Surveyor lc quaternary pump

Peptides (IMAC flow through) and phosphopeptides were separately fractionated by high pH reversed phase chromatography using a 250 mm by 4.6 mm column packed with 5 μm C18 particles (Gemini, Phenomenex) on a Surveyor LC quaternary pump (Thermo Scientific). The samples were resuspended in high pH reversed phase buffer, injected, and separated over a 60 min gradient (buffer A: 20 mM ammonium formate, pH 10; buffer B: 20 mM ammonium formate, pH 10, in 80% ACN). Flow rate was held at 0.8 mL/min except during re-equilibration when it was increased to 1 mL/min. Fractions were collected each min from 10 to 36 min and combined into 20 fractions for the peptides and 10 fractions for the phosphopeptides, which were frozen and dried to completion. The combined TMT-labeled sample was also fractionated using the same method. The eosinophil, platelet-depleted eosinophil, and platelet samples were fractionated using the same gradient but concatenated to 16 fractions.

Labeled peptides were fractionated by strong cation exchange on a polysulfoethyl A column (0.4 mm × 200 mm) with mobile phases A (5 mM KH₂PO₄, pH 2.7, and 30% acetonitrile); B (5 mM KH₂PO₄, pH 2.7, 350 mM KCl, and 30% acetonitrile); C (5 mM KH₂PO₄, pH 6.5, 500 mM KCL, and 20% acetonitrile); and D (water). Peptides were eluted over the following gradient on a Surveyor LC quaternary pump (Thermo Scientific) at 3 ml/min: 0–2 minutes, 100% A; 2–5 minutes, 0%–10% B; 5–35 minutes, 10%–60% B; 35–41 minutes, 60%–100% B; this gradient was followed by washes with C and D prior to reequilibration with mobile phase A. Sixteen fractions were collected and desalted. A small portion, 5%, of each was retained for protein analysis, while the remaining material was pooled into 6 fractions for acetyl lysine enrichment.
These pooled fractions were dissolved in 50 mM HEPES, pH 7.6, 100 mM NaCl, and each fraction was combined with approximately 50 μl pan-acetyl lysine antibody agarose conjugate. The samples were rotated overnight at 4°C and then rinsed 8 times with cold 50 mM HEPES, pH 7.6, and 100 mM NaCl. Rinses were followed by elution with 0.1% TFA, and eluted peptides were desalted prior to analysis.