Reliable Detection of Deamidated Peptides from Lens Crystallin Proteins Using Changes in Reversed-Phase Elution Times and Parent Ion Masses

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.
Keywords: proteomics • mass spectrometry • bioinformatics • human lens • deamidation • ion trap • post-translational modification • crystallin • reversed-phase chromatography • mass correction • insolubility • cataracts