Identification of phosphorylated peptides from complex mixtures using negative‐ion orifice‐potential stepping and capillary liquid chromatography/electrospray ionization mass spectrometry

Abstract

A rapid method for identifying and characterizing sites of phosphorylation of peptides and proteins is described. High-performance capillary liquid chromatography (HPLC) coupled with electrospray ionization mass spectrometry (ESI-MS) is used to distinguish non-phosphorylated and phosphorylated peptides originating from mixtures as complex as enzyme digests. The method relies on the ability to produce a fragment ion characteristic and unique to phosphopeptides (m/z 79, PO3) by stepping the orifice potential of the mass spectrometer as a function of mass. At low m/z values, a high orifice potential is applied to induce extensive fragmentation of the peptide, leading to the formation of the m/z 79 phosphate-derived ion. This method is analogous to that described by Carr et al. for the identification of glycopeptides from enzymatic digestion of glycoproteins (S.A. Carr, M.J. Huddleston, M.F. Bean, Protein Science 2, 183 (1993)). The method was first evaluated and validated for a mixture of non-, mono- and di-phosphorylated synthetic peptides. Both mono- and di-phosphorylated peptides were found to generate fragment ions characteristic of PO3 whereas the non-phosphorylated peptide did not. Application of the method was extended to identifying phosphopeptides generated from an endoprotease Lys-C digestion of beta-casein. Both the expected mono- and tetra-phosphorylated Lys-C peptides were observed and identified rapidly in the LC/SEI-MS analysis. The procedure was used additionally to identify the site(s) of phosphorylation of the cytosolic non-receptor tyrosine kinase, pp60(c-src).

Identification of protein kinase A phosphorylation sites on NBD1 and R domains of CFTR using electrospray mass spectrometry with selective phosphate ion monitoring

“…3B). Previous studies using selective ion monitoring (SIM) have analyzed proteolytic digests of phosphoproteins containing much lower concentrations of salts (Huddleston et al, 1993;Ding et al, 1994;Lombard0 et al, 1995). Both urea and phosphate were required for solubility of the NBD1-R protein, necessitating a reversed phase HPLC (RP-HPLC) step prior to LC-ESI/MS analysis and possibly contributing to the higher ion current background for the PO,-ion.…”

“…Ten percent of the effluent (-25 pmol) was infused into the mass spectrometer. For full-scan monitoring in positive-ion mode, the instrument was scanned from 400-2,000 m/z in 5 s. For detection of phosphopeptides by selective ion monitoring (Huddleston et al, 1993;Ding et al, 1994), the instrument was cycled between fullscan mode (scan time = 5 s) with a cone voltage of -100 V and single ion monitoring (SIM) at m/z = 79 (dwell time = 1 s ) with a cone voltage of -200 V. Phosphorylated kemptide (Leu-ArgArg-Ala-Ser-Leu-Gly), prepared as described above in PKA buffer, was used to assess instrument performance in SIM. Molecular weight calculations were performed using MacBioSpec Software, version 1.0 from Perkin Elmer SCIEX (Thornhill, Ontario).…”

Identification of in Vitro Phosphorylation Sites in the Growth Cone Protein SCG10

“…Phosphopeptide Identification-Phosphopeptides were identified from the tryptic and Glu-C digests using the stepped orifice voltage technique, previously reported by Huddleston et al (30) and Ding et al (31). An aliquot of protein digest (5-25 pmol) was separated on the capillary C18 column and analyzed by electrospray ionization-MS. Peptides were ionized in the negative ion mode, and phosphopeptides were identified based on their ability to form a prominent PO 3 Ϫ ion, indicative of phosphorylation, at m/z 79.…”

Determination of the relative energies of activation for the dissociation of aromatic versus aliphatic phosphopeptides by ESI-FTICR-MS and IRMPD

“…Several methods of phosphopeptide mapping have also been proposed, taking advantage of the relative speed, sensitivity, and adaptability of MS. These methods include the use of phosphatases [17][18][19][20], 31 P monitoring by inductively couple plasma (ICP)-MS [21], collision induced dissociation (CID) [22][23][24][25], precursor ion scanning [24, 26 -31], neutral loss scanning [32], nozzle-skimmer dissociation [33,34], metastable decomposition [35][36][37], and electron capture [38].…”