Single peptide-based protein identification in human proteome through MALDI-TOF MS coupled with amino acids coded mass tagging

Anal Chem. 2003 Mar 15;75(6):1316-24. doi: 10.1021/ac020482s.

Abstract

Identification of proteins with low sequence coverage using mass spectrometry (MS) requires tandem MS/MS peptide sequencing. It is very challenging to obtain a complete or to interpret an incomplete tandem MS/MS spectrum from fragmentation of a weak peptide ion signal for sequence assignment. Here, we have developed an effective and high-throughput MALDI-TOF-based method for the identification of membrane and other low-abundance proteins with a simple, one-dimensional separation step. In this approach, several stable isotope-labeled amino acid precursors were selected to mass-tag, in parallel, the human proteome of human skin fibroblast cells in a residue-specific manner during in vivo cell culturing. These labeled residues can be recognized by their characteristic isotope patterns in MALDI-TOF MS spectra. The isotope pattern of particular peptides induced by the different labeled precursors provides information about their amino acid compositions. The specificity of peptide signals in a peptide mass mapping is thus greatly enhanced, resolving a high degree of mass degeneracy of proteolytic peptides derived from the complex human proteome. Further, false positive matches in database searching can be eliminated. More importantly, proteins can be accurately identified through a single peptide with its m/z value and partial amino acid composition. With the increased solubility of hydrophobic proteins in SDS, we have demonstrated that our approach is effective for the identification of membrane and low-abundant proteins with low sequence coverage and weak signal intensity, which are often difficult for obtaining informative fragment patterns in tandem MS/MS peptide sequencing analysis.

Publication types

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

MeSH terms

  • Amino Acids
  • Fibroblasts / chemistry
  • Fibroblasts / cytology
  • Humans
  • Molecular Probes
  • Peptide Mapping*
  • Proteins / analysis*
  • Proteomics / methods*
  • Radioactive Tracers
  • Skin / cytology
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / methods*

Substances

  • Amino Acids
  • Molecular Probes
  • Proteins
  • Radioactive Tracers