Tandem mass spectrometry with ultrahigh mass accuracy clarifies peptide identification by database retrieval.
A platform was developed to analyze MS/MS spectra from large peptides with low part-per-million mass accuracy, including a commercial-grade software suite. Termed Middle Down Proteomics, this platform identified 7454 peptides from 2-20 kDa (1472 unique) from 555 proteins after 23 LC-MS/MS injections of Lys-C digests of HeLa-S3 nuclear proteins. Along with greatly increased confidence for both peptide identification (expectation values from 10(-89) to 10(-4)) and characterization (up to 18% of peptides were modified in some LC-MS/MS runs), fragmentation data with <2 ppm accuracy enabled error tolerant and routine multiplexed database searching-all clearly demonstrated in this study.
Boyne
Michael T
MT
Department of Chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, Illinois 61801, USA.
Garcia
Benjamin A
BA
Li
Mingxi
M
Zamdborg
Leonid
L
Wenger
Craig D
CD
Babai
Shannee
S
Kelleher
Neil L
NL
D000465:Algorithms; D000595:Amino Acid Sequence; D030562:Databases, Protein; D006367:HeLa Cells; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000032:analysis; D040901:Proteomics / Q000379:methods; D015203:Reproducibility of Results; D012984:Software; D053719:Tandem Mass Spectrometry / Q000379:methods*
D010455:Peptides; D011506:Proteins