Advances in mass spectrometry for proteome analysis

doi:10.1016/S0958-1669(00)00114-2

Current Opinion in Biotechnology

Volume 11, Issue 4, 1 August 2000, Pages 384-390

https://doi.org/10.1016/S0958-1669(00)00114-2 Get rights and content

Abstract

The most demanding problems in proteomics continue to challenge modern mass spectrometry. Recent developments in instrument design have led to lower limits of detection, while new ion activation techniques and improved understanding of gas-phase ion chemistry have enhanced the capabilities of tandem mass spectrometry for peptide and protein structure elucidation. Future developments must address the understanding of protein–protein interactions and the characterisation of the dynamic proteome.

Introduction

Proteomics concerns the characterization of the full complement of proteins in a specific organism or cell type. The outline of a stereotypical proteome analysis is well established: (ideally) unbiased recovery of all protein constituents; fractionation of the protein mixture; generation of structural data by mass spectrometry (usually following enzymatic hydrolysis of the proteins); and matching of analytical data against those predicted for constituents of a database of known proteins or anticipated expression products. Whereas the conventional choice of two-dimensional electrophoresis as the means for protein mixture fractionation is open to discussion, there is no significant debate concerning the central role of mass spectrometry (MS) as the structural technique of choice; it is presently unrivalled with respect to the yield of structurally characteristic data at high sensitivities of analysis. The inherently exquisite sensitivity of MS with respect to the detection of ions, however, disguises the frustratingly low efficiency of overall analysis. It may be possible to detect single gas-phase ions but peptide and protein analyses are still generally performed at the femtomole level or higher because of the poor efficiency of ion formation and transmission. Furthermore, the use of tandem MS to generate structural information in order to refine and improve the specificity of database searching directs attention to the understanding and control of the gas-phase ion chemistry underlying the tandem techniques. How do we optimize the generation of structural data and what level of structural detail is required for effective database searching? To what extent can demands on the protein fractionation step that precedes MS analysis be mitigated by the fact that MS, and particularly tandem MS, can accomodate mixtures and provide diagnostic data for individual components? Consideration of issues such as these prompts consideration of recent and anticipated developments in MS and related techniques applied to proteome analysis. This review presents a discussion of some of these developments. It is intended to be neither detailed nor comprehensive, but rather to emphasize that the role of MS in proteomics (Figure 1) is rapidly developing and that our present routine capabilities are likely to appear modest, if not naı̈ve, in very few years from now.

Section snippets

Sample presentation to the mass spectrometer

Sample preparation and presentation to the mass spectrometer play a key role in determining the successful outcome of any proteomics experiment. Two-dimensional gel electrophoresis is most commonly employed despite the recognition of discrimination against more hydrophobic proteins (such as membrane proteins) and that this denaturing technique necessarily destroys protein–protein interactions. Recognizing these limitations, a number of research groups have moved towards the use of

Mass spectrometry instrument developments for protein and peptide analysis

The MALDI technique generates gas-phase ions from analytes deposited on surfaces and this essential attribute has prompted multiple investigations of the direct desorption of proteins or peptides from electrophoresis gels or from membrane blots 15, 16, 17. Strupat and co-workers [15] described the development of a method for obtaining the molecular masses both of proteins blotted onto membranes and of proteolytic peptides arising from on-membrane digestion. Desorption from the membrane was

Integrating analytical data output with database searching

Improved and extended capabilities of mass spectrometric analysis (as summarized in the previous section) have a direct bearing on the success of protein recognition via database searching. The first phase of such searching is usually based on the masses of proteolytic fragments alone; a number of workers have investigated the effects of mass accuracy 41, 42. Two factors may limit the success of this approach: firstly, peptide masses alone may provide insufficient specificity of searching;

Conclusions

Such approaches described above in which the analyte is modified to achieve appropriate characterization complement developments of the instrumental techniques. This report has reviewed some significant recent developments and highlighted areas in which future progress may be expected. The analytical challenge remains considerable, particularly in considering the need for quantitative definition of a dynamic proteome.

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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ReviewAdvances in mass spectrometry for proteome analysis

Abstract

Introduction

Section snippets

Sample presentation to the mass spectrometer

Mass spectrometry instrument developments for protein and peptide analysis

Integrating analytical data output with database searching

Conclusions

References and recommended reading

Anal Biochem

J Chromatogr B Biomed Sci Appl

Trends Biotechnol

J Am Soc Mass Spectrom

J Am Soc Mass Spectrom

J Am Soc Mass Spectrom

Anal Biochem

J Am Soc Mass Spectrom

Identification of proteins in complexes by solid-phase microextraction/multistep elution/capillary electrophoresis/tandem mass spectrometry

Anal Chem

Data-dependent modulation of solid-phase extraction capillary electrophoresis for the analysis of complex peptide and phosphopeptide mixtures by tandem mass spectrometry: application to endothelial nitric oxide synthase

Anal Chem

A generic strategy to analyze the spatial organization of multi- protein complexes by cross-linking and mass spectrometry

Anal Chem

Integrated microanalytical technology enabling rapid and automated protein identification

Anal Chem

Analytical properties of the nanoelectrospray ion source

Anal Chem

Separation and identification of peptides from gel-isolated membrane proteins using a microfabricated device for combined capillary electrophoresis/nanoelectrospray mass spectrometry

Anal Chem

Microfabricated devices for capillary electrophoresis-electrospray mass spectrometry

Anal Chem

Microfabricated polymer devices for automated sample delivery of peptides for analysis by electrospray ionization tandem mass spectrometry

Anal Chem

A micromachined chip-based electrospray source for mass spectrometry

Anal Chem

Miniaturized time-of-flight mass spectrometer for peptide and oligonucleotide analysis

J Mass Spectrom

Characterization of a serial array of miniature cylindrical ion trap mass analyzers

Rapid Commun Mass Spectrom

IR-MALDI-mass analysis of electroblotted proteins directly from the membrane: comparison of different membranes, application to on-membrane digestion, and protein identification by database searching

Anal Chem

Analysis of proteins by direct-scanning infrared-MALDI mass spectrometry after 2D-PAGE separation and electroblotting

Anal Chem

High sensitivity mass spectrometric methods for obtaining intact molecular weights from gel-separated proteins

Electrophoresis

Review
Advances in mass spectrometry for proteome analysis