FIGURE 2.

Solid-phase β-elimination with concurrent Michael addition of the phosphoserine/proline- and phosphothreonine-containing peptides. P5 (RGApSPVE) and P7 (KRpTIRR) were enriched on ZipTip_μ-C18 pipette tips from 40 μL aliquots of a 50-fmol/μL solution and incubated in the 66-mM barium hydroxide/33-mM 2-aminoethanethiol hydrochloride reagent mixture for 1 h at 55°C. Equimolar amounts of P7 were taken to dryness, reacted for 1 h at 55°C in 40 μL alkaline nucleophile solution or for 30 min at 55°C in 40 μL elimination base, followed by ZipTip_μ-C18 pipette tip purification. The samples were eluted in matrix directly onto the target. MALDI-TOF spectra of (A) unmodified peptide P5, (B) after the concurrent reaction, (C) unmodified peptide P7, and (D) after the concurrent reaction. (B, inset) The MALDI-TOF spectrum of P5 after incubation for 1 h at 37°C. Note residual β-elimination product at m/z 697.4, owing to incompleteness of the addition reaction. (A) *Unidentified, minor species. (C, inset) The MALDI-TOF spectrum of P7 after 30 min incubation at 55°C in 66 mM barium hydroxide. (D, inset) MALDI-TOF spectrum of P7 after concurrent in-solution derivatization. Note incompleteness of thiol adduct formation (arrow).