Enhancing TOF/TOF-based de novo sequencing capability for high throughput protein identification with amino acid-coded mass tagging.
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.
Shui
Wenqing
W
Department of Chemistry, Fudan University, Shanghai 200433, China.
Liu
Yinkun
Y
Fan
Huizhi
H
Bao
Huimin
H
Liang
Shufang
S
Yang
Pengyuan
P
Chen
Xian
X
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*
D011506:Proteins; D003903:Deuterium