Subfemtomole MS and MS/MS peptide sequence analysis using nano-HPLC micro-ESI fourier transform ion cyclotron resonance mass spectrometry.
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).
Martin
S E
SE
Department of Chemistry, University of Virginia, Charlottesville 22904-4319, USA.
Shabanowitz
J
J
Hunt
D F
DF
Marto
J A
JA
D000465:Algorithms; D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D017357:Cyclotrons; D005583:Fourier Analysis; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000737:chemistry; D010449:Peptide Mapping; D010455:Peptides / Q000032:analysis*; D011817:Rabbits; D017421:Sequence Analysis; D021241:Spectrometry, Mass, Electrospray Ionization
D009124:Muscle Proteins; D010455:Peptides