CD8+ T cell stimulation, tandem mass tag (TMT) labeling, hydrophilic interaction liquid chromatography (HILIC) fractionation, and immobilized metal affinity chromatography (IMAC) phosphopeptide enrichment

Freshly isolated human effector CD8⁺ T cells (15 × 10⁶) from three donors (67.2, 50.5, and 45.7% effector CD57⁺CD8⁺ T cells, respectively) were stimulated for 10 min at 37°C in complete RPMI 1640-Hepes medium with isotype (10 μg/ml) or a mixture of CD2-specific antibodies (T11.1, T11.2, and HIK27 clones, all from Sanquin) each at 5 μg/ml. Cells were pelleted and lysed in 250 μl of 1% NP-40 lysis buffer [150 mM NaCl, 5 mM EDTA, and 20 mM tris-HCl (pH 8.0)]. Protein (220 μg) from postnuclear lysates was reduced in 5 mM DTT, alkylated in 20 mM iodoacetamide, and precipitated by methanol-chloroform extraction. Protein pellets were resuspended in 100 mM triethylammonium bicarbonate and digested with 4.4 μg of trypsin (Promega) overnight at 37°C. Desalted peptides (SOLA cartridges, Thermo Fisher Scientific) were labeled with six channels of a TMT10plex (Thermo Fisher Scientific) according to the manufacturer’s instructions, except that 220 μg of total protein/0.8 mg of TMT reagent was used. Excess TMT was quenched with 40 mM tris-HCl. The labeled peptides were pooled, desalted on Sep-Pak Plus cartridges (Waters), and dried down in a vacuum centrifuge. Phosphopeptides were enriched by HILIC/IMAC as described previously (81). Briefly, peptides were prefractionated on a HILIC column (Amide 80, 4.6 mm by 250 mm, Tosoh Bioscience LLC). Thirty fractions were collected and concatenated into 10 pools. Fractions were each incubated with 30 μl of a 50% slurry of PHOS-Select Iron affinity gel (Sigma-Aldrich) for 30 min at room temperature. Samples were washed twice with 0.5 ml of 250 mM acetic acid/30% ACN and then with double-distilled water before being eluted with 100 μl of 400 mM ammonium hydroxide.