title: Improved molecular weight-based processing of intact proteins for interrogation by quadrupole-enhanced FT MS/MS.
abstract: Complete coverage of protein primary structure is demonstrated for 37 yeast protein forms between 6 and 30 kDa in an improved platform for Top Down mass spectrometry (MS). Tandem mass spectrometry (MS/MS) for protein identification with 100% sequence coverage is achieved in a highly automated fashion with 15-300-fold less sample amounts than an initial report of a proteome fractionation approach employing preparative gel electrophoresis with an acid-labile surfactant to facilitate reversed phase separation in a second dimension. Using a quadrupole-enhanced Fourier Transform Ion Cyclotron Resonance Mass Spectrometer (FTICRMS) improves the dynamic range for protein detection by approximately 50-fold and MS/MS by approximately 30-fold. The technology development illustrated here typifies an accelerating effort to detect whole proteins in a more general and higher throughput fashion for improved biomarker identification and detection of diverse post-translational modifications. Capillary RPLC is used in both off-line and on-line modes, with one on-line LC/FTMS sample providing 25 observed protein forms from 11 to 22 kDa.
authors.affiliation: Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Du
authors.forename: Fanyu
authors.initials: F
authors.lastname: Meng
authors.forename: Steven M
authors.initials: SM
authors.lastname: Patrie
authors.forename: Leah M
authors.initials: LM
authors.lastname: Miller
authors.forename: Neil L
authors.initials: NL
authors.lastname: Kelleher
mesh_terms: D000595:Amino Acid Sequence; D005583:Fourier Analysis; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D020543:Proteome / Q000737:chemistry*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry
chemical_list: D020543:Proteome; D029701:Saccharomyces cerevisiae Proteins