Pooled lysates from healthy human donors are initially divided into “Enriched” and “Control” samples. Disulfide bonds are reduced by tris(2-carboxyethyl)phosphine (TCEP), and free thiols are blocked by N-ethylmaleimide (NEM). Following cleavage of the palmitoyl thioester bond by hydroxylamine (HA) in the enriched sample, previously palmitoylated thiols are biotinylated using the sulfhydryl-reactive EZ-Link HPDP-biotin. Control samples omit HA cleavage, and remain unbiotinylated. Following enrichment via streptavidin-agarose beads and elution by beta-mercaptoethanol (2-ME), enriched and control samples are run in parallel lanes of an SDS-PAGE gel. These lanes are cut into equal-sized bands and subjected to tryptic digest in either heavy (H₂¹⁸O) or light (H₂¹⁶O) water, providing the isotopic label. After digestion, samples are mixed and measured by LC-MS/MS; evaluating the isotopic heavy/light intensity ratio gives rise to an enriched pool representing the population of palmitoylated proteins in the cell.

(a) Representative scatter plot of two of four enriched pools of palmitoylated proteins from primary T cells; x- and y-axes represent heavy-to-light isotopic ratios (log10). In total, 280 proteins were found to be palmitoylated. Highlighted are palmitoylation candidates selected for confirmation by Western blot (red). Also shown: “canonical” palmitoylated proteins, reported in at least two previous palmitome studies (blue). (b) Confirmation of selected palmitoylation candidates by Western blot using the indicated antibodies. Interestingly, only the truncated Isoform 2 of ZDHHC18, a palmitoylacyl transferase previously unreported as palmitoylated, is palmitoylated, suggesting the possibility of isoform-dependent, differential activation states. (c) Summary and evaluation of palmitoylated proteins in primary T cells. Enriched and unenriched pools were evaluated by: (i) prediction of palmitoylation motifs by CSS-Palm algorithm, (ii) previous reports in earlier palmitome studies across various mammalian cell lines, and (iii) prediction of transmembrane domains by the TMHMM algorithm. A clear enrichment is seen in all three criteria. (d) Biological functions of primary T cell palmitoylation candidates identified in this study (Gene Ontology annotations). For clarity, groups with fewer than 10 members were pooled under the “Other” category.