ReviewMass spectrometry and proteomics
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
References (31)
- et al.
Using mass spectrometry for quantitative proteomics
Proteomics: A Trends Guide
(2000) Mass spectrometry. From genomics to proteomics
Trends Genet
(2000)- et al.
Rapidly switchable MALDI and electrospray quadrupole-time-of-flight mass spectrometry for protein identification
J Am Soc Mass Spectrom
(2000) - et al.
An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database
J Am Soc Mass Spectrom
(1994) - et al.
Linking genome and proteome by mass spectrometry: large-scale identification of yeast proteins from two dimensional gels
Proc Natl Acad Sci USA
(1996) - et al.
Mass spectrometric approaches for the identification of gel-separated proteins
Electrophoresis
(1995) - et al.
MALDI quadrupole time-of-flight mass spectrometry: a powerful tool for proteomic research
Anal Chem
(2000) - et al.
The characteristics of peptide collision-induced dissociation using a high-performance MALDI-TOF/TOF tandem mass spectrometer
Anal Chem
(2000) - et al.
High throughput proteome-wide precision measurements of protein expression using mass spectrometry
J Am Chem Soc
(1999) - et al.
Protein identification with a single accurate mass of a cysteine-containing peptide and constrained database searching
Anal Chem
(2000)
Probability-based protein identification by searching sequence databases using mass spectrometry data
Electrophoresis
Error-tolerant identification of peptides in sequence databases by peptide sequence tags
Anal Chem
A strategy for rapid, high-confidence protein identification
Anal Chem
Role of accurate mass measurement (+/−10 ppm) in protein identification strategies employing MS or MS/MS and database searching
Anal Chem
Fluorescence detection of proteins in sodium dodecyl sulfate-polyacrylamide gels using environmentally benign, nonfixative, saline solution
Electrophoresis
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