title: Top-down proteomics on a chromatographic time scale using linear ion trap fourier transform hybrid mass spectrometers.
abstract: Proteomics has grown significantly with the aid of new technologies that consistently are becoming more streamlined. While processing of proteins from a whole cell lysate is typically done in a bottom-up fashion utilizing MS/MS of peptides from enzymatically digested proteins, top-down proteomics is becoming a viable alternative that until recently has been limited largely to offline analysis by tandem mass spectrometry. Here we describe a method for high-resolution tandem mass spectrometery of intact proteins on a chromatographic time scale. In a single liquid chromatography-tandem mass spectrometry (LC-MS/MS) run, we have identified 22 yeast proteins with molecular weights from 14 to 35 kDa. Using anion exchange chromatography to fractionate a whole cell lysate before online LC-MS/MS, we have detected 231 metabolically labeled (14N/15N) protein pairs from Saccharomyces cerevisiae. Thirty-nine additional proteins were identified and characterized from LC-MS/MS of selected anion exchange fractions. Automated localization of multiple acetylations on Histone H4 was also accomplished on an LC time scale from a complex protein mixture. To our knowledge, this is the first demonstration of top-down proteomics (i.e., many identifications) on linear ion trap Fourier transform (LTQ FT) systems using high-resolution MS/MS data obtained on a chromatographic time scale.
authors.affiliation: Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
authors.forename: Bryan A
authors.initials: BA
authors.lastname: Parks
authors.forename: Lihua
authors.initials: L
authors.lastname: Jiang
authors.forename: Paul M
authors.initials: PM
authors.lastname: Thomas
authors.forename: Craig D
authors.initials: CD
authors.lastname: Wenger
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Roth
authors.forename: Michael T
authors.initials: MT
authors.lastname: Boyne
authors.forename: Patricia V
authors.initials: PV
authors.lastname: Burke
authors.forename: Kurt E
authors.initials: KE
authors.lastname: Kwast
authors.forename: Neil L
authors.initials: NL
authors.lastname: Kelleher
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods; D005583:Fourier Analysis; D008970:Molecular Weight; D040901:Proteomics; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis*; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013997:Time Factors
chemical_list: D029701:Saccharomyces cerevisiae Proteins