title: Elucidating Proteoform Families from Proteoform Intact-Mass and Lysine-Count Measurements.
abstract: Proteomics is presently dominated by the "bottom-up" strategy, in which proteins are enzymatically digested into peptides for mass spectrometric identification. Although this approach is highly effective at identifying large numbers of proteins present in complex samples, the digestion into peptides renders it impossible to identify the proteoforms from which they were derived. We present here a powerful new strategy for the identification of proteoforms and the elucidation of proteoform families (groups of related proteoforms) from the experimental determination of the accurate proteoform mass and number of lysine residues contained. Accurate proteoform masses are determined by standard LC-MS analysis of undigested protein mixtures in an Orbitrap mass spectrometer, and the lysine count is determined using the NeuCode isotopic tagging method. We demonstrate the approach in analysis of the yeast proteome, revealing 8637 unique proteoforms and 1178 proteoform families. The elucidation of proteoforms and proteoform families afforded here provides an unprecedented new perspective upon proteome complexity and dynamics.
authors.affiliation: Department of Chemistry, University of Wisconsin , 1101 University Avenue, Madison, Wisconsin 53706, United States.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Shortreed
authors.affiliation: Department of Chemistry, University of Wisconsin , 1101 University Avenue, Madison, Wisconsin 53706, United States.
authors.forename: Brian L
authors.initials: BL
authors.lastname: Frey
authors.affiliation: Department of Chemistry, University of Wisconsin , 1101 University Avenue, Madison, Wisconsin 53706, United States.
authors.forename: Mark
authors.initials: M
authors.lastname: Scalf
authors.affiliation: Department of Chemistry, University of Wisconsin , 1101 University Avenue, Madison, Wisconsin 53706, United States.
authors.forename: Rachel A
authors.initials: RA
authors.lastname: Knoener
authors.affiliation: Department of Chemistry, University of Wisconsin , 1101 University Avenue, Madison, Wisconsin 53706, United States.
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Cesnik
authors.affiliation: Department of Chemistry, University of Wisconsin , 1101 University Avenue, Madison, Wisconsin 53706, United States.|Genome Center of Wisconsin, University of Wisconsin , 425G Henry Mall, Room 3420, Madison, Wisconsin 53706, United States.
authors.forename: Lloyd M
authors.initials: LM
authors.lastname: Smith
mesh_terms: D002853:Chromatography, Liquid; D008239:Lysine / Q000737:chemistry* / Q000378:metabolism; D013058:Mass Spectrometry; D008970:Molecular Weight; D046912:Multiprotein Complexes / Q000302:isolation & purification* / Q000378:metabolism; D025941:Protein Interaction Mapping; D040901:Proteomics / Q000379:methods*; D012269:Ribosomal Proteins / Q000302:isolation & purification* / Q000378:metabolism; D054683:Ribosome Subunits, Large, Eukaryotic / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D046912:Multiprotein Complexes; D012269:Ribosomal Proteins; D029701:Saccharomyces cerevisiae Proteins; D008239:Lysine
keywords: NeuCode; PTM; database search; proteoform; proteoform family; proteomics; top-down