Physicochemical properties determining the detection probability of tryptic peptides in Fourier transform mass spectrometry. A correlation study

Anal Chem. 2004 Oct 1;76(19):5872-7. doi: 10.1021/ac049571q.

Abstract

Sequence verification and mapping of posttranslational modifications require nearly 100% sequence coverage in the "bottom-up" protein analysis. Even in favorable cases, routine liquid chromatography-mass spectrometry detects from protein digests peptides covering 50-90% of the sequence. Here we investigated the reasons for limited peptide detection, considering various physicochemical aspects of peptide behavior in liquid chromatography-Fourier transform mass spectrometry (LC-FTMS). No overall correlation was found between the detection probability and peptide mass. In agreement with literature data, the signal increased with peptide hydrophobicity. Surprisingly, the pI values exhibited an opposite trend, with more acidic tryptic peptides detected with higher probability. A mixture of synthesized peptides of similar masses confirmed the hydrophobicity dependence but showed strong positive correlation between pI and signal response. An explanation of this paradoxal behavior was found through the observation that more acidic tryptic peptide lengths tend to be longer. Longer peptides tend to acquire higher average charge state in positive mode electrospray ionization than more basic but shorter counterparts. The induced-current detection in FTMS favors ions in higher charge states, thus providing the observed pI-FTMS signal anticorrelation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cattle
  • Chemical Phenomena
  • Chemistry, Physical
  • Chromatography, Liquid
  • Fourier Analysis*
  • Humans
  • Ions / chemistry
  • Mass Spectrometry / methods*
  • Peptide Fragments / analysis*
  • Peptide Fragments / chemistry*
  • Peptide Fragments / metabolism
  • Probability
  • Trypsin / metabolism*

Substances

  • Ions
  • Peptide Fragments
  • Trypsin