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.