Protocol

Analysis of Proteomes Using the Molecular Scanner

This protocol was adapted from "The Use of Mass Spectrometry in Proteomics," Chapter 8, in Proteins and Proteomics (ed. Simpson). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2003.

INTRODUCTION

The molecular scanner offers a flexible and powerful visualization tool that can create a fully annotated 2D gel electrophoresis map. Proteins separated by 2D gel electrophoresis are simultaneously digested while undergoing electrotransfer from the gel to a membrane. The peptides are subjected to peptide mass fingerprint (PMF) analysis to identify proteins directly from the PVDF membranes by MALDI-TOF-MS scanning. An ensemble of dedicated tools is then used to create, analyze, and visualize a proteome as a multidimensional image. The molecular scanner method reduces to a minimum the sample handling prior to mass analysis and decreases the sample size to a few tens of micrometers, that is, the size of the MALDI-TOF-MS laser beam impact. The process can be divided into four parts: separation and digestion of proteins, acquisition of PMF data, processing of the MS data and protein identification, and creation of multidimensional proteome maps.