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A new approach for analyzing complicated peptide mixtures by LC/MS
Author(s) -
Berger Scott Jason,
Chakraborty Asish B.,
Gebler John C.
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.lb66-d
Subject(s) - peptide , mass spectrometry , chemistry , fragmentation (computing) , chromatography , peptide sequence , reproducibility , analytical chemistry (journal) , biochemistry , biology , gene , ecology
LC/MS peptide maps are useful tools for characterizing proteins and their modifications. In this work, we have employed a new methodology (LC/MSE), which combines peptide MS with global multiplexed peptide fragmentation MS analysis in a single experiment. We have evaluated this approach by producing tryptic peptide maps of yeast enolase. Digests were analyzed by capillary LC/Q‐Tof MS. The Q‐Tof collision cell was alternately cycled between low energy (peptide MS mode) and elevated energy (MSE fragmentation mode) during data acquisition. MSE fragment data was associated with peptide MS data by time‐resolved mass alignment to produce the precursor‐fragment ion groupings that were used for peptide identification. From the MS data, high peptide map coverage (98% of sequence from peptides >3 AA) was reproducibly observed. The MS response of different enolase peptides varied 175‐fold within a single run. Replicate analysis revealed excellent analytical reproducibility (RT of 0.6% CV, avg. mass accuracy 5.1 ppm, and peptide intensity 8% CV) with this approach. The ability to deconvolute the multiplexed MSE fragmentation data was confirmed, and the relationship between peptide intensity and MS/MS quality was found to be reflective of both peptide precursor abundance and individual peptide sequence.