Resin-Assisted Capture Coupled with Isobaric Tandem Mass Tag Labeling for Multiplexed Quantification of Protein Thiol Oxidation

Abstract

Reversible oxidative modifications on protein thiols have recently emerged as important mediators of cellular function. Herein we describe the detailed procedure of a quantitative redox proteomics method that utilizes resin-assisted capture (RAC) in combination with tandem mass tag (TMT) isobaric labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to allow multiplexed stochiometric quantification of oxidized protein thiols at the proteome level. The site-specific quantitative information on oxidized cysteine residues provides additional insight into the functional impacts of such modifications. The workflow is adaptable across many sample types, including cultured cells (e.g., mammalian, prokaryotic) and whole tissues (e.g., heart, lung, muscle), which are initially lysed/homogenized and with free thiols being alkylated to prevent artificial oxidation. The oxidized protein thiols are then reduced and captured by a thiol-affinity resin, which streamlines and simplifies the workflow steps by allowing the proceeding digestion, labeling, and washing procedures to be performed without additional transfer of proteins/peptides. Finally, the labeled peptides are eluted and analyzed by LC-MS/MS to reveal comprehensive stoichiometric changes related to thiol oxidation across the entire proteome. This method greatly improves the understanding of the role of redox-dependent regulation under physiological and pathophysiological states related to protein thiol oxidation.

Figure 1:. Sample processing workflow.

The sample processing workflow is adaptable for investigating thiol oxidation in various sample types and biological systems. The workflow allows for investigation of oxidation at both the protein and peptide levels (e.g., SDS-PAGE, western blot) as well as deep coverage for quantitative, site-specific identification of individual cysteine sites using HPLC coupled with mass spectrometry. Sample processing can be completed in as little as three days, including the completion of several critical steps for the generation of quality, consistent data. Sample multiplexing via TMT labeling allows for the processing of multiple samples in parallel at the same time. The representative 10-plex TMT labeling scheme illustrates how samples can be arranged considering the potential crosstalk from the total-thiol channel. With the isotopic impurities of the TMT reagents, the signal intensity of one channel with high intensity (such as total-thiol) can contribute to another channel with low signal intensity and influence its quantification. In the scheme, a pooled total-thiol channel (a combination of control and experimental samples) is expected to contain high levels of Cys-peptides and is labeled with 131N, which will have a signal in channel 130N. Thus, channel 130N is not used in the experiment. The amount of channel crosstalk created by TMT labels can be found in the manufacturer's certificates of analysis for a corresponding batch of the reagent. This figure has been adapted from Guo et al., Nature Protocols, 2014. Abbreviations: NEM = N-ethylmaleimide; DTT = dithiothreitol; SDS-PAGE = sodium dodecylsulfate polyacrylamide gel electrophoresis; SPE = solid-phase extraction; LC-MS/MS = liquid chromatography-tandem mass spectrometry; TMT = tandem mass tag.

Figure 2:. Analysis of peptides from RAC enrichment.

(A) SDS-PAGE analysis of oxidized peptides from RAW 264.7 cells treated with the chemical oxidant, diamide, for 30 min at increasing concentrations (0.1 and 0.5 mM) and total peptide thiols. This sub-figure and sub-figure D are adapted from Guo et al., Nature Protocols, 2014 . Peptides were visualized by silver staining. (B) RAW 264.7 cells were treated with exogenous oxidants (hydrogen peroxide and diamide) at increasing concentrations. The resulting SSG-enriched protein eluate was separated by SDS-PAGE and subsequently probed by western blot for individual proteins (GAPDH, TXN, PRDX3, and ANXA1). This sub-figure is adapted from Su et al., Free Radical Biology and Medicine, 2014. (C) Representative MS/MS spectrum data of a cysteine-containing peptide viewed in Xcalibur software. The inset MS/MS image shows the corresponding reporter ion intensities for the same peptide in each TMT channel. In this experiment, the total-thiol sample was assigned to the TMT label 131N, which has the highest intensity of all channels used in the experiment. (D) Stoichiometry of iTRAQ-labeled, enriched, oxidized peptides as measured by LC-MS/MS. The total-thiol channel was used as a reference to calculate the stoichiometry of oxidation based on the ratio of reporter ion intensity of each sample compared to that of the total-thiol channel. Abbreviations: RAC = resin-assisted capture; SDS-PAGE = sodium dodecylsulfate polyacrylamide gel electrophoresis; Ctrl = control; GAPDH = glyceraldehyde 3- phosphate dehydrogenase; TXN = thioredoxin; PRDX3 = thioredoxin-dependent peroxide reductase; ANXA1 = annexin A1; LC-MS/MS = liquid chromatography-tandem mass spectrometry; MS/MS = tandem mass spectrometry; TMT = tandem mass tag; iTRAQ = isobaric tag for relative and absolute quantitation.