Selective, sensitive, and rapid phosphopeptide identification in enzymatic digests using ESI-FTICR-MS with infrared multiphoton dissociation

Rapid screening for phosphopeptides within complex proteolytic digests involving electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FITCR-MS) in the negative ion mode with infrared multiphoton dissociation (IRMPD) accompanied by improved phosphopeptide sensitivity and selectivity is demonstrated with the tryptic digests of the naturally phosphorylated proteins bovine alpha- and beta -casein. All peptides in a complex proteolytic digest can be examined simultaneously for phosphorylation with a 4-s IR laser pulse at 7-11 W where phosphopeptide signature ions form upon irradiation, as the low energy of activation phosphate moiety cleavage transpires without the dissociation of the unphsophorylated peptide population. The tyrosine phosphorylated peptide HGLDN-pY-R, its nonphosphorylated analogue HGLDNYR, the kinase domain of insulin receptor unphosphorylated TRDIYETDYYRK, monophosphorylated TRDTYED-pY-YRK, and triphosphorylated TRDI-pY-ETD-pY-pY-RK were also used as model peptides in this research. The sensitivity and selectivity of phosphopeptides is shown to greatly improve when the volatile base piperidine is used to adjust the pH of the ESI buffer, which results in greater than a 3-fold increase in relative ion abundance of a mono-phosphorylated peptide (HGLDN-pY-R) over that of its unphosphorylated analogue. The addition of triethylamine provides comparatively equal ion intensities of modified and unmodified peptides, whereas ammonium hydroxide significantly suppresses phosphopeptide relative ion abundance when used to adjust the pH of the ESI buffer. With the addition of 20 mM piperidine in the ESI buffer, 30.6% of the amino acid sequence of the enzymatically digested beta -casein, including the tetraphosphorylated tryptic fragment, was identified at a 500 pM (0.5 fmol/muL) concentration when electrosprayed at a rate of 3.3 nL/s.