title: Enhancing TOF/TOF-based de novo sequencing capability for high throughput protein identification with amino acid-coded mass tagging.
abstract: Because of the intrinsic physical properties of single- or double-charged ions, MALDI-based CID on these peptide precursor ions tends to be incomplete, resulting in a large number of MS/MS spectra unassigned or ambiguously identified. Consequently, the TOF/TOF high throughput capability may not be fully explored and utilized. Here, we describe a novel method for de novo sequence assignment of those MALDI TOF/TOF MS/MS spectra with incomplete or weak fragment ion series. In this approach, the deuterium-labeled lysine and leucine precursors were used in parallel to mass-tag the proteome of a metastatic human hepatocellular carcinoma (HCC) cell line during in vivo cell culturing. These stable isotope precursor markers not only position at terminal but at internal MS/MS fragment ions with the characteristic isotope pattern induced by multiple mass tagging in parallel. This enhanced signal specificity evidently resolved ambiguities in those sparse poor-quality TOF/TOF spectra by providing critical sequential links among MS/MS fragment ions. Our data-dependent approach was able to reduce many false-positives in current genome sequence-based peptide sequencing. With developing new algorithms accordingly, our approach is amenable for automation that will lead to more comprehensive and reliable identification for proteomes.
authors.affiliation: Department of Chemistry, Fudan University, Shanghai 200433, China.
authors.forename: Wenqing
authors.initials: W
authors.lastname: Shui
authors.forename: Yinkun
authors.initials: Y
authors.lastname: Liu
authors.forename: Huizhi
authors.initials: H
authors.lastname: Fan
authors.forename: Huimin
authors.initials: H
authors.lastname: Bao
authors.forename: Shufang
authors.initials: S
authors.lastname: Liang
authors.forename: Pengyuan
authors.initials: P
authors.lastname: Yang
authors.forename: Xian
authors.initials: X
authors.lastname: Chen
mesh_terms: D006528:Carcinoma, Hepatocellular / Q000737:chemistry; D045744:Cell Line, Tumor; D003903:Deuterium; D006801:Humans; D007553:Isotope Labeling; D013058:Mass Spectrometry / Q000379:methods*; D011506:Proteins / Q000032:analysis*; D040901:Proteomics / Q000379:methods; D020539:Sequence Analysis, Protein / Q000379:methods*
chemical_list: D011506:Proteins; D003903:Deuterium