Phosphopeptide Enrichment and Sequential Modification Analysis

In a method adapted from Ficarro et al.^{5 (link)} iron-chelated IMAC beads were prepared from Ni-NTA superflow agarose beads (Qiagen, #1018611) that were stripped of nickel with 100 mM EDTA and incubated in an aqueous solution of 10 mM FeCl₃ (Sigma, 451649). Dried phosphopeptide fractions (high scale: 12; medium scale: 4) were reconstituted in 50% acetonitrile/0.1% trifluoroacetic acid and then diluted 1:1 with 100% acetonitrile/0.1% trifluoroacetic acid to obtain a final 80% acetonitrile/0.1% TFA peptide solution at a concentration of 0.5 μg/μl. Peptide mixtures were enriched for phosphorylated peptides with 10 μL IMAC beads for each sample for 30 min. Enriched IMAC beads were loaded on Empore C18 silica-packed stage tips (3M, 2315) as has been described previously^{11 (link)}. Stage tips were equilibrated with 2 × 100 μL washes of methanol, 2 × 50 μL washes of 50% acetonitrile/0.1% formic acid, and 2 × 100 μL washes of 1% formic acid. Samples were then loaded onto stage tips and washed twice with 50 μL of 80% acetonitrile/0.1% trifluoroacetic acid and 100 μL of 1% formic acid. Phosphorylated peptides were eluted from IMAC beads with 3 × 70 μL washes of 500 mM dibasic sodium phosphate, pH 7.0, (Sigma, S9763) and washed twice with 100 μL of 1% formic acid before being eluted from stage tips with 60 μL 50% acetonitrile/0.1% formic acid. All washes were performed on a tabletop centrifuge at a maximum speed of 3,500g.
For subsequent serial enrichment for ubiquitinated (K(GG)) and lysine-acetylated (K(Ac)) peptides, flow-throughs after IMAC enrichment were retained and combined in a stepwise fashion to generate 6 large-scale and 2 medium-scale fractions: For large-scale analysis every 6^th IMAC supernatant fraction was combined (1,7; 2,8; ...), and for medium scale analysis every 2^nd IMAC supernatant fraction was combined (1,3; 2,4).