Theoretical and experimental prospects for protein identification based solely on accurate mass measurement

We discuss the theoretical and experimental potential and limitations of protein identification by mass measurement of proteolytic peptides and database searching. For peptides differing in composition by one (or two or three) amino acids, a surprisingly high number turn out to have isomers: 10% (or 29% or 53%), considering the 20 common amino acids with equal relative abundance. Even if isomers differing by leucine/isoleucine are excluded, the latter numbers are 14% and 38%--those isomeric peptides cannot be distinguished based on mass alone, and tandem mass spectrometry and/or other additional constraints are needed. However, for nominally isobaric peptides, the mass accuracy and resolving power of broadband Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry theoretically and experimentally suffice to resolve virtually all peptide doublets differing by up to two amino acids--including the smallest mass difference of 3.4 mDa. We demonstrate experimental resolution of another pair of peptides differing by 11 mDa, even when present in a complex mixture of hundreds of other peptides.