Phosphoric acid enhances the performance of Fe(III) affinity chromatography and matrix-assisted laser desorption/ionization tandem mass spectrometry for recovery, detection and sequencing of phosphopeptides.
An integrated analytical strategy for enrichment, detection and sequencing of phosphorylated peptides by matrix-assisted laser desorption/ionization (MALDI) tandem mass spectrometry (MS/MS) is reported. o-Phosphoric acid was found to enhance phosphopeptide ion signals in MALDI-MS when used as the acid dopant in 2,5-dihydroxybenzoic acid (2,5-DHB) matrix. The effect was largest for multiply phosphorylated peptides, which exhibited an up to ten-fold increase in ion intensity as compared with standard sample preparation methods. The enhanced phosphopeptide response was observed during MALDI-MS analysis of several peptide mixtures derived by proteolytic digestion of phosphoproteins. Furthermore, the mixture of 2,5-DHB and o-phosphoric acid was an excellent eluant for immobilized metal affinity chromatography (IMAC). Singly and multiply phosphorylated peptide species were efficiently recovered from Fe(III)-IMAC columns, reducing sample handling for phosphopeptide mapping by MALDI-MS and subsequent phosphopeptide sequencing by MALDI-MS/MS. The enhanced response of phosphopeptide ions in MALDI facilitates MS/MS of large (>3 kDa) multiply phosphorylated peptide species and reduces the amount of analyte needed for complete characterization of phosphoproteins.
Stensballe
Allan
A
Department of Biochemistry & Molecular Biology, University of Southern Denmark, DK-5230 Odense M, Denmark.
Jensen
Ole Nørregaard
ON
D000595:Amino Acid Sequence; D000818:Animals; D001412:Bacillus subtilis / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry; D002417:Cattle; D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods*; D007501:Iron / Q000737:chemistry*; D008894:Milk Proteins / Q000737:chemistry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry; D010748:Phosphopeptides / Q000032:analysis* / Q000737:chemistry*; D010756:Phosphoric Acids / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000295:instrumentation / Q000379:methods*
D001426:Bacterial Proteins; D008894:Milk Proteins; D010446:Peptide Fragments; D010748:Phosphopeptides; D010756:Phosphoric Acids; C061124:lactophorin protein, Bos taurus; D007501:Iron; C030242:phosphoric acid