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Title: De novo sequencing of unique sequence tags for discovery of post-translational modifications of proteins

De novo sequencing has a promise to discover the protein post-translation modifications; however, such approach is still in their infancy and not widely applied for proteomics practices due to its limited reliability. In this work, we describe a de novo sequencing approach for discovery of protein modifications through identification of the UStags (Anal. Chem. 2008, 80, 1871-1882). The de novo information was obtained from Fourier-transform tandem mass spectrometry for peptides and polypeptides in a yeast lysate, and the de novo sequences obtained were filtered to define a more limited set of UStags. The DNA-predicted database protein sequences were then compared to the UStags, and the differences observed across or in the UStags (i.e., the UStags’ prefix and suffix sequences and the UStags themselves) were used to infer the possible sequence modifications. With this de novo-UStag approach, we uncovered some unexpected variances of yeast protein sequences due to amino acid mutations and/or multiple modifications to the predicted protein sequences. Random matching of the de novo sequences to the predicted sequences were examined with use of two random (false) databases, and ~3% false discovery rates were estimated for the de novo-UStag approach. The factors affecting the reliability (e.g., existence of de novomore » sequencing noise residues and redundant sequences) and the sensitivity are described. The de novo-UStag complements the UStag method previously reported by enabling discovery of new protein modifications.« less
Authors:
; ; ; ; ;
Publication Date:
OSTI Identifier:
946651
Report Number(s):
PNNL-SA-60782
26194; KP1504020; TRN: US200903%%543
DOE Contract Number:
AC05-76RL01830
Resource Type:
Journal Article
Resource Relation:
Journal Name: Analytical Chemistry, 80(20):7742-7754; Journal Volume: 80; Journal Issue: 20
Research Org:
Pacific Northwest National Laboratory (PNNL), Richland, WA (US), Environmental Molecular Sciences Laboratory (EMSL)
Sponsoring Org:
USDOE
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; AMINO ACIDS; MASS SPECTROSCOPY; MODIFICATIONS; MUTATIONS; PEPTIDES; POLYPEPTIDES; POST-TRANSLATION MODIFICATION; PROTEINS; RELIABILITY; RESIDUES; SENSITIVITY; YEASTS; Environmental Molecular Sciences Laboratory