Metal Ligand Affinity Pipettes and Bioreactive Alkaline Phosphatase Probes

Abstract

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. 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. Posttranslational modification is an important aspect of expression of biologically active proteins. Primary translation products may undergo modifications, ranging from quite simple (N-terminal acetylation or C-terminal amidation) to more complex additions (glycosylation, phosphorylation, farnesylation) or proteolytic cleavages to generate the final active product. Developing the capabilities for determining the nature of the modifications and their positions within a protein sequence is a challenging problem that requires considerable time and effort when standard biochemical methods are used as tools. For example, radiolabeling and two-dimensional gel electrophoresis are often used to determine the extent and positions of protein phosphorylation. This combination of methods is time consuming and requires isotopic techniques with their attendant problems. Moreover, in vivo radiolabeling is not a generally accepted practice for studies with human mass methods are of in the problem of modifications, protein of mass the of proteins and by mass of with mass in and of proteins by mass of of with mass methods often two-dimensional gel used matrix-assisted laser mass used matrix-assisted laser mass or to proteins and of proteins by two-dimensional gel electrophoresis and by laser of spectrometry and of for the of proteins immobilized affinity mass for proteins and of of by mass of the translation of proteins as a for the by mass spectrometry and to to of protein with as a for of proteins and for mass and of phosphoproteins from by affinity with mass