title: Toward efficient analysis of >70 kDa proteins with 100% sequence coverage.
abstract: For complete characterization of larger proteins, primary structural analysis by mass spectrometry must be made more efficient. A straightforward approach is illustrated here using two proteins of 159 and 199 kDa with five and nine Lys residues, respectively. These proteins were degraded by Lys-C to mixtures of peptides ranging in size from 5 to 48 kDa, whose multiply charged ions (from electrospray ionization) are far more amenable than the intact proteins to direct interrogation in a Fourier-transform mass spectrometer. For the 199 kDa PchF of approximately 60% purity, an unfractionated Lys-C digest gave 106 isotopic distributions from 71 components (most of which were below 6 kDa); 15% sequence coverage was obtained. For the > 90% pure PchE (159 kDa), complete sequence coverage was obtained from six Lys-C peptides of 5, 8, 26, 32, 40 and 48 kDa, with all but the largest of these measured at isotopic resolution on a 4.7 Tesla instrument. Practical strategies for implementing this characterization strategy on a proteomic scale are considered.
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School Boston, MA, USA.
authors.forename: A J
authors.initials: AJ
authors.lastname: Forbes
authors.forename: M T
authors.initials: MT
authors.lastname: Mazur
authors.forename: H M
authors.initials: HM
authors.lastname: Patel
authors.forename: C T
authors.initials: CT
authors.lastname: Walsh
authors.forename: N L
authors.initials: NL
authors.lastname: Kelleher
mesh_terms: D000595:Amino Acid Sequence; D005583:Fourier Analysis; D013058:Mass Spectrometry / Q000379:methods; D008954:Models, Biological; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010449:Peptide Mapping; D010636:Phenols / Q000032:analysis / Q000737:chemistry; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D020543:Proteome; D013844:Thiazoles
chemical_list: D010636:Phenols; D011506:Proteins; D020543:Proteome; D013844:Thiazoles; C025316:pyochelin