Capillary electrophoresis and electron capture dissociation Fourier transform ion cyclotron resonance mass spectrometry for peptide mixture and protein digest analysis

Recent advances in peptide fragmentation techniques and mass spectrometry have opened up the possibility of combining peptide sepn. techniques, such as capillary electrophoresis (CE) and capillary liq. chromatog., with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and electron capture dissocn. (ECD) in order to characterize peptide mixts. and protein digests. The results presented in this study show that CE/ECD-FT-ICR MS can be employed for peptide characterization in mixts. of std. peptides and in peptides resulting from the enzymic digestion of proteins. Furthermore, the technique has potential for the identification and localization of post-translational modifications in peptides and proteins. [on SciFinder (R)]

Empirical multi-dimensional space for scoring peptide spectrum matches in shotgun proteomics

Unige Anna Laskay, Yury Tsybin

Data-dependent tandem mass spectrometry (MS/MS) is one of the main techniques for protein identification in shotgun proteomics. In a typical LC MS/MS workflow, peptide product ion mass spectra (MS/MS spectra) are compared with those derived theoretically from a protein sequence database. Scoring of these matches results in peptide identifications. A set of peptide identifications is characterized by false discovery rate (FDR), which determines the fraction of false identifications in the set. The total number of peptides targeted for fragmentation is in the range of 10 000 to 20 000 for a several-hour LC MS/MS run. Typically,

Amer Chemical Soc2014

Periodic Sequence Distribution of Product Ion Abundances in Electron Capture Dissociation of Amphipathic Peptides and Proteins

Hisham Ben Hamidane, Yury Tsybin

The rules for product ion formation in electron capture dissociation (ECD) mass spectrometry of peptides and proteins remain unclear. Random backbone cleavage probability and the nonspecific nature of ECD toward amino acid sequence have been reported, contrary to preferential channels of fragmentation in slow heating-based tandem mass spectrometry. Here we demonstrate that for amphipathic peptides and proteins, modulation of ECD product ion abundance (PIA) along the sequence is pronounced. Moreover, because of the specific primary (and presumably secondary) structure of amphipathic peptides, PIA in ECD demonstrates a clear and reproducible periodic sequence distribution. On the one hand, the period of ECD PIA corresponds to periodic distribution of spatially separated hydrophobic and hydrophilic domains within the peptide primary sequence. On the other hand, the same period correlates with secondary structure units, such as a-helical turns, known for solution-phase structure. Based on a number of examples, we formulate a set of characteristic features for ECD of amphipathic peptides and proteins: (1) periodic distribution of PIA is observed and is reproducible in a wide range of ECD parameters and on different experimental platforms; (2) local maxima of PIA are not necessarily located near the charged site; (3) ion activation before ECD not only extends product ion sequence coverage but also preserves ion yield modulation; (4) the most efficient cleavage (e.g. global maximum of ECD PIA distribution) can be remote from the charged site; (5) the number and location of PIA maxima correlate with amino acid hydrophobicity maxima generally to within a single amino acid displacement; and (6) preferential cleavage sites follow a selected hydrogen spine in an a-helical peptide segment. Presently proposed novel insights into ECD behavior are important for advancing understanding of the ECD mechanism, particularly the role of peptide sequence on PIA. An improved ECD model could facilitate protein sequencing and improve identification of unknown proteins in proteomics technologies. In structural biology, the periodic/preferential product ion yield in ECD of a-helical structures potentially opens the way toward de novo site-specific secondary structure determination of peptides and proteins in the gas phase and its correlation with solution-phase structure. (J Am Soc Mass Spectrom 2009, 20, 1182-1192) (C) 2009 Published by Elsevier Inc. on behalf of American Society for Mass Spectrometry

Springer-Verlag2009

Radical Stability Directs Electron Capture and Transfer Dissociation of Beta-Amino Acids in Peptides

Hisham Ben Hamidane, Yury Tsybin, Aleksey Vorobyev

We report on the characteristics of the radical-ion-driven dissociation of a diverse array of beta-amino acids incorporated into alpha-peptides, as probed by tandem electron-capture and electron-transfer dissociation (ECD/ETD) mass spectrometry. The reported results demonstrate a stronger ECD/ETD dependence on the nature of the amino acid side chain for beta-amino acids than for their alpha-form counterparts. In particular, only aromatic (e.g., beta-Phe), and to a substantially lower extent, carbonyl-containing (e.g., beta-Glu and beta-Gln) amino acid side chains, lead to N-C-beta bond cleavage in the corresponding beta-amino acids. We conclude that radical stabilization must be provided by the side chain to enable the radical-driven fragmentation from the nearby backbone carbonyl carbon to proceed. In contrast with the cleavage of backbones derived from alpha-amino acids. ECD of peptides composed mainly of beta-amino acids reveals a shift in cleavage priority from the N-C-beta to the C-alpha-C bond. The incorporation of CH2 groups into the peptide backbone may thus drastically influence the backbone charge solvation preference. The characteristics of radical-driven beta-amino acid dissociation described herein are of particular importance to methods development, applications in peptide sequencing, and peptide and protein modification (e.g., deamidation and isomerization) analysis in life science research. mainly of beta-amino acids reveals a shift in cleavage priority from the N-C-beta to the C-alpha-C bond. The incorporation of CH2 groups into the peptide backbone may thus drastically influence the backbone charge solvation preference. The characteristics of radical-driven beta-amino acid dissociation described herein are of particular importance to methods development, applications in peptide sequencing, and peptide and protein modification (e.g., deamidation and isomerization) analysis in life science research.