Figure 1. Mass reconstruction of a tandem mass spectrum from a phosphopeptide.

Figure 2. Rigorous immunodepletion of serum prior to affinity purification eliminates the detection of target protein prior to stimulation of protein phosphorylation. A phosphospecific antibody was generated to the pSer19 form of myosin light chain 2, and the serum subjected to an increasing number of immunodepletion steps using the nonmodified peptide conjugated to thiol‐specific resin. The material eluting from this column was recovered, the protein recovered (as estimated by OD 280 nm), and the process repeated until detection of the target protein was eliminated (0 time) prior to the addition of ionophore (2 h) to stimulate the phosphorylation at Ser19.

Figure 3. (a) Mass reconstruction from a tandem mass spectrum of a synthetic phosphopeptide from the protein TRA2‐beta (C‐Ahx‐ ¹⁹⁴SITKRPH(pT)PTPGI ₂₀₆‐amide); (b) Antiserum after 30 days of immunization; (c) Antiserum after 60 days of immunization; (d) Antiserum after 90 days of immunization, and affinity purification.