Targeted Mass Spectrometry-Based Approach for Protein-Ligand Binding Analyses in Complex Biological Mixtures Using a Phenacyl Bromide Modification Strategy.

The characterization of protein folding stability changes on the proteomic scale is useful for protein-target discovery and for the characterization of biological states. The Stability of Proteins from Rates of Oxidation (SPROX) technique is one of several mass spectrometry-based techniques recently established for the making proteome-wide measurements of protein folding and stability. A critical part of proteome-wide applications of SPROX is the identification and quantitation of methionine-containing peptides. Demonstrated here is a targeted mass spectrometry-based proteomics strategy for the detection and quantitation of methionine-containing peptides in SPROX experiments. The strategy involves the use of phenacyl bromide (PAB) for the targeted detection and quantitation of methionine-containing peptides in SPROX using selective reaction monitoring (SRM) on a triple quadrupole mass spectrometer (QQQ-MS). As proof-of-principle, the known binding interaction of Cyclosporine A with cyclophilin A protein in a yeast cell lysate is successfully detected and quantified using a targeted SRM workflow. Advantages of the described workflow over other SPROX protocols include a 20-fold reduction in the amount of total protein needed for analysis and the ability to work with the endogenous proteins in a given sample (e.g., stabile isotope labeling with amino acids in cell culture is not necessary).

Proteomic characterization of the cellular response to nitrosative stress mediated by s-nitrosoglutathione reductase inhibition.

The S-nitrosoglutathione-metabolizing enzyme, GSNO reductase (GSNOR), has emerged as an important regulator of protein S-nitrosylation. GSNOR ablation is protective in models of asthma and heart failure, raising the idea that GSNOR inhibitors might hold therapeutic value. Here, we investigated the effects of a small molecule inhibitor of GSNOR (GSNORi) in mouse RAW 264.7 macrophages. We found that GSNORi increased protein S-nitrosylation in cytokine-stimulated cells, and we utilized stable isotope labeling of amino acids in cell culture (SILAC) to quantify the cellular response to this "nitrosative stress". The expression of several cytokine-inducible immunomodulators, including osteopontin, cyclooxygenase-2, and nitric oxide synthase isoform 2 (NOS2), were decreased by GSNORi. In addition, selective targets of the redox-regulated transcription factor, nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-including heme oxygenase 1 (HO-1) and glutamate cysteine ligase modulatory subunit-were induced by GSNORi in a NOS2- and Nrf2-dependent manner. In cytokine-stimulated cells, Nrf2 protected from GSNORi-induced glutathione depletion and cytotoxicity and HO-1 activity was required for down-regulation of NOS2. Interestingly, GSNORi also affected a marked increase in NOS2 protein stability. Collectively, these data provide the most complete description of the global effects of GSNOR inhibition and demonstrate several important mechanisms for inducible response to GSNORi-mediated nitrosative stress.