Reversible cysteine modifications play important physiological roles such as modulating enzymatic catalysis, maintaining redox homeostasis and conducting cellular signaling. These roles can be critical in the context of disease. Oxidative modifications such as S-nitrosylation (SNO) are signatures of neurodestruction in conditions of oxidative stress however are also indicators of neuroprotection and normal signaling in cellular environments with low concentrations of reactive oxygen and nitrogen species. SNO is a dynamic and low abundance modification and requires sensitive and selective analytical methods for its detection in biological tissues. Here we present an enhanced multiplexing strategy to study SNO in complex mixtures arising from tissues. This method, termed oxidized cysteine-selective cPILOT (OxcyscPILOT), allows simultaneous analysis of SNO-modified peptides in 12 samples. OxcyscPILOT has three primary steps: (1) blocking of free thiols by a cysteine-reactive reagent, (2) enrichment of peptides containing SNO on a solid phase resin, and (3) isotopic labeling and isobaric tagging of enriched peptides on the solid phase resin. This approach offers the advantage of allowing total protein abundance levels to be measured simultaneously with endogenous SNO levels and measurement of SNO levels across four biological replicates in a single analysis. Furthermore, the relative amount of SNO on a specific cysteine site can also be determined. A well-known model of Alzheimer's disease, the APP/PS-1 transgenic mouse model, was selected for demonstration of the method as several SNO-modified proteins have previously been reported in brain and synaptosomes from AD subjects. OxcyscPILOT analysis resulted in identification of 138 SNO-modified cysteines in brain homogenates that correspond to 135 proteins. Many of these SNO-modified proteins were only present in wild-type or AD mice, whereas 93 proteins had SNO signals in both WT and AD. Pathway analysis links SNO-modified proteins to various biological pathways especially metabolism and signal transduction, consistent with previous reports in the literature. The OxcyscPILOT strategy provides enhanced multiplexing capability to current redox proteomics methods to study oxidative modifications of cysteine.

Cysteine-selective proteomics approaches simplify complex protein mixtures and improve the chance of detecting low abundant proteins. It is possible that cysteinyl-peptide/protein enrichment methods could be coupled to isotopic labeling and isobaric tagging methods for quantitative proteomics analyses in as few as two or up to 10 samples, respectively. Here we present two novel cysteine-selective proteomics approaches: cysteine-selective dimethyl labeling (cysDML) and cysteine-selective combined precursor isotopic labeling and isobaric tagging (cPILOT). CysDML is a duplex precursor quantification technique that couples cysteinyl-peptide enrichment with on-resin stable-isotope dimethyl labeling. Cysteine-selective cPILOT is a novel 12-plex workflow based on cysteinyl-peptide enrichment, on-resin stable-isotope dimethyl labeling, and iodoTMT tagging on cysteine residues. To demonstrate the broad applicability of the approaches, we applied cysDML and cPILOT methods to liver tissues from an Alzheimer’s disease (AD) mouse model and wild-type (WT) controls. From the cysDML experiments, an average of 850 proteins were identified and 594 were quantified, whereas from the cPILOT experiment, 330 and 151 proteins were identified and quantified, respectively. Overall, 2259 unique total proteins were detected from both cysDML and cPILOT experiments. There is tremendous overlap in the proteins identified and quantified between both experiments, and many proteins have AD/WT fold-change values that are within ~20% error. A total of 65 statistically significant proteins are differentially expressed in the liver proteome of AD mice relative to WT. The performance of cysDML and cPILOT are demonstrated and advantages and limitations of using multiple duplex experiments versus a single 12-plex experiment are highlighted.

Sepsis is commonly caused by community-acquired pneumonia (CAP) and may develop into severe sepsis, characterized by multiple organ failure. The risk of severe sepsis among CAP patients and subsequent mortality increases sharply after the age of 65. The molecular mechanisms associated with this age-related risk are not fully understood. To better understand factors involved with increased incidence and mortality of severe sepsis in the elderly, we used a nested case-control study of patients enrolled in a multicenter observational cohort of 2320 participants with CAP. We identified a total of 39 CAP patients 50–65 and 70–85 years old who did or did not develop severe sepsis. Plasma samples were obtained on presentation to the emergency department and prior to therapeutic interventions. A semiquantitative plasma proteomics workflow was applied which incorporated tandem immunoaffinity depletion, iTRAQ labeling, strong cation exchange fractionation, and nanoflow liquid chromatography coupled to high-resolution mass spectrometry. In total, 772 proteins were identified, of which 58 proteins exhibit statistically significant differences in expression levels among patients with severe sepsis as a function of age. Differentially expressed proteins are involved in pathways such as acute phase response, coagulation signaling, atherosclerosis signaling, lipid metabolism, and production of nitric oxide and reactive oxygen species. This study provides insight into factors that may explain age-related differences in incidence of severe sepsis in the elderly.

Oxidative modifications can have significant effects on protein structure in solution. Here, the structures and stabilities of oxidized ubiquitin ions electrosprayed from an aqueous solution (pH 2) are studied by ion mobility spectrometry-mass spectrometry (IMS-MS). IMS-MS has proven to be a valuable technique to assess gas phase and in many cases, solution structures. Herein, in vitro oxidation is performed by Fenton chemistry with Fe(II)/hydrogen peroxide. Most molecules in solution remain unmodified, whereas ∼20% of the population belongs to an M+16 Da oxidized species. Ions of low charge states (+7 and +8) show substantial variance in collision cross section distributions between unmodified and oxidized species. Novel and previously reported Gaussian conformers are used to model cross section distributions for +7 and +8 oxidized ubiquitin ions, respectively, in order to correlate variances in observed gas-phase distributions to changes in populations of solution states. Based on Gaussian modeling, oxidized ions of charge state +7 have an A-state conformation which is more populated for oxidized relative to unmodified ions. Oxidized ubiquitin ions of charge state +8 have a distribution of conformers arising from native-state ubiquitin and higher intensities of A- and U-state conformers relative to unmodified ions. This work provides evidence that incorporation of a single oxygen atom to ubiquitin leads to destabilization of the native state in an acidic solution (pH ∼2) and to unfolding of gas-phase compact structures.

Current strategies for identification and quantification of 3-nitrotyrosine (3NT) post-translationally modified proteins (PTM) generally rely on biotin/avidin enrichment. Quantitative approaches have been demonstrated which employ isotopic labeling or isobaric tagging in order to quantify differences in the relative abundances of 3NT-modified proteins in two or potentially eight samples, respectively. Here, we present a novel strategy which uses combined precursor isotopic labeling and isobaric tagging (cPILOT) to increase the multiplexing capability of quantifying 3NT-modified proteins to 12 or 16 samples using commercially available tandem mass tags (TMT) or isobaric tags for relative and absolute quantification (iTRAQ), respectively. This strategy employs “light” and “heavy” labeled acetyl groups to block both N-termini and lysine residues of tryptic peptides. Next, 3NT is reduced to 3-aminotyrosine (3AT) using sodium dithionite followed by derivatization of light and heavy labeled 3AT-peptides with either TMT or iTRAQ multiplex reagents. We demonstrate the proof-of-principle utility of cPILOT with in vitro nitrated bovine serum albumin (BSA) and mouse splenic proteins using TMT0, TMT6, and iTRAQ8 reagents and discuss limitations of the strategy.

Adriamycin (ADR) is a potent anticancer drug used to treat a variety of cancers. Patients treated with ADR have experienced side effects such as heart failure, cardiomyopathy, and “chemobrain”, which have been correlated to changes in protein expression in the heart and brain. In order to better understand cellular responses that are disrupted following ADR treatment in immune tissues, this work focuses on spleen. Significantly reduced spleen sizes were found in ADR-treated mice. Global isotopic labeling of tryptic peptides and nanoflow reversed-phase liquid chromatography-tandem mass spectrometry (LC–MS/MS) were employed to determine differences in the relative abundances of proteins from ADR-treated mice relative to controls. Fifty-nine proteins of the 388 unique proteins identified showed statistically significant differences in expression levels following acute ADR treatment. Differentially expressed proteins are involved in processes such as cytoskeletal structural integrity, cellular signaling and transport, transcription and translation, immune response, and Ca2+ binding. These are the first studies to provide insight to the downstream effects of ADR treatment in a peripheral immune organ such as spleen using proteomics.

Drosophila melanogaster is used as a model system to investigate protein changes associated with the aging process under conditions that alter organism lifespan. Changes in the proteome are assessed at various ages in populations of Oregon-R adult males that have mean lifetimes of 47 and 111 days at 28 and 18 °C, respectively. Peptide hits detected from strong-cation-exchange and reversed-phase liquid chromatography coupled to tandem mass spectrometry analysis are employed to examine patterns in relative protein expression. Thirty-three proteins were identified as having similar patterns of expression at both temperatures investigated when scaling the organism age to lifespan. In addition, the proteins ferritin 2 light chain homologue and larval serum protein 1β were identified in relatively high abundance and displayed distinctly different patterns of expression between the two temperatures. Overall, the results support the notion that aspects of the aging process may be preprogrammed at the protein level.Research highlights▶ Populations of Oregon-R adult males have mean lifetimes of 47 and 111 days at 28 and 18 °C, respectively. ▶ Thirty-three proteins have similar age-related patterns of expression at 28 and 18 °C. ▶ Ferritin 2 light chain homologue and larval serum protein 1β have distinct temperature-dependent proteome profiles. ▶ Aspects of aging may be preprogrammed at the protein level.

Here, we present the proteomics dataset of young and middle-aged Caenorhabditis elegans (C. elegans) exposed to Pseudomonas aeruginosa (P. aeruginosa strain PA01), which is related to the article "Proteomic Identification of Virulence-Related Factors in Young and Aging C. elegans infected with Pseudomonas aeruginosa" (C. D. King et. al, in-revisions). This dataset was generated to better understand the effects of aging on molecular mechanisms involved in host response to pathogen exposure. Protein from C. elegans of different age and exposure to P. aeruginosa PA01 or control E. coli OP50 were extracted and tryptically digested. Peptides were labeled with the reagents tandem mass tag (TMT6-plex), separated, and detected by using offline strong-cation exchange and online liquid chromatography – mass spectrometry (SCX – LC – MS/MS & MS3). A separate mixture of peptides were labeled on N-terminal amines and lysines with dimethylation. Dimethylated peptides were analyzed using LC – MS/MS and a portion of the results were used to verify fold-change direction for TMT6-plex experiments. Raw data can be found online at www.CHORUSproject.org, a cloud-based data repository (see specifications table for details).