Review
Mass spectrometry and proteomics

https://doi.org/10.1016/S1367-5931(00)00121-6Get rights and content

Abstract

Proteomics is the systematic analysis of the proteins expressed by a cell or tissue, and mass spectrometry is its essential analytical tool. In the past two years, incremental advances in standard proteome technology have increased the speed of protein identification with higher levels of automation and sensitivity. Furthermore, new approaches have provided landmark advances in determining functionally relevant properties of proteins, including their quantity and involvement within protein complexes.

Introduction

A core component of proteomics is the ability to systematically identify every protein expressed in a cell or tissue as well as to determine the salient properties of each protein (e.g. abundance, state of modification, involvement in multi-protein complexes, etc.). The technology for such analyses integrates separation science for the separation of proteins and peptides, analytical science for the identification and quantification of the analytes, and bioinformatics for data management and analysis. Its initial implementation consisted of the combination of high-resolution two-dimensional gel electrophoresis (2DE), using IEF (isoelectric focusing)/SDS-PAGE gel, for the separation, detection and quantification of individual proteins present in a complex sample with mass spectrometry and sequence database searching for the identification of the separated proteins. A commonly used method is schematically illustrated in Figure 1. This technique and variations thereof (for review see [1]) have been used to identify and catalog large numbers of proteins present in a complex sample and to represent them in a proteome database, a process we refer to here as ‘descriptive proteomics’. For example, Shevchenko et al. [2] systematically identified 150 yeast proteins from 2D gels. Numerous such annotated databases are now accessible. The same techniques have also been used as a global discovery tool to detect dynamic changes in the proteome of a cell or tissue in response to external or internal perturbations. Because the detection of dynamic changes requires accurate quantification of each detected component, we use the term ‘quantitative proteomics’.

In this report we summarize the most significant developments related to proteomics and mass spectrometry as they have been reported from January 1999 to April 2000. Advances in core mass spectrometry technology have led to further refinements of the 2DE-based proteomics methods. They have also catalyzed alternative approaches to the traditional gel-based methods, such as the introduction of accurate protein quantification based on isotope dilution theory and the systematic analysis of protein complexes.

Section snippets

Advances in MS technology for proteome analysis

In this section we summarize advances in MS instruments, their control and operation, and progress in the searching tools used for the identification of proteins by correlating mass spectrometric data with sequence databases.

The performance of existing types of mass spectrometers for proteomics research has incrementally improved as new types of mass spectrometers were introduced. The instruments most commonly used throughout the review period can be grouped into two categories: single stage

Advances in descriptive proteomics

At the beginning of the review period, essentially all proteome projects were based on a combination of 2DE for protein separation, visualization and quantification and mass spectrometry for protein identification. This approach has been advanced by the developments in MS described above, by incremental improvements to 2DE, and by innovative combinations of gel electrophoresis and MS. Improvements to 2DE include the introduction of new fluorescent staining methods providing higher sensitivity

Quantitative proteomics

To add a quantitative dimension to non-2DE-based proteome analyses, the venerable technique of stable-isotope labeling [26] has been adapted for protein analysis. The method involves the addition to a sample of chemically identical but stable isotopically labeled internal standards (e.g. using 2H, 13C, 15N, etc.). Because ionization efficiency is highly variable for different peptides, the only suitable internal standard for a candidate peptide is that very peptide labeled with stable isotopes.

Analysis of protein complexes

Most cellular functions are not performed by individual proteins but rather by protein assemblies, also termed multi-protein complexes. It is rightly assumed that proteins which specifically interact also partake in the same function. The identification of specifically interacting proteins is, therefore, a critical component of the proteomics because it directly relates to protein function within biological processes. In general, the methods described above for the analysis of protein mixtures

Conclusions

A main strength of proteomics is the ability to analyze the dynamics of biological processes by the systematic analysis of expressed proteins. The technical advances described in this review, in particular the ability to measure accurately the quantitative changes induced by perturbations on large numbers of proteins and the ability to analyze functional protein complexes, add significantly to our ability to study biological processes and systems from a global standpoint. The coming year will

Acknowledgements

This work was supported by grants from the National Institutes of Health (HG00041, RR11823, T32HG00035, CA84698, A141109), National Science Foundation (BIR 9214821) and Merck Genome Research Institute.

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

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