An alternative to tandem mass spectrometry: isoelectric point and accurate mass for the identification of peptides.
The traditional approach to the identification of peptides in complex biological samples integrally involves the use of tandem mass spectrometry to generate a unique fragmentation pattern in order to accurately assign its identity to a particular protein. In this article we describe the theoretical basis for a new paradigm for the identification of peptides and proteins. This methodology employs the use of accurate mass and peptide isoelectric point (pI) as identification criteria, and represents a change in focus from current tandem mass spectrometry-dominated approaches. A mathematical derivation of the false positive rate associated with accurate mass and pI measurements is presented to demonstrate the utility of the technique. The equations for calculation of the experimental false positive rate allow for the determination of the validity of the data. The false positive rate issue examined in detail here is not restricted to accurate mass-based approaches, but also has application to the tandem mass spectrometry community as well. The theoretical proteomes of Escherichia coli and Rattus norvegicus are used to evaluate the efficacy of this approach. The power of the technique is demonstrated by analyzing a series of peptides with the same monoisotopic masses but with differing isoelectric points. Finally, the speed of algorithm when combined with the experimental peptide analysis has the potential to rapidly accelerate the protein identification process.
Cargile
Benjamin J
BJ
Mass Spectrometry Research Group, Research Triangle Institute, 3040 Cornwallis Road, Research Triangle Park, North Carolina 27709-2194, USA.
Stephenson
James L
JL
D000465:Algorithms; D000595:Amino Acid Sequence; D000818:Animals; D019295:Computational Biology / Q000379:methods; D003198:Computer Simulation; D030562:Databases, Protein; D029968:Escherichia coli Proteins / Q000032:analysis; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D013058:Mass Spectrometry / Q000379:methods / Q000706:statistics & numerical data; D008962:Models, Theoretical; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D010455:Peptides / Q000032:analysis*; D011506:Proteins / Q000032:analysis; D020543:Proteome / Q000032:analysis; D051381:Rats; D015203:Reproducibility of Results
D029968:Escherichia coli Proteins; D010446:Peptide Fragments; D010455:Peptides; D011506:Proteins; D020543:Proteome