title: 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.
authors.affiliation: MDS Proteomics A/S, Staermosegaardsvej 6, DK-5230 Odense M, Denmark.
authors.forename: Michael L
authors.initials: ML
authors.lastname: Nielsen
authors.forename: Keiryn L
authors.initials: KL
authors.lastname: Bennett
authors.forename: Brett
authors.initials: B
authors.lastname: Larsen
authors.forename: Marc
authors.initials: M
authors.lastname: Moniatte
authors.forename: Matthias
authors.initials: M
authors.lastname: Mann
mesh_terms: D002138:Calibration; D030562:Databases, Protein; D010455:Peptides / Q000032:analysis*; D040901:Proteomics; D012680:Sensitivity and Specificity; D020539:Sequence Analysis, Protein / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000295:instrumentation; D013997:Time Factors
chemical_list: D010455:Peptides