Phosphoproteome Analysis in Medical Research
Authors: Butt Elke , Marcus Katrin 
Published: March 2008

Abstract
Originally published in: Proteomics in Drug Research. Edited by Michael Hamacher, Katrin Marcus, Kai Stühler, André van Hall, Bettina Warscheid and Helmut E. Meyer. Copyright © 2006 Wiley‐VCH Verlag GmbH & Co. KGaA Weinheim. Print ISBN: 3‐527‐31226‐9

The field of proteomics has undergone tremendous advances over the past few years and enabled the detailed analysis of proteins in very complex systems, such as cells, tissues, or body fluids (at a given time point), including the capture of structural diversity of proteins, splicing, processing, and posttranslational modification (e.g., phosphorylation, glycosylation). Several developments in the area of proteome research have revolutionized science, as biological processes can be probed at the molecular level using sophisticated analytical tools including two‐dimensional polyacrylamide gel electrophoresis (2D PAGE) and mass spectrometry (MS). The purpose of this review is to highlight how proteomics techniques have been used to answer specific questions related to signal transduction in platelets although the methods can be adopted in a wide variety of systems. Here, we restrict our discussion to the study of phosphoproteins. Our basic approach is to examine only those proteins that differ by some variable from the control sample. In this way, the number of proteins to be processed is greatly reduced. In eukaryotic cells, proteins are mainly phosphorylated on serine, threonine and tyrosine residues. Three examples of typical experiments to measure changes in protein phosphorylation in response to specific treatment will be discussed. In these studies the protein samples were separated by 2D PAGE and the phosphorylated proteins were directly assigned by detection of [
32
P]‐orthophosphate via autoradiography. Analysis of the phosphorylated proteins was done by MS. To concurrently identify the phosphoserine and phosphothreonine substrates of cAMP‐ and cGMP‐dependent protein kinases associated with the inhibition of platelet aggregation, human platelets were analyzed by incubating the cells with [
32
P] before separation on 2‐D gels. A similar approach was used to characterize the substrate specificities of different MAPK kinases by comparing the phosphoproteome of knock out mice with wild type animals after extracellular stress. The use of antibodies directed against phosphoamino acids represents an alternative procedure to detect phosphorylated proteins. This method was used for the additional detection of tyrosine‐phosphorylated proteins after the activation of platelets by thrombin. The data will help to build the basis for future identification of new drug targets and therapeutic strategies against thrombotic diseases such as stroke and myocardial infarction.

The sections in this article are

Introduction

Phosphoproteomics of Human Platelets

Cortactin

Myosin Regulatory Light Chain

Protein Disulfide Isomerase

Identification of
c
AMP
‐ and
c
GMP
‐Dependent Protein Kinase Substrates in Human Platelets

Identification of a New Therapeutic Target for Anti‐Inflammatory Therapy by Analyzing Differences in the Phosphoproteome of Wild Type and Knock Out Mice

Conclusions

Acknowledgements