Comparative Informatics Analysis to Evaluate Site-Specific Protein Oxidation in Multidimensional LC–MS/MS Data

Redox proteomics has yielded molecular
insight into diseases of
protein dysfunction attributable to oxidative stress, underscoring
the need for robust detection of protein oxidation products. Additionally,
oxidative protein surface mapping techniques utilize hydroxyl radicals
to gain structural insight about solvent exposure. Interpretation
of tandem mass spectral data is a critical challenge for such investigations,
because reactive oxygen species target a wide breadth of amino acids.
Additionally, oxidized peptides may be generated in a wide range of
abundances since the reactivity of hydroxyl radicals with different
amino acids spans 3 orders of magnitude. Taken together, these attributes
of oxidative footprinting pose both experimental and computational
challenges to detecting oxidized peptides that are naturally less
abundant than their unoxidized counterparts. In this study, model
proteins were oxidized electrochemically and analyzed at both the
intact protein and peptide levels. A multidimensional chromatographic
strategy was utilized to expand the dynamic range of oxidized peptide
measurements. Peptide mass spectral data were searched by the “hybrid”
software packages Inspect and Byonic, which incorporate de
novo elements of spectral interpretation into a database
search. This dynamic search capacity accommodates the challenge of
searching for more than 40 oxidative mass shifts that can occur in
a staggering variety of possible combinatorial occurrences. A prevailing
set of oxidized residues was identified with this comparative approach,
and evaluation of these sites was informed by solvent accessible surface
area gleaned through molecular dynamics simulations. Along with increased
levels of oxidation around highly reactive “hotspot”
sites as expected, the enhanced sensitivity of these measurements
uncovered a surprising level of oxidation on less reactive residues.