Preparation of Human Lymphocyte Proteins to Profile Targets of Oxidative Stress

This article provides detailed methods for preparing membrane and cytosolic proteins isolated from human CD4⁺ T cells to identify biomarkers of oxidative stress (OS). Proteins important for T-cell metabolism, differentiation, and redox homeostasis can be identified using this untargeted approach. Proteins of interest contain cysteine thiols hypothesized to be susceptible to irreversible modification from reactive electrophiles generated under conditions of OS. Membrane-bound targets include receptors, ion channels, and enzymes that serve fundamental roles in sensing the redox microenvironment of T cells during activation and signaling pathways controlling responses to biochemical cues from their surroundings. Cytosolic targets include enzymes, chaperones, and transcription factors that can be separated from membrane proteins providing increased sequence coverage. This procedure includes detailed steps for isolating CD4⁺ T cells from peripheral blood samples and separating their membrane and cytosolic protein fractions for subsequent identification by LC-MS/MS. Proteomics data obtained from this procedure allows the relative abundance of proteins responsible for redox regulation of CD4⁺ T-cell activation and differentiation programs to be established. These data can be used to determine whether additional enrichment steps are necessary for discovering, characterizing, and quantifying novel irreversible modifications to specific thiols from proteins of interest that are expressed in low abundance. This protocol can be used for ex vivo studies where oxidation is experimentally induced to identify proteins diagnostic of OS associated with specific chemical exposures. Results from these studies can help determine the biochemical mechanisms of disease influenced by the human exposome and increased OS. © 2026 Wiley Periodicals LLC. Basic Protocol 1: Separation of human peripheral blood mononuclear cells from whole blood Basic Protocol 2: Isolation of CD4⁺ T cells by negative selection Basic Protocol 3: Fractionation of CD4⁺ T-cell membrane and cytosolic proteins Basic Protocol 4: Bradford assay and concentration of protein fractions.