Phosphopeptide Characterization by Mass Spectrometry using Reversed-Phase Supports for Solid-Phase β-Elimination/Michael Addition

Abstract

We have adapted the Ba 2ϩ ion-catalyzed concurrent Michael addition reaction to solid-phase derivatization on ZipTip C18 pipette tips using 2-aminoethanethiol as a nucleophile. This approach provides several advantages over the classical in-solution-based techniques, including ease of operation, completeness of reaction, improved throughput, efficient use of dilute samples, and amenability to automation. Phosphoseryl and phosphothreonyl peptides, as well as phosphoserine peptides with adjoining prolines, were used to optimize the reaction conditions, which proved highly compatible with the integrity of the samples. The analyte was recovered from the silica-based C18 resin at minimal sample loss. The use of the protocol for improved phosphopeptide detection by signal enhancement was demonstrated with low-level amounts of proteolytic digests from model proteins and experimental samples, an effect found especially prominent with multiple phosphorylated species. The reaction products proved highly suitable for structural characterization by collisionally induced dissociation (CID), and the resultant increased spectral information content, greatly facilitating mapping of the site of phosphorylation. In select cases, the method enables phosphorylation site localization within known protein sequences on the basis of single-stage data alone. The solid-phase strategy presented here provides a simple, versatile, and efficient tool for phosphopeptide structural characterization equipment readily available in most biological laboratories.

Recovery of RNA Polymerase III Transcription from the Glycerol-repressed State

“…Purification of GST-kinases from yeast and their use in vitro kinase assays was as published (6,13). Expression and native purification of full-length Maf1and Maf1-id was as described (27). The Maf1-id protein yield at 20 mg/liter was 4-fold higher than that obtained for the full-length protein.…”

Phosphopeptide Enrichment by Covalent Chromatography after Derivatization of Protein Digests Immobilized on Reversed-Phase Supports

“…As discussed in an earlier report and in the companion article, 30,31 the solid-phase derivatization format offers some notable advantages over the classical in-solutionbased methods, the predominant sample-preparation technique in current proteomic studies. In brief, the solid-phase strategy obviates the need for sample dry-down, commonly practiced to concentrate peptide mixtures before de-rivatization in solution-based proteomics studies solution.…”

“…38 The process of sample adsorption concentrates the analyte on the support, allowing the reaction to proceed in situ, in general, at higher efficiency and faster kinetics than in solution. 30,31,39,40 Protein digests can be enriched effectively on the solid phase from dilute solutions, in which chemical reactions inherently proceed at slow reaction rates. As demonstrated in this report, in the companion article, and in an earlier communication, 41 the solid-phase format proved particularly advantageous for combining distinct chemistries into serial reaction schemes.…”

“…30 GelCode Blue-stained bands, from 5 to 50 pmol protein loaded onto the gels, were destained twice with 200 l 25 mM ammonium bicarbonate in 50% aqueous acetonitrile for 30 min at 37°C. Bands were dried briefly in a SpeedVac and incubated for 15 min at 37°C in 100 l 2 mM TCEP/25 mM ammonium bicarbonate; the supernatant was then removed, leaving the bands diffused with the reductant.…”

“…We have used this minimal sample handling format earlier for phosphopeptide detection in MALDI mass maps of an unfractionated protein digest and to prepare digest for phosphorylation-site determination by chemical-targeted proteolysis. 30,31 Benefits gained from of this approach include ease of operation, completeness of derivatization, improved throughput, and efficient use of dilute samples. It is noteworthy that these advantages had been recognized already one decade ago in conjunction with off-line and on-line derivatization on various sorbents and have since then been exploited by use of chromatographic trapping columns to improve significantly the accuracy, sensitivity and throughput of the quantitative analysis of bioorganic compounds.…”