TY - JOUR
T1 - Phosphopeptide characterization by mass spectrometry using reversed-phase supports for solid-phase β-elimination/michael addition
AU - Nika, Heinz
AU - Lee, Jaehoon
AU - Willis, Ian M.
AU - Hogue Angeletti, Ruth
AU - Hawke, David H.
PY - 2012/7
Y1 - 2012/7
N2 - We have adapted the Ba2+ ion-catalyzed concurrent Michael addition reaction to solid-phase derivatization on ZipTipC18 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.
AB - We have adapted the Ba2+ ion-catalyzed concurrent Michael addition reaction to solid-phase derivatization on ZipTipC18 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.
KW - Derivatization
KW - Phosphopeptide identification
KW - Phosphorylation site determination
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UR - http://www.scopus.com/inward/citedby.url?scp=84865773882&partnerID=8YFLogxK
U2 - 10.7171/jbt.2012-2302-002
DO - 10.7171/jbt.2012-2302-002
M3 - Article
C2 - 22951960
AN - SCOPUS:84865773882
SN - 1524-0215
VL - 23
SP - 51
EP - 68
JO - Journal of Biomolecular Techniques
JF - Journal of Biomolecular Techniques
IS - 2
ER -