Technology
Metal Ligand Affinity Pipettes and Bioreactive Alkaline Phosphatase Probes: Tools for Characterization of Phosphorylated Proteins and Peptides*

https://doi.org/10.1074/mcp.T300008-MCP200Get rights and content
Under a Creative Commons license
open access

An alkaline phosphatase-bioreactive probe, in which the enzyme is covalently bound to the mass spectrometry target, has been developed for studies of phosphoproteins. The bioreactive probe was used in combination with affinity capture and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to study hydrolysis of several phosphoproteins found in human saliva. Human salivary proteins were extracted from diluted human saliva with immobilized metal-affinity pipettes, which under defined conditions bound the phosphoproteins of interest preferentially over histatins. Phosphoproteins were eluted directly from the affinity pipettes to the bioreactive probe with diluted ammonium hydroxide, which provided conditions appropriate for hydrolysis by the alkaline phosphatase covalently bound to the probe surface. Results indicate the combination of metal-affinity pipette extraction, alkaline phosphatase-bioreactive probes, and matrix-assisted laser desorption/ionization mass spectrometry is an effective way to find and characterize phosphoproteins, known and unknown, in complex mixtures. Facile hydrolysis of human salivary phosphoproteins by the bioreactive probes was readily observed.

Cited by (0)

Published, MCP Papers in Press, January 6, 2004, DOI 10.1074/mcp.T300008-MCP200

*

This work was funded in part by National Institute of Environmental Health Sciences, National Institutes of Health under Contract No. N44-ES-35511. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.