Subfemtomole MS and MS/MS peptide sequence analysis using nano-HPLC micro-ESI fourier transform ion cyclotron resonance mass spectrometry

Anal Chem. 2000 Sep 15;72(18):4266-74. doi: 10.1021/ac000497v.

Abstract

Subfemtomole peptide sequence analysis has been achieved using microcapillary HPLC columns, with integrated nanoelectrospray emitters, coupled directly to a Fourier transform ion cyclotron resonance mass spectrometer. Accurate mass (+/-0.010 Da) peptide maps are generated from a standard six-protein digest mixture, whose principle components span a concentration dynamic range of 1000:1. Iterative searches against approximately 189000 entries in the OWL database readily identify each protein, with high sequence coverage (20-60%), from as little as 10 amol loaded on-column. In addition, a simple variable-flow HPLC apparatus provides for on-line tandem mass spectrometric analysis of tryptic peptides at the 400-amol level. MS/MS data are searched against approximately 280000 entries in a nonredundant protein database using SEQUEST. Accurate precursor and product ion mass information readily identifies primary amino acid sequences differing by asparagine vs aspartic acid (deltam = 0.98 Da) and glutamine vs lysine (deltam = 0.036 Da).

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Algorithms
  • Amino Acid Sequence
  • Animals
  • Chromatography, High Pressure Liquid
  • Cyclotrons
  • Fourier Analysis
  • Molecular Sequence Data
  • Muscle Proteins / chemistry
  • Peptide Mapping
  • Peptides / analysis*
  • Rabbits
  • Sequence Analysis
  • Spectrometry, Mass, Electrospray Ionization

Substances

  • Muscle Proteins
  • Peptides