Targeted quantitation of site-specific cysteine oxidation in endogenous proteins using a differential alkylation and multiple reaction monitoring mass spectrometry approach.
Reactive oxygen species (ROS) are both physiological intermediates in cellular signaling and mediators of oxidative stress. The cysteine-specific redox-sensitivity of proteins can shed light on how ROS are regulated and function, but low sensitivity has limited quantification of the redox state of many fundamental cellular regulators in a cellular context. Here we describe a highly sensitive and reproducible oxidation analysis approach (OxMRM) that combines protein purification, differential alkylation with stable isotopes, and multiple reaction monitoring mass spectrometry that can be applied in a targeted manner to virtually any cysteine or protein. Using this approach, we quantified the site-specific cysteine oxidation status of endogenous p53 for the first time and found that Cys182 at the dimerization interface of the DNA binding domain is particularly susceptible to diamide oxidation intracellularly. OxMRM enables analysis of sulfinic and sulfonic acid oxidation levels, which we validate by assessing the oxidation of the catalytic Cys215 of protein tyrosine phosphatase-1B under numerous oxidant conditions. OxMRM also complements unbiased redox proteomics discovery studies as a verification tool through its high sensitivity, accuracy, precision, and throughput.
double daggerBuck Institute for Age Research, Novato, California 94945, USA.
Jason M
JM
Held
Steven R
SR
Danielson
Jessica B
JB
Behring
Christian
C
Atsriku
David J
DJ
Britton
Rachel L
RL
Puckett
Birgit
B
Schilling
Judith
J
Campisi
Christopher C
CC
Benz
Bradford W
BW
Gibson
D000478:Alkylation; D000595:Amino Acid Sequence; D002460:Cell Line; D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011499:Protein Processing, Post-Translational; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1 / Q000737:chemistry / Q000235:genetics; D040901:Proteomics / Q000379:methods; D017382:Reactive Oxygen Species / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry / Q000235:genetics
D017382:Reactive Oxygen Species; D016159:Tumor Suppressor Protein p53; C516607:PTPN1 protein, human; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D003545:Cysteine