Daniel Figeys

Identification of proteins by capillary electrophoresis-tandem mass spectrometry evaluation of an on-line solid-phase extraction device

Capillary electrophoresis-tandem mass spectrometry has been used successfully for the analysis of complex peptide mixtures. The method is limited by a relatively high concentration limit of detection and by matrix effects. Here we describe on-line coupling of a solid-phase microextraction device to a capillary electrophoresis-tandem mass spectrometry system. The performance of the integrated instrument was evaluated for the identification of proteins by their amino acid sequence. We report that the concentration limit of detection was improved at least 1000 fold to the low attomole/microliter range and that matrix effects were minimized by extensive sample clean-up during solid-phase extraction. We demonstrate that the implementation of a solid-phase extraction device significantly enhances capillary electrophoresis-tandem mass spectrometry as a method for the identification of low abundance proteins isolated from high-resolution two-dimensional polyacrylamide gels.

Capillary electrophoresis of peptides and proteins at neutral pH in capillaries covalently coated with polyethyleneimine.

Two procedures for the derivatization of the inner wall of fused-silica capillaries for the analysis of peptides and proteins by capillary electrophoresis (CE) at neutral pH are presented. In the first procedure, polyethyleneimine (PEI) is covalently attached to the capillary wall. In the second procedure, PEI is additionally cross-linked. We present analysis of standard peptides and proteins by CE using the coated capillaries. These coatings will have application for the separation of protein complexes at neutral pH, prior to analysis by electrospray mass spectrometry.

Towards an Integrated Analytical Technology for the Generation of Multidimensional Protein Expression Maps

A proteome has been defined as the protein complement expressed by the genome of an organism, or, in multicellular organisms, as the protein complement expressed by a tissue or differentiated cell [1]. In the most commonly used approach to proteome analysis the proteins extracted from the cell or tissue to be analyzed are separated by high resolution two dimensional gel electrophoresis (2DE), detected in the gel by staining or, if radiolabeled, by autoradiography, and identified by their amino acid sequence. The ease, sensitivity and speed with which gel separated proteins can be identified by the use of recently developed mass spectrometric techniques (MS) has led to a dramatic increase in interest in proteome studies (reviewed in [2]). One of the most attractive features of such analyses is that complex biological systems can potentially be studied as complete systems, rather than as a conglomerate of individual components.

Strategies and Methods for Proteome Analysis

A proteome has been defined as the protein complement expressed by the genome of an organism (Wilkins, et al. 1996). In multicellular organisms the proteome is the protein complement expressed by a tissue or differentiated cell. The most common approach to proteome analysis involves separation of proteins by one- or two-dimensional gel electrophoresis (IEF/SDS-PAGE), enzymatic cleavage of selected proteins, tandem mass spectrometry (MS/MS) of peptides, and finally data interpretation by computer routines which also search databases (Fig. 1.1). In most cases the availability of protein and DNA sequences in public databases eliminates the need for complete protein sequence analysis. Protein sequences are more rapidly identified by partial sequence analysis using tandem mass spectrometry which allows rapid, complete gene identification. As reliable as protein identification by mass spectrometry has become there are still many obstacles that prevent proteome analysis from becoming as automated and routine as genome analysis.

Solid-Phase Extraction-Capillary Zone Electrophoresis-Mass Spectrometry Analysis of Low-Abundance Proteins

Co-ordinated sequencing efforts have already produced the complete genomic DNA sequences of more than 18 prokaryotic and two eukaryotic (Saccharomyces cerevisiae and Caenorhabditis elegans) species (Goffeau et al. 1996; Blattner et al. 1997; Consortium 1998; see www.tigr.org for more details). However, neither the genomic sequence nor the sequences of expressed genes is sufficient to precisely describe biological processes. This is mainly due to the fact that the level of expression, activity, location in the cell, etc. of the components that constitute such processes are intricately controlled at various levels, including post-transcriptional ones (Gygi and Aebersold 1999). An accurate description of biological processes also requires analysis of the system at the protein level. Proteins are the molecules that control and execute most biological functions, and the level of expression, cellular location and state of activity of a protein are not apparent from its sequence alone.

Chapter 2:The Coupling of Microfabricated Fluidic Devices with Electrospray Ionization Mass Spectrometers

The early days of proteomics were driven by the need to develop novel analytical techniques for the rapid and sensitive identification of gel-separated proteins. At that time, two-dimensional (2D) gel electrophoresis was the predominant technique for separating complex protein mixtures. Furthermore,...