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A Novel Silica‐Based Immobilized Metal Affinity Chromatography (IMAC) Technology for the Enrichment of Phosphopeptides
Author(s) -
Turner Jeffrey L.,
Wildsmith Justin,
Boland Judy,
MoellerGaa Jessica,
Ray Kevin,
Dapron John G.,
Scott Graham B. I.
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a528
Subject(s) - phosphopeptide , nitrilotriacetic acid , phosphoprotein , chemistry , chromatography , affinity chromatography , mass spectrometry , phosphorylation , combinatorial chemistry , biochemistry , organic chemistry , enzyme , chelation
Protein phosphorylation is a dynamic post‐translational modification that plays a critical role in the regulation of numerous cellular events including signal transduction, gene expression, and apoptosis. Tryptic phosphopeptides, however, are often found in low natural abundance and ionize poorly, complicating their identification by mass spectrometry. Immobilized metal affinity chromatography (IMAC) is a tool that has been developed to aid in the isolation and subsequent identification of phosphorylated molecules. We have developed and optimized a novel silica matrix for IMAC applications, utilizing a proprietary nitrilotriacetic acid analog complexed with gallium. The advantages of this matrix, such as enhanced sensitivity and selectivity, as compared to similar phosphopeptide enrichment technologies, are highlighted when tested against a complex tryptic digest of a phosphoprotein mixture. Additionally, the versatility of the gallium based phosphopeptide enrichment becomes apparent in the examination of phosphoproteins from blood plasma that has been depleted of the most abundant proteins. Despite scrutinous testing against the most complex samples, the technology is shown to be both effective and robust.