Targeted 18O-labeling for improved proteomic analysis of carbonylated peptides by mass spectrometry

Mikel R. Roe 0 3 Thomas F. McGowan 0 1 LaDora V. Thompson 0 2 Timothy J. Griffin 0 3 0 Address reprint requests to Dr. T. J. Griffin, Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota , Minneapolis, MN 55455, USA 1 Center for Mass Spectrometry and Proteomics, University of Minnesota , Minneapolis, MN, USA 2 Department of Physical Medicine and Rehabilitation, University of Minnesota , Minneapolis, MN, USA 3 Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota , Minneapolis, MN, USA Proteomic characterization of carbonylated amino acid sites currently relies on confidently matching tandem mass spectra (MS2) to peptides within a sequence database. Although effective to some degree, reliable proteomic characterization of carbonylated peptides using this approach remains a challenge needing new, complementary solutions. To this end, we developed a method based on partial 18O-labeling of reactive carbonyl modifications, which produces a unique isotope signature in mass spectra of carbonylated peptides and enables their detection without reliance on matching MS2 spectra to a peptide sequence. Key to our method were optimized measures for eliminating trypsin-catalyzed incorporation of 18O at peptide C-termini, and for stabilizing the incorporated 18O within the carbonyl modification to prevent its loss during liquid chromatography separation. Applying our method to a rat skeletal muscle homogenate treated with the carbonyl modification 4-hyroxynonenal (4-HNE), we demonstrated its compatibility with solid-phase hydrazide enrichment of carbonylated peptides from complex mixtures. Additionally, we demonstrated the value of 18O isotope signatures for confirming HNE-modified peptide sequences matched via sequence database searching, and identifying modified peptides missed by MS2 and/or sequence database searching. Combining our 18O-labeling method with a customized automated software script, we systematically evaluated for the first time the efficiency of MS2 and sequence database searching for identifying HNE-modified peptides. We estimated that less than half of the modified peptides selected for MS2 were successfully identified. Collectively, our method and software should provide valuable new tools for investigators studying protein carbonylation via mass spectrometry-based proteomics. (J Am Soc Mass Spectrom 2010, 21, 1190 -1203) 2010 American Society for Mass Spectrometry - Ttone and aldehyde moieties into proteins, known he post-translational introduction of reactive keas protein carbonylation, is a classic marker of oxidative stress that correlates well with both the aging process itself as well as various age-associated diseases, ranging from Alzheimers disease and Parkinsons disease to amyotrophic lateral sclerosis and diabetes [1]. While a definitive role in disease etiology has yet to be established, the deleterious effect carbonylation has on protein function provides a putative biochemical mechanism through which this irreversible modification may contribute towards the initiation and propagation of disease [2]. To further characterize the basic biology of protein carbonylation and thus better define its potential pathologic role, the specific proteins and amino acids carbonylated throughout disease progression need to be identified [2]. Characterizing protein carbonylation on a proteomewide scale is a core objective in the emerging field of redox proteomics, which seeks to characterize proteins susceptible to oxidative or nitrosative modifications [3]. Tandem mass spectrometry (MS2)-based proteomics enables both the identification of carbonylated proteins and the localization of the corresponding carbonyl to a specific amino acid, thus providing a powerful tool in redox proteomics. However, such studies for protein carbonylation are not routine, as several challenges complicate the process. One challenge is due to the complexity of carbonyl modifications, which involves a number of mechanisms generating various chemically unique reactive carbonyls of differing masses that target several amino acids. For example, carbonyls may be directly introduced into the side chains of Lys, Arg, Pro, and Thr via metal catalyzed oxidation, and into the side chain of Glu and the N-termini of peptides via -amidation of the protein backbone [4, 5]. Alternatively, reactive carbonyl intermediates derived from protein glycation and lipid peroxidation target the side chains of Lys and Arg, and Cys, His, Lys, and Arg, respectively [6 9]. Importantly, most of these reactive carbonyl moieties (in the form of aldehydes or, to a lesser extent, ketones) retain their reactivity following conjugation, and are thus susceptible to subsequent Schiff-base bond formation. Another challenge lies in the relatively low abundance of carbonylated proteins within complex biological mixtures. To address this challenge, front-end enrichment methods that target this substechiometric protein population have been developed. Primarily, these methods rely on covalent chemistry-based enrichment methods exploiting the reactivity of hydrazides with reactive carbonyls, enabling the global analysis of carbonylated proteomes [2]. One common approach is to enrich carbonylated proteins labeled with reagents such as biotin-hydrazide, or variations thereof, via avidin-affinity chromatography before their identification by mass spectrometry. This approach has proven useful for characterizing the carbonyl proteomes of various mammalian-derived protein lysates generated from plasma [10], tissue homogenates [1113], mitochondrial extracts [14], and tissue-derived cell lines [15, 16]. An important caveat regarding the biotinhydrazide approach is that carbonylation of the proteins identified is inferred based on their enrichment by avidin alone, as the specific carbonylated residue is very rarely identified due to signal suppression from the remaining non-carbonylated peptides in the sample. Also, biotin-hydrazide itself readily fragments into a number of abundant ions, which can preclude identification of biotin-hydrazide labeled peptides [17]. Efforts to unequivocally identify sites of carbonylation to specific residues have thus relied on methods for enriching carbonylated peptides, followed by MS2 analysis and matching to peptide sequences via automated sequence database searching. One promising approach, involving the avidin-affinity enrichment of biotinylated peptides, rather than labeled proteins, has been used to successfully localize sites of carbonylation within both simple and complex protein mixtures [18, 19]. However, the aforementioned fragmentation of biotinylation reagents in MS2 spectra and increased hydrophobicity from the label complicate this method [17]. As an alternative to label-based enrichment approaches, we developed a label-free solid-phase hydrazide (SPH) reagent that directly and reversibly captures carbonylated peptides (...truncated)