Peptide End Sequencing by Orthogonal MALDI Tandem Mass Spectrometry

Abstract

Highly sensitive peptide fragmentation and identification in sequence databases is a cornerstone of proteomics. Previously, a two-layered strategy consisting of MALDI peptide mass fingerprinting followed by electrospray tandem mass spectrometry of the unidentified proteins has been successfully employed. Here, we describe a high-sensitivity/high-throughput system based on orthogonal MALDI tandem mass spectrometry (o-MALDI) and the automated recognition of fragments corresponding to the N- and C-terminal amino acid residues. Robotic deposition of samples onto hydrophobic anchor substrates is employed, and peptide spectra are acquired automatically. The pulsing feature of the QSTAR o-MALDI mass spectrometer enhances the low mass region of the spectra by approximately 1 order of magnitude. Software has been developed to automatically recognize characteristic features in the low mass region (such as the y₁ ion of tryptic peptides), maintaining high mass accuracy even with very low count events. Typically, the sum of the N-terminal two ions (b₂ ion), the third N-terminal ion (b₃ ion), and the two C-terminal fragments of the peptide (y₁ and y₂) can be determined. Given mass accuracy in the low ppm range, peptide end sequencing on one or two tryptic peptides is sufficient to uniquely identify a protein from gel samples in the low silver-stained range. Keywords: proteomics • peptide sequencing • database searching • high-throughput protein identification • tandem mass spectrometry