Direct MALDI-MS/MS of Phosphopeptides Affinity-Bound to Immobilized Metal Ion Affinity Chromatography Beads

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• 作者：Christina S. Raska ; Carol E. Parker ; Zbigniew Dominski ; William F. Marzluff ; Gary L. Glish ; R. Marshall Pope ; and Christoph H. Borchers
• 刊名：Analytical Chemistry
• 出版年：2002
• 出版时间：July 15, 2002
• 年：2002
• 卷：74
• 期：14
• 页码：3429 - 3433
• 全文大小：77K
• 年卷期：v.74,no.14(July 15, 2002)
• ISSN：1520-6882

文摘

Immobilized metal ion affinity chromatography (IMAC) isa useful method to selectively isolate and enrich phosphopeptides from a peptide mixture. Mass spectrometryis a very suitable method for exact molecular weightdetermination of IMAC-isolated phosphopeptides, due toits inherent high sensitivity. Even exact molecular weightdetermination, however, is not sufficient for identificationof the phosphorylation site if more than one potentialphosphorylation site is present on a peptide. The previousmethod of choice for sequencing the affinity-bound peptides was electrospray tandem mass spectrometry (ESI-MS/MS). This method required elution and salt removalprior to MS analysis of the peptides, which can lead tosample loss. Using a matrix-assisted laser desorption/ionization (MALDI) source coupled to an orthogonalinjection quadrupole time-of-flight (QqTOF) mass spectrometer with true MS/MS capabilities, direct sequencingof IMAC-enriched peptides has been performed on IMACbeads applied directly to the MALDI target. The utility ofthis new method has been demonstrated on a protein withunknown phosphorylation sites, where direct MALDI-MS/MS of the tryptic peptides bound to the IMAC beadsresulted in the identification of two novel phosphopeptides. Using this technique, the phosphorylation sitedetermination is unambiguous, even with a peptidecontaining four potentially phosphorylated residues. Direct analysis of phosphorylated peptides on IMAC beadsdoes not adversely affect the high-mass accuracy of anorthogonal injection QqTOF mass spectrometer, makingit a suitable technique for phosphoproteomics.