA. gxs52@po.cwru.edu
Anderson
Vernon E
VE
D000818:Animals; D045304:Cytochromes c / Q000032:analysis* / Q000737:chemistry*; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006736:Horses; D007553:Isotope Labeling / Q000379:methods; D010103:Oxygen Isotopes / Q000032:analysis / Q000737:chemistry; D010449:Peptide Mapping / Q000379:methods*; D010455:Peptides / Q000032:analysis* / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D010103:Oxygen Isotopes; D010455:Peptides; D045304:Cytochromes c