Mass spectrometry: Critical mass

During the past decade, dramatic progress in the field of mass spectrometry has resulted in a large increase in the number of commercially available mass spectrometry (MS) instruments principally used for biochemical analysis. Advances in mass spectrometry instrumentation, driven primarily by proteomics and the need to identify low abundance proteins (<50 femtomoles) has resulted in the current generation of instruments having approximately 50 times more sensitivity combined with substantially greater resolution than mass spectrometers available only four or five years ago.

MS is becoming a pivotal bioanalytical tool in many biotechnology and biochemistry laboratories. Two ionisation methods, matrix assisted laser desorption/ionisation (MALDI) and electrospray ionisation (ESI) MS, were discovered in the late 1980s and rapidly incorporated into commercially available mass spectrometers, allowing the characterisation of a myriad array of small and large molecules with high sensitivity, speed, accuracy, and efficiency.

MS-based techniques are becoming a permanent component of studies involving functional genomics, proteomics, early drug discovery, clinical diagnostics, and combinatorial chemistry. According to Assoc Prof Michael Guilhaus at the Bioanalytical Mass Spectrometry Facility (BMSF), located at the University of NSW, the number of scientists using MS to analyse and identify biological molecules, especially proteins and peptides, is increasing at an exponential rate.

Two complementary methods are frequently used for proteomics applications - MALDI, which derives a peptide mass fingerprint from enzymatic digestion of a protein, and micro-LC/ESI low energy collision-induced dissociation, which produces a string of amino acid sequences. Spectra from both methods are used in combination with protein database searches.

Guilhaus says the biggest trend in MS instrumentation and technology is the push for more sensitivity. "In real estate it is 'location, location, location' and in mass spectrometry it is 'sensitivity, sensitivity, sensitivity'. There is no value in resolution, accuracy and precision if you can't detect the molecule that you are looking for," he says.

Mass spectrometers are amongst the most sensitive instruments in biochemical analysis, Guilhaus says, with detection limits reaching down to attomole levels (millions of molecules). However this is woefully insensitive compared to cells themselves where function may be driven by the expression of just a few molecules. There is a wide, even bewildering range of equipment available for MS, from a variety of vendors, and Guilhaus says that there is no "best solution" with many instruments having complementary capabilities.

Tandem mass spectrometers, such as the hybrid quadrupole TOF systems are very important, according to Guilhaus, as they give high quality data with high sensitivity but without requiring exceptional skill. In addition they have high throughput capabilities and require minimal maintenance.

Of the other major instruments out there, Guilhaus says that ion trap instruments offer ease of use, automation and flexibility for tandem MS applications. High-end MALDI instruments provide rapid and easy access to molecular weights (particularly important for peptide maps) and triple quadrupole instruments have unique advantages for quantitative analysis with high duty-cycle efficiency in linked scans.

Of all the techniques available, Fourier transform MS is still probably the most powerful technique available, Guilhaus says, but it has high capital and maintenance costs and is not yet particularly user-friendly. Guilhaus also believes that new technologies like TOF-TOF and linear ion trap instruments bear watching. "The strategy for instrument acquisition at the BMSF is for productivity. In general we seek the best instrument for the job and prefer to dedicate that instrument to a particular type of task that it is good at," says Guilhaus. He explains that multifunctional instruments tend not to work as well for the facility because of the compromises in performance and the time lost changing the configuration.

MS these days is combined with a variety of different technologies, particularly in proteomics. While it is often run in combination with chromatographic techniques such as GC and HPLC, the advent of proteomics has incorporated differential display gel analysis and chip analysis, as well as protein and peptide separation and prefractionation to extend the coverage of analysis or specifically select classes of proteins. Affinity based quantitative measurements can be used to determine the relative abundance of proteins and peptides.

Sophisticated algorithms and software have also been developed to allow accurate and rapid determination of proteins using protein sequence databases, validation and determination of possible protein-protein interactions. "The focus now is to apply cutting-edge proteomic technologies so that a robust analysis of complex biological systems becomes inexpensive and widespread," says proteomics specialist Valerie Wasinger, who recently joined the UNSW mass spectrometry team.

What the BMSF does

The Bioanalytical Mass Spectrometry Facility (BMSF) at the University of NSW has grown from a single GC/MS instrument in the Faculty of Medicine in the 1970s to being classified as a Major Australian Research Facility in 2000 with 12 front-line instruments covering the major MS technologies and applications. Last year, the BMSF received over $4 million in support, including some of the $2 million awarded to UNSW as part of the DETYA 2001 Systemic Infrastructure Initiative.

Under the leadership of recently appointed director Assoc Prof Michael Guilhaus, a team of MS specialists at the BMSF covers instrumentation, small molecules and biomarkers, protein fractionation and characterisation, and biological mass spectrometry, providing training, education, and analytical services. "The mission of the BMSF is to underpin research principally in the Faculties of Medicine, Science and Engineering at UNSW," says Guilhaus.

"In a wider context, the BMSF acts as an advanced mass spectrometry node for the national biotechnology and biomedical research network." But as well as providing MS services to UNSW and the biotechnology community, the BMSF is actively engaged in research of its own. "A unique feature of the BMSF is that it is engaged in the development of new and improved mass spectrometry instrumentation," says Guilhaus.

Guilhaus is internationally recognised for his work developing time-of-flight (TOF) mass analyser technology, now used in the Micromass Q-TOF and Applied Biosystems Q-STAR mass spectrometers. In 1997, he was awarded the Curt Brunnee Award for Outstanding Contribution to Instrumentation and Theory in Mass Spectrometry, from the International Mass Spectrometry Society.

Among the comprehensive list of MS instruments and systems at the BMSF are automated GC/MS instruments, benchtop LC/MS, triple quadrupole and hybrid quadrupole-TOF LC/MS/MS systems that are based on Guilhaus' technology, as well as prototypes aimed at next generation of benchtop MALDI and GC/MS TOF instruments under development.

The facility has also set up a state-of-the-art electrophoresis laboratory to complement its liquid chromatography and MS capabilities. The BMSF is actively engaged in research on large molecules and proteomics, and small molecules and biomarkers. Guilhaus says that research originates at the facility as well as through collaborations with external research groups and industry, and covers a wide range of applications.

"Large molecule/proteomic research focuses on the fractionation and analysis of biopolymers especially proteins in complex mixtures. As well as identification, (post) translational modification and associated implications for various pathologies are investigated," explains Mark Raftery, one of two project leaders for large molecule and proteomics research (the other is Valerie Wasinger).

"The capability of mass spectrometry to characterise very large molecules, with molecular mass of up to hundreds of KDa, has dramatically increased since about 1990," says Raftery. He attributes this to the development of MALDI and ESI techniques, which have allowed powerful analysis of polymers including biopolymers.

"In the area of biopolymers the greatest current interest is in protein/peptide analysis sometimes referred to as proteomics," Raftery says. Among its proteomics projects the BMSF is working on isolation and characterisation of a variety of oxidation products and oxidative modifications to proteins including apolipoproteins including novel atherosclerosis-associated proteins isolated from human lesions.

In another project, the structures, reactivity and mechanisms of action and regulation of blood factors including plasminogen and von Willebrand factor are being investigated using peptide mapping and MS techniques. One aspect of Wasinger's work, in collaboration with Gradipore, is assessing methods to remove albumin from plasma to allow easier characterisation of less abundant plasma proteins.

BMSF deputy director Assoc Prof George Smythe heads up the small molecules and biomarkers group. "There is a vital role for MS in medical research and environmental chemistry. The BMSF participates in ongoing collaborative projects with researchers, principally investigating physiological and diagnostic roles of small molecule biomarkers and metabolites," he explains. According to Smythe, the major focus of the research is on inflammatory and oxidative pathways - especially in newborn and age-related diseases. "Clearly, many of the disease processes involved in the current investigations are multi-facetted and the major expansion of the BMSF into proteomics and novel instrument sampling techniques will enable considerable interaction between the specific disciplines of the expanded facility," Smythe says.

Research performed by the group includes a project applying MS and proteomics to investigate the role of oxidative and inflammatory processes in Alzheimer's Disease, with the hope of identifying diagnostic markers for the disease. They are also investigating the utility of MALDI-TOF MS for rapid, specific identification of clinically relevant bacteria. A number of other projects investigate a range of metabolic processes, including growth hormone pathways, inflammatory and apoptotic processes and nitric oxide metabolism in bone fractures and tissue healing.

While the BMSF is actively involved in research, it also provides analytical services on a contract basis. It has a partnership with the Australian Protein Analysis Facility, based at Macquarie University, to provide services on a contract basis as required. Training and education also play a big role at the BMSF. Guilhaus says that the more user-friendly nature of MS instruments means that scientists can perform their own measurements once trained.

"At the BMSF we are moving towards a more hands-on approach for our collaborators and analytical services. We offer training for postgraduates and research staff and assist in the analysis and interpretation of data," he says. A four-day proteomics workshop will be hosted by the BMSF in late September.

The BMSF's web address is www.bmsf.unsw.edu.au

We are grateful to the BMSF team for its collaboration on this article.