Reliable detection of deamidated peptides from lens crystallin proteins using changes in reversed-phase elution times and parent ion masses

Surendra Dasari, Phillip Wilmarth, D. Leif Rustvold, Michael A. Riviere, Srinivasa R. Nagalla, Larry David

Research output: Contribution to journalArticle

27 Citations (Scopus)

Abstract

Identifying deamidated peptides using low-resolution mass spectrometry is difficult because traditional database search programs cannot accurately detect modified peptides when the mass differences are only 0.984 Da. In this study, we utilized differential reversed-phase elution behavior of deamidated and corresponding unmodified peptide forms to significantly improve deamidation detection on a low-resolution LCQ ion trap instrument. We also improved the mass measurements of unmodified and deamidated peptide forms by averaging survey scans across each chromatogram peak. Tryptic digests of a series of normal (3-day old, 2-year old, 18-year old, 35-year old, and 70-year old) and cataractous (93-year old) human lens samples were used to produce large numbers of potentially deamidated peptides. The complex peptide mixtures were separated by strong cation exchange (SCX) chromatography followed by reversed-phase (RP) chromatography. Synthetic peptides were used to show that unmodified and deamidated peptides coeluted during the SCX separation and were completely resolved with the RP conditions used. Retention time shifts (RTS) and mass differences (ΔM) of deamidated lens peptides and their corresponding unmodified forms were manually determined for the 70-year old lens sample. These values were used to assign correct or incorrect deamidation identifications from SEQUEST searches where deamidation was specified as a variable modification. Manual validation of SEQUEST identifications from synthetic peptides, 3-day old, and 70-year old samples had an overall 42% deamidation detection accuracy. Filtering SEQUEST identifications using RTS and ΔM constraints resulted in >93% deamidation detection accuracy. An algorithm was developed to automate this method, and 72 Crystallin deamidation sites, 18 of which were not previously reported in human lens tissue, were detected.

Original languageEnglish (US)
Pages (from-to)3819-3826
Number of pages8
JournalJournal of Proteome Research
Volume6
Issue number9
DOIs
StatePublished - Sep 2007

Fingerprint

Crystallins
Lenses
Ions
Peptides
Proteins
Chromatography
Cations
Reverse-Phase Chromatography
Phase behavior
Complex Mixtures
Mass spectrometry
Mass Spectrometry
Databases
Tissue

Keywords

  • Bioinformatics
  • Cataracts
  • Crystallin
  • Deamidation
  • Human lens
  • Insolubility
  • Ion trap
  • Mass correction
  • Mass spectrometry
  • Post-translational modification
  • Proteomics
  • Reversed-phase chromatography

ASJC Scopus subject areas

  • Genetics
  • Biotechnology
  • Biochemistry

Cite this

Reliable detection of deamidated peptides from lens crystallin proteins using changes in reversed-phase elution times and parent ion masses. / Dasari, Surendra; Wilmarth, Phillip; Rustvold, D. Leif; Riviere, Michael A.; Nagalla, Srinivasa R.; David, Larry.

In: Journal of Proteome Research, Vol. 6, No. 9, 09.2007, p. 3819-3826.

Research output: Contribution to journalArticle

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abstract = "Identifying deamidated peptides using low-resolution mass spectrometry is difficult because traditional database search programs cannot accurately detect modified peptides when the mass differences are only 0.984 Da. In this study, we utilized differential reversed-phase elution behavior of deamidated and corresponding unmodified peptide forms to significantly improve deamidation detection on a low-resolution LCQ ion trap instrument. We also improved the mass measurements of unmodified and deamidated peptide forms by averaging survey scans across each chromatogram peak. Tryptic digests of a series of normal (3-day old, 2-year old, 18-year old, 35-year old, and 70-year old) and cataractous (93-year old) human lens samples were used to produce large numbers of potentially deamidated peptides. The complex peptide mixtures were separated by strong cation exchange (SCX) chromatography followed by reversed-phase (RP) chromatography. Synthetic peptides were used to show that unmodified and deamidated peptides coeluted during the SCX separation and were completely resolved with the RP conditions used. Retention time shifts (RTS) and mass differences (ΔM) of deamidated lens peptides and their corresponding unmodified forms were manually determined for the 70-year old lens sample. These values were used to assign correct or incorrect deamidation identifications from SEQUEST searches where deamidation was specified as a variable modification. Manual validation of SEQUEST identifications from synthetic peptides, 3-day old, and 70-year old samples had an overall 42{\%} deamidation detection accuracy. Filtering SEQUEST identifications using RTS and ΔM constraints resulted in >93{\%} deamidation detection accuracy. An algorithm was developed to automate this method, and 72 Crystallin deamidation sites, 18 of which were not previously reported in human lens tissue, were detected.",
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