Mass spec and the soft cell

If mass spectrometry didn't exist, biologists would surely have had to invent it. Mass spectrometry has put the pep into peptide sequencing -- there's no quicker nor more accurate way of doing it.

Mass spectrometry has given molecular geneticists a double-headed arrow. At the gene's end, the DNA specifies the protein's peptide sequence -- but betwixt DNA and protein, Darwin's elves can play tricks with the basic recipe, by such means as shuffling exons, or a little post-hoc enzymatic cleavage.

The paucity of genes identified by the Human Genome Project, when compared to the plethora of proteins in human cells, suggests that the average human gene specifies at least half a dozen different proteins or peptide fragments.

From a spot on a 2D electrophoresis gel, a new-generation mass spectrometer can often determine which variant you're dealing with at the cellular end. Or, from a newly determined peptide sequence of a cellular protein, MS points back to the DNA source code, linking function to gene.

Now you're doing functional genomics, says Prof Michael Guilhaus, director of the Bioanalytical Mass Spectrometry Facility (BMSF) at the University of New South Wales (UNSW).

Guilhaus says there is a complex interaction between genome projects to map genes in various organisms, and determining what the proteins specified by genes actually do.

"There are lots of techniques for mapping genes, but proteins are the functional components or the nano-machines that make cells -- and life -- work," he says. "MS has really come into high prominence as a tool for characterising proteins as they occur in situ, in living tissues.

"What has changed is its ability to analyse large molecules, through new techniques like electrospray ionisation (ESI) and matrix-assisted laser desorption/ionisation (MALDI).

"These allow you to analyse all sorts of molecules that were formerly not amenable to MS because they couldn't be ionised in the gas phase.

"It's very important for molecular biologists to know the protein's amino acid sequence -- its primary structure -- because it tells you a lot about its function; a hydrophobic sequence may anchor it in a cell membrane, or different domains may stick a number of proteins together to assemble an ion channel."

The basic approach involves cleaving the protein into small peptide fragments with special enzymes, then 'flying' the fragments through the mass spectrometer to determine their mass. From the observed masses of the peptides, special software then identifies likely protein candidates among those expected to be expressed by known genes. Databases for this work are growing rapidly and mostly available online.

Another very powerful capability of mass spectrometry is de novo sequencing of the amino acids in peptide fragments. Here a special 'tandem' mass spectrometer selects a peptide fragment and fragments it into smaller units which collectively reveal part of the amino acid sequence. Knowledge of a few consecutive amino acids can often allow the whole protein to be identified, again with the aid of databases.

The holy trinity

Guilhaus describes genome databases, protein databases, and mass spectrometry as a "holy trinity" of modern biology.

"Databases for determining protein structure from the genomics end are becoming more vast, more accessible, and more easily searchable," he says.

Once a peptide mass fingerprint from a protein has been obtained in one species, there is now a reasonable chance of finding a match in a database, perhaps from another species.

Even if the match is slightly imperfect, it may be close enough to say that the two proteins are doing similar things -- that there's homology at the level of protein sequence, and thus, a likely similarity in function.

"The technology is new and exciting," Guilhaus says. "It's more sensitive, more robust, and more friendly to use. MS is much more sensitive and selective than it was 20 years ago."

While the interplay of genomics and proteomics is the main game, Guilhaus says the phenomenal sensitivity of today's mass spectrometers is revolutionising the study of metabolites.

These small molecules are produced by metabolic processes mediated by proteins. Through biochemical feedback, metabolites also have an important role in protein expression and post translational modification (see 'Cancer suspects', below).

Guilhaus says MS, in combination with separation and fractionation methods, is the only technique sensitive enough to characterise proteins and protein changes in very complex mixtures.

MBSF researchers are using MS to study, among other things, the role of protein oxidation in the development of the toxic protein deposits, or plaques in the brains of patients with Alzheimer's disease.

MS is now routinely used to make fine discriminations between chemically identical molecules, on the basis of different isotopes (see 'Analysis and detection', below).

Sensitive techniques

In the 1980s, Guilhaus and his colleagues at UNSW developed an extremely sensitive and selective MS technique called orthogonal acceleration time-of-flight mass spectrometry, or oa-TOF MS. This technology forms the heart of a new generation of hybrid quadrupole TOF MS instruments that is now rapidly advancing life science research.

"We developed a breakthrough in the TOF part to make it work with continuous ion sources like electrospray," Guilhaus says. "The new instruments have much more speed, sensitivity and resolution than earlier ones and they are a perfect tool for proteomics."

Where now for mass spectrometry?

Guilhaus says today's most sensitive mass spectrometers can detect just below a femtomole of protein in real samples. "You need about 100 million molecules before you can begin to identify a protein. But sometimes a cell will express only a few molecules of a particular protein, so you need to harvest a lot of cells to get to the detection threshold.

"Yet cells function beautifully with only a handful of molecules of certain proteins. A cell is around 10 million times more sensitive than a mass spectrometer, so the challenge is to catch up with that sensitivity.

"Another challenge, and one of the big issues in molecular analysis whether you're doing small molecules or large, is to detect molecules that may be at very low abundance in a diverse matrix of molecules, such as rare metabolites.

"Its natural abundance may be a billion times lower than that of the most numerous molecule in the matrix, such as the abundant blood-plasma protein albumin.

"Some mass spectrometers already employ advanced separation or fractionation tools, but they have to become even more selective. Local companies like Gradipore and Proteome Systems are working in this area, with new gels and membrane separation systems."

Guilhaus says better bioinformatics systems are also required to enhance the information yield from mass spectrometry, and make it more accessible. "We need to improve the way we integrate the various types of data, which means we need faster processing speeds, more intelligent search algorithms that can cross databases, and detect similarities or correlations between data types. "It's going to be a challenge to make it work."

Cancer suspects

Dr George Smythe, a member of Guilhaus' research team at the Bioanalytical Mass Spectrometry Facility, has developed a MS-based technique for diagnosing a rare cancer of the adrenal gland that causes high blood pressure.

The cancer, phaeochromocytoma, is so rare that Smythe has detected only 20 cases in the past 10 years. But he receives around 50 blood and urine samples to test each week because, he says, it has "a high suspicion index". With early detection and surgery, the cure rate is 99.9 per cent; undiagnosed, it can be rapidly lethal.

He uses a technique called isotope-dilution mass spectrometry to measure levels of seven different metabolites of transmitter compounds secreted by the adrenal medulla, in single run. No single metabolite is informative by itself -- the key to diagnosis is the relative levels of all seven.

Smythe says the patient's sample is diluted with a standard sample with a known C12/C13 or hydrogen/deuterium ratio. Changed isotope ratios then indicate how the patient's metabolite levels deviate from the standard, yielding a 'fingerprint' that, very rarely, may produce a diagnosis of phaeochromocytoma. Smythe says the technique goes beyond proteomics, into 'metabolinomics'.

"Once we've identified all the proteins in a cell, we then have to determine what the proteins are doing, in terms of cell function," he says.

"Metabolites are the key to understanding body function -- all those neurotransmitters, salts and other small molecules keep us going. Diagnostically, they're more important because they tell us what's going on, they work more rapidly, and they have longer half-lives than proteins.

"Increasingly, people working on cellular proteins are using MS to work backwards from metabolites to determine what is happening."

Analysis and detection

Last year, the partners in Australia's GrainGene consortium -- CSIRO Plant Industry, the Australian Wheat Board, and the Grains and Research Development Corporation -- released a new drought-tolerant wheat variety, Drysdale.

Researchers used MS to analyse the photosynthetic products of different cultivars, to identify breeding lines whose photosynthesis products reflect the natural abundance of the 12C and 13C isotopes of carbon in atmospheric carbon dioxide. Cultivars that discriminate against the rarer, slightly heavier 13C isotope use water less efficiently.

Drysdale's more efficient use of soil moisture is predicted to confer a yield advantage of 10 per cent over other cultivars in drought and reduced rainfall years.

Sports drug-testing agencies use MS to detect athletes illegally using the performance-enhancing hormone testosterone. The fixed 12C/13C ratio in synthetic testosterone contrasts with the variable ratio in 'natural' testosterone, due to diet.

Quadrupole mass spectrometry is the most commonly used system for detecting drugs on the International Olympic Committee's list of prohibited substances.

According to Damian Pomeroy, chromatography specialist with Varian Australia in Melbourne, it is almost impossible to do a full-scan analysis for all prohibited substances at low concentrations without considerable clean-up treatment of an athlete's urine sample.

It is not possible to unequivocally confirm the presence of a prohibited substance, such as an anabolic steroid, by selected-ion monitoring alone. The IOC now requires its accredited drug-testing laboratories to use high-resolution or tandem MS to identify anabolic steroids at levels approaching the limits of detection.

Varian researchers have been developing an analysis technique, based on Varian's Saturn 2200 GC MS/MS Ion Trap mss spectrometer, to detect 25 compounds -- either anabolic steroids themselves, or their metabolic by-products.

Used in samples involving complex matrixes, in which the compounds being sought may be present at only parts per billion levels, the technique selectively eliminates interference arising from the matrix 'background', to improve detection limits.

Mass spectrometry research and services in Australia

Below is a list of some of the key mass spec research groups and analytical service providers in Australia.

Australian Institute of Marine Science Conducts research for AIMS (mariculture, marine bioproducts), James Cook University and commercial ventures. Key contact: Rick Willis

Bioanalytical Mass Spectrometry Facility (UNSW) Research (large molecules and proteomics, small molecules and biomarkers, instrumentation) and wide range of analytical services. Key contact: Michael Guilhaus

Centre of Excellence in Mass Spectrometry (Curtin University) Thermal Ionisation Mass Spectrometry (TIMS), Sensitive High Resolution Ion Microbe (SHRIMP), Argon Mass Spectrometry (K-Ar, Ar-Ar), Inductively Coupled Plasma Mass Spectrometry (ICP-MS), Isotope Ratio Monitoring - Gas Chromatography Mass Spectrometry (GCMS), Stable Isotope Mass Spectrometry Key contact: John de Laeter

La Trobe University Research areas include photoionisation mass spectrometry, electrospray mass spectrometry Key contact: John Traeger

Macquarie University ESI MS, GC/MS, LC/MS Key contact: Daniel Jardine

RMIT University Provides mass spec services, support, and research to investigators within the Department of Applied Chemistry. Requests for mass spectrometry services are also taken from outside sources. Key contact: Frank Anastolic

University of Adelaide Electrospray ionisation triple quadrupole mass spectrometer is located at the Australian Wine Research Institute. Precise determination of molecular weight for proteins of up to 80KD or more, combined with HPLC system to provide LC/MS. Nanospray ionisation source allows low level sequencing of tryptic peptides by MS/MS. Jointly operated by the Department of Plant Science and The Australian Wine Research Institute. Key contact: Yoji Hayasaka

University of Melbourne Supporting the activities of many active research groups, as well as undergraduate teaching. The Mass Spectrometry Unit aims to provide high-quality mass spectral data using modern mass spectrometric methods. Key contact: Richard O'Hair

University of NSW Range of projects involving application of modern analytical instruments and methods to chemical analysis, and also in the design, modelling and fabrication of new instrumentation for mass spectrometry. The research is focused on but not limited to time-of-flight mass spectrometry. Key contact: Michael Guilhaus

University of Newcastle Group carries out experimental and theoretical research in chemistry. Key contact: Ellak von Nagy-Felsobuki

ABCUniversity of Queensland LC/MS for exploring novel methods for improving the delivery of pharmacologically active substances, such as drugs and peptide hormones. Key Contact: Istvan Toth

University of Sydney (Downard laboratory) Developing advanced mass spectrometry-based approaches and related technologies to study proteins and their interactions with other macromolecules to advance discoveries in proteomics. Key contact: Kevin Downard

University of Sydney (Chemistry) Analytical services Key contact: Xiaomin Song

ABCUniversity of Sydney (Pharmacy) Research, and services (instruments include ion trap MS/MS, LC/MS/MS). Key contact: Bruce Tattam

University of Tasmania Organic Mass Spectrometry Facility. Recent research applications include work for faculties of plant science, chemistry, biochemistry, pharmacy, zoology, agricultural science, and Royal Hobart Hospital, Forestry CRC and others. Key contact: Noel Davies

University of Western Australia Solid probe electron impact mass spectrometry, gas chromatography mass spectrometry (EI GC/MS), low and high resolution (up to ~20,000) solids probe EI, chemical ionisation (CI), EI GC/MS, CI GC/MS, fast atom bombardment (FAB or LSIMS) and electrospray mass spectrometry in both +ve and -ve ion. Key contact: Tony Reeder

University of Wollongong Research (development and application of new mass spectrometry-based methods for studying modified proteins and DNA), services (low resolution, high resolution, specialist tandem services) Key contact: Margaret Sheil

Victorian College of Pharmacy Analytical services supporting research projects. Low resolution mass spectrometric analysis using ESI+/- or APCI+/- ionisation, accurate mass determination using EI or ESI ionisation, LC/MS using ESI or APCI ionisation. Key contact: Stuart Thomson

Other links:

Australia & New Zealand Society for Mass Spectrometry Home page for the society includes information on upcoming events and links to research in Australia and abroad.

i-mass International web-based mass spec resources.

Spectroscopy Now Publisher Wiley's web site for spectroscopy users.

Usenet mass spectrometry newsgroup Contains the sci.techniques.mass-spec archives and links.

Sources: Australia and New Zealand Society for Mass Spectrometry, RMIT Applied Chemistry