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Supplementary Dataset 20 - GluC PICS2 with E coli proteome-derived tryptic peptide library

ABSTRACT: GluC PICS2 with E coli proteome-derived tryptic peptide library

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Escherichia Coli (ncbitaxon:562)

SUBMITTER: Amy Weeks

PROVIDER: MSV000091266 | MassIVE | Sat Feb 11 19:07:00 GMT 2023

SECONDARY ACCESSION(S): PXD040063

REPOSITORIES: MassIVE

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Project description:We describe the creation of a mass spectral library composed of all identifiable spectra derived from the tryptic digest of the NISTmAb IgG1?. The library is a unique reference spectral collection developed from over six million peptide-spectrum matches acquired by liquid chromatography-mass spectrometry (LC-MS) over a wide range of collision energy. Conventional one-dimensional (1D) LC-MS was used for various digestion conditions and 20- and 24-fraction two-dimensional (2D) LC-MS studies permitted in-depth analyses of single digests. Computer methods were developed for automated analysis of LC-MS isotopic clusters to determine the attributes for all ions detected in the 1D and 2D studies. The library contains a selection of over 12,600 high-quality tandem spectra of more than 3,300 peptide ions identified and validated by accurate mass, differential elution pattern, and expected peptide classes in peptide map experiments. These include a variety of biologically modified peptide spectra involving glycosylated, oxidized, deamidated, glycated, and N/C-terminal modified peptides, as well as artifacts. A complete glycation profile was obtained for the NISTmAb with spectra for 58% and 100% of all possible glycation sites in the heavy and light chains, respectively. The site-specific quantification of methionine oxidation in the protein is described. The utility of this reference library is demonstrated by the analysis of a commercial monoclonal antibody (adalimumab, Humira®), where 691 peptide ion spectra are identifiable in the constant regions, accounting for 60% coverage for both heavy and light chains. The NIST reference library platform may be used as a tool for facile identification of the primary sequence and post-translational modifications, as well as the recognition of LC-MS method-induced artifacts for human and recombinant IgG antibodies. Its development also provides a general method for creating comprehensive peptide libraries of individual proteins.

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Supplementary Dataset 20 - GluC PICS2 with E coli proteome-derived tryptic peptide library

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