title: The Negative Mode Proteome with Activated Ion Negative Electron Transfer Dissociation (AI-NETD).
abstract: The field of proteomics almost uniformly relies on peptide cation analysis, leading to an underrepresentation of acidic portions of proteomes, including relevant acidic posttranslational modifications. Despite the many benefits negative mode proteomics can offer, peptide anion analysis remains in its infancy due mainly to challenges with high-pH reversed-phase separations and a lack of robust fragmentation methods suitable for peptide anion characterization. Here, we report the first implementation of activated ion negative electron transfer dissociation (AI-NETD) on the chromatographic timescale, generating 7,601 unique peptide identifications from Saccharomyces cerevisiae in single-shot nLC-MS/MS analyses of tryptic peptides-a greater than 5-fold increase over previous results with NETD alone. These improvements translate to identification of 1,106 proteins, making this work the first negative mode study to identify more than 1,000 proteins in any system. We then compare the performance of AI-NETD for analysis of peptides generated by five proteases (trypsin, LysC, GluC, chymotrypsin, and AspN) for negative mode analyses, identifying as many as 5,356 peptides (1,045 proteins) with LysC and 4,213 peptides (857 proteins) with GluC in yeast-characterizing 1,359 proteins in total. Finally, we present the first deep-sequencing approach for negative mode proteomics, leveraging offline low-pH reversed-phase fractionation prior to online high-pH separations and peptide fragmentation with AI-NETD. With this platform, we identified 3,467 proteins in yeast with trypsin alone and characterized a total of 3,730 proteins using multiple proteases, or nearly 83% of the expressed yeast proteome. This work represents the most extensive negative mode proteomics study to date, establishing AI-NETD as a robust tool for large-scale peptide anion characterization and making the negative mode approach a more viable platform for future proteomic studies.
authors.affiliation: From the ‡Department of Chemistry, §Genome Center, and.
authors.forename: Nicholas M
authors.initials: NM
authors.lastname: Riley
authors.affiliation: From the ‡Department of Chemistry, §Genome Center, and.
authors.forename: Matthew J P
authors.initials: MJ
authors.lastname: Rush
authors.affiliation: From the ‡Department of Chemistry, §Genome Center, and.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Rose
authors.affiliation: From the ‡Department of Chemistry, §Genome Center, and.
authors.forename: Alicia L
authors.initials: AL
authors.lastname: Richards
authors.affiliation: From the ‡Department of Chemistry, §Genome Center, and.
authors.forename: Nicholas W
authors.initials: NW
authors.lastname: Kwiecien
authors.affiliation: §Genome Center, and.
authors.forename: Derek J
authors.initials: DJ
authors.lastname: Bailey
authors.affiliation: §Genome Center, and.
authors.forename: Alexander S
authors.initials: AS
authors.lastname: Hebert
authors.affiliation: §Genome Center, and.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Westphall
authors.affiliation: From the ‡Department of Chemistry, §Genome Center, and ¶Department of Biomolecular Chemistry University of Wisconsin, Madison, Wisconsin, 53706 jcoon@chem.wisc.edu.
authors.forename: Joshua J
authors.initials: JJ
authors.lastname: Coon
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods; D004583:Electrons; D006863:Hydrogen-Ion Concentration; D020543:Proteome; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D020543:Proteome; D029701:Saccharomyces cerevisiae Proteins