ReviewProtein complexes gain momentum
Introduction
More than a decade after both electrospray ionisation (ESI) and matrix-assisted laser desorption/ionisation (MALDI) emerged as gentle ionisation methods, mass spectrometry (MS) has become a powerful tool for analysing the structure of biomolecules and, more recently, for describing their interactions [1]. Primary structural information is obtained by performing controlled fragmentation, a technique that underpins most proteomic strategies. For noncovalent biomolecular complexes, cross-linking strategies or maintenance of the complex within the mass spectrometer allow the molecular weight of the complex to be recorded, thus defining the stoichiometry of the interacting components. The speed with which such analyses can be performed enables dynamic reactions to be monitored in real time, adding an exciting new dimension to the investigation of transient associations in macromolecular complexes. In this review, we present recent highlights of mass spectrometric approaches to the study of protein interactions and the spatial arrangement of proteins in complexes.
Section snippets
Mass spectrometry methods for the analysis of biomolecules
For MALDI-MS, the sample is mixed with a large excess of matrix material; it is then deposited on a metal surface and the solvent is allowed to evaporate. Upon irradiation with short UV or IR laser pulses, ions are desorbed from this target in vacuo. In more recent variants of this technique, the solid, or sometimes liquid, target is at atmospheric or intermediate pressure. MALDI produces predominantly singly charged ions and consequently is often combined with specific protease digestion to
Proteomic strategies for the identification of interacting proteins
MS is the key technology in the systematic analysis of an organism's entirety of proteins (the proteome) [11•]. Two major applications in this area are expression proteomics, in which up and down regulation of protein levels are monitored in response to different factors, and functional proteomics, targeted at the characterisation of cellular components and multiprotein complexes. An impressive example of the latter application identified more than 24 proteins that interact with the 26S yeast
Defining the stoichiometry of protein complexes
An emerging method, which is being employed to identify subunit stoichiometry and transient associations, is to preserve complexes intact in the mass spectrometer, measure their mass and use high energy collisions to effect their dissociation. These approaches are established as powerful methods for examining the stoichiometry of complexes and monitoring changes in response to different solution conditions, for example, temperature, pH, ligands, cofactors and other proteins. Recent highlights
Determining the topological arrangement of protein subunits
The spatial organisation of multicomponent complexes can be investigated by MS using two different strategies. The first involves the covalent cross-linking of the interacting surfaces. For example, a yeast nuclear pore complex was purified using an affinity-tagged component. A six-membered subcomplex was then partially cross-linked and the products were separated by SDS-PAGE and digested [21•]. Mass analysis by MALDI and specific proteases revealed nearest-neighbour relationships between
Monitoring dynamic interactions
An important asset of MS, often overlooked, is the rapid time frame for recording experiments. A timescale of less than 1 s for acquisition of a spectrum enables the formation of assembly intermediates to be studied in real time. For example, to study the assembly of a hexameric molecular chaperone complex, GimC, the two different subunits were mixed in the appropriate ratio and species formation was monitored by MS [24]. The results showed that an α-subunit dimer was formed initially,
Future prospects
Given the ability to ionise intact ribosomes [7] and viruses, such as the MS2 bacteriophage composed of 180 identical copies of capsid protein [26], the question arises as to the limitations of the MS approach. The MS analysis of these complexes typically gives rise to signals around 20 000–25 000 m/z, corresponding to ions carrying between approximately 100 and 130 positive charges. The practical limitations arise in defining the m/z values of the charge states, primarily due to the incomplete
Update
This year's Nobel prize in chemistry, awarded in part to John B Fenn (Virginia Commonwealth University, Richmond, Virginia, USA) and Koichi Tanaka (Shimadzu Corporation, Kyoto, Japan), has recognised the development of the two complementary soft ionisation/desorption techniques highlighted in this review. This breakthrough, about a decade ago, has transformed MS, enabling its widespread use in all fields of chemistry and especially biochemistry. From the earliest applications of ESI and MALDI
Acknowledgements
We acknowledge with thanks helpful discussions with Helena Hernández, and financial support from the Wellcome Trust, BBSRC and the Royal Society.
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 (28)
Protein complexes and analysis of their assembly by mass spectrometry
Curr Opin Struct Biol
(2000)Electrospray ionisation mass spectrometry: a technology for studying noncovalent macromolecular complexes
Int J Mass Spectrom
(2000)Timing the flight of biomolecules: a personal perspective
Int J Mass Spectrom
(2000)- et al.
MS as a novel approach to probe cooperativity in multimeric enzymatic systems
Anal Biochem
(2001) - et al.
Screening transthyretin amyloid fibril inhibitors: characterization of novel multiprotein, multiligand complexes by mass spectrometry
Structure
(2002) - et al.
Probing molecular interactions in intact antibody: antigen complexes, an electrospray time-of-flight mass spectrometry approach
Biophys J
(2001) - et al.
Calcium-induced noncovalently linked tetramers of MRP8 and MRP14 are confirmed by electrospray ionisation-mass analysis
J Am Soc Mass Spectrom
(2000) - et al.
Observation of large, non-covalent globin subassemblies in the approximately 3600 kDa hexagonal bilayer hemoglobins by electrospray ionisation time-of-flight mass spectrometry
J Mol Biol
(2001) - et al.
Subunit exchange of multimeric protein complexes. Real-time monitoring of subunit exchange between small heat shock proteins by using electrospray-mass spectrometry
J Biol Chem
(2002) - et al.
Nano-electrospray ionisation mass spectrometry: addressing analytical problems beyond routine
Fresenius J Anal Chem
(2000)
The effect of the source pressure on the abundance of ions of noncovalent protein assemblies in an electrospray ionisation orthogonal time-of-flight instrument
Rapid Commun Mass Spectrom
Influence of pressure in the first pumping stage on analyte desolvation and fragmentation in nano-ESI MS
Anal Chem
A tandem mass spectrometer for improved transmission and analysis of large macromolecular assemblies
Anal Chem
Detection and selective dissociation of intact ribosomes in the mass spectrometer
Proc Natl Acad Sci USA
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