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

Abstract

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 three 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, three 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 forty 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.

Organic and inorganic mercurials have distinct effects on cellular thiols, metal homeostasis, and Fe-binding proteins in Escherichia coli

“…cysteine) that Hg prefers. ROS-induced protein modifications were not searched in the proteomics datasets, due to difficulty in distinguishing true in vivo induced modifications versus artifacts arising from sample processing without special considerations [56], which was not a research goal of this proteomics project.…”

The O-glycomap of Lubricin, a Novel Mucin Responsible for Joint Lubrication, Identified by Site-specific Glycopeptide Analysis

“…The raw files containing centroid MS 2 spectra were searched against UniProt (AC# Q92954, released February 19, 2014), NCBI (released August 13, 2013; 251,429 human entries), and Swiss-Prot (version 2013 05; 20,257 human entries) human protein databases using the in-house version of the Mascot software (v.2.2.04, Matrix Science Inc., Boston, MA). The raw file was also converted to mzXML and mgf formats by Software from Seattle Proteome Center (ReAdW 4.3.1), and searches were carried out using the online GPM ( 32 ) and Byonic software (Protein Matrics, San Carlos, CA) ( 33 ). The search parameters for GPM software were set as follows: peptide tolerance, 4 ppm; MS 2 tolerance, 0.5 Da; enzyme, trypsin; one missed cleavage allowed; fixed carbamidomethyl modification of cysteines; and variable modifications of HexNAc (203.0794 Da) of Ser.…”

Global Protein Oxidation Profiling Suggests Efficient Mitochondrial Proteome Homeostasis During Aging

“…A clear advantage of the used untargeted proteomic approach is no a priori limitation to a particular protein modification. However, previous untargeted proteome analyses have concentrated almost exclusively on the identification of post-translational modifications, rather than on their quantification (61,62). Based on these previous qualitative studies it is well-known that an extremely diverse mixture of protein oxidation products occurs during aging, which we aimed to analyze quantitatively, too.…”