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J Proteome Res. 2005 Jan-Feb;4(1):83-90.

Enhancing TOF/TOF-based de novo sequencing capability for high throughput protein identification with amino acid-coded mass tagging.

Author information

1
Department of Chemistry, Fudan University, Shanghai 200433, China.

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.

PMID:
15707361
DOI:
10.1021/pr049850u
[Indexed for MEDLINE]

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