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

Summary

Protein thiol oxidation has significant implications under normal physiological and pathophysiological conditions. We describe the details of a quantitative redox proteomics method, which utilizes resin-assisted capture, isobaric labeling, and mass spectrometry, enabling site-specific identification and quantification of reversibly oxidized cysteine residues of proteins.

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.

Introduction

Under homeostatic conditions, cells generate reactive oxygen, nitrogen, or sulfur species that help to facilitate processes, such as metabolism and signaling^1,2,3, extending to both prokaryotes and eukaryotes. Physiological levels of these reactive species are necessary for proper cellular function, also known as 'eustress'^1,4. In contrast, an increase in oxidants that leads to an imbalance between oxidants and antioxidants can cause oxidative stress, or 'distress'¹, which leads....

Protocol

All procedures described in the protocol related to animal or human samples/tissues were approved by and followed the institutional guidelines of the human and animal research ethics committee.

1. Sample homogenization/lysis

1. Frozen tissue samples
   1. Mince frozen tissue (~30 mg) on a glass microscope slide on dry ice using a prechilled razor blade and forceps. Transfer the minced tissue to a prechilled 5 mL round-bottom polystyrene tube containing 700 µL of buffer A (see Table 1) and incubate on ice for 30 min, protected from light.
   2. Disrupt the tissue for 30 s or until completely homogeni....

Results

Completion of the protocol will result in highly specific enrichment of formerly oxidized cysteine-containing peptides, often with >95% specificity^27,35,36. However, several key steps of the protocol require special attention, e.g., the initial blocking of free thiols prior to sample lysis/homogenization, which prohibits artificial oxidation and non-specific enrichment of artificially oxidized thiols²⁵

Discussion

Resin-assisted capture has been utilized across a variety of sample types and biological systems for the investigation of oxidative modifications of cysteine residues^25,29,30. This method allows for the evaluation of samples at multiple levels and readouts, including proteins and peptides using SDS-PAGE and western blot analysis, as well as individual cysteine sites using mass spectrometry. Regardless of the sample type or the f.......

Materials

Name	Company	Catalog Number	Comments
2-(Pyridyldithio)ethylamine hydrochloride	Med Chem Express	HY-101794	Reagent for in-house resin synthesis
2.0 mL LoBind centrifuge tubes	Eppendorf	22431048
5.0 mL LoBind centrifuge tubes	Eppendorf	30108310
5.0 mL round bottom tubes	Falcon	352054
Acetone	Fisher Scientific	A949-1
Acetonitrile	Sigma Aldrich	34998
Activated Thiol–Sepharose 4B	Sigma Aldrich	T8512	Potential replacement for thiol-affinity resin
Amicon Ultra 0.5 mL centrifugal filter	Millipore Sigma	UFC5010BK
Ammonium bicarbonate	Sigma Aldrich	09830
Bicinchonicic acid (BCA)	Thermo Scientific	23227	Protein Assay Reagent
Centrifuge	Eppendorf	5810R
Centrifuge	Eppendorf	5415R
Dithiothreitol (DTT)	Thermo Scientific	20291
EDTA	Sigma Aldrich	E5134
HEPES buffer	Sigma Aldrich	H4034
Homogenizer	BioSpec Products	985370
Iodoacetimide (IAA)	Sigma Aldrich	I1149
N-ethylmaleimide	Sigma Aldrich	4259
NHS-Activated Sepharose 4 Fast Flow	Cytiva	17-0906-01	Reagent for in-house resin synthesis
QIAvac 24 Plus vacuum manifold	Qiagen	19413
Sodium chloride	Sigma Aldrich	S3014
Sodium dodecyl sulfate (SDS)	Sigma Aldrich	L6026
Sonicator	Branson	1510R-MT
Spin columns	Thermo Scientific	69705
Strata C18-E reverse phase columns	Phenomenex	8B-S001-DAK	Peptide desalting
Thermomixer	Eppendorf	5355
Thiopropyl Sepharose 6B	GE Healthcare	17-0420-01	Thiol-affinity resin; *Production of Thiopropyl Sepharose 6B resin has been discontinued by the manufacturer (see protocol for details).
TMT isobaric labels (16 plex)	Thermo Scientific	A44522	Peptide labeling reagent; available in multiple formats
Triethylammonium bicarbonate buffer (TEAB)	Sigma Aldrich	T7408
Trifluoroacetic acid (TFA)	Sigma Aldrich	T6508
Triton X-100	Sigma Aldrich	T8787
Trypsin	Promega	V5820
Urea	Sigma Aldrich	U5378
Vacufuge Plus speedvac	Eppendorf	22820001	vacuum concentrator
Vortex mixer	Scientific Industries	SI-0236