Mass Spectrometric Techniques
Denis Hochstrasser, M.D., professor of medical biochemistry at Geneva University Hospital (Switzerland), described the need for instrumentation that can perform multidimensional protein separation and analysis and provide accurate results rapidly and inexpensively.
One of the main questions in clinical proteomics at present is where to focus biomarker discovery efforts. Plasma may not be the best place to start, in Dr. Hochstrasser's view, even with fairly sophisticated tools, due to the complexity of its protein make-up.
He suggested beginning the search for disease-related biomarkers in the affected tissuesfor example, for stroke, looking at the spinal fluid for markers of cell death as evidence of brain tissue injury. Once a marker is identified, an immunoassay can then be developed for clinical diagnostic use that detects and measures the marker in plasma.
Dr. Hochstrasser anticipates that clinical labs will require instrumentation and expertise in a combination of immunochemistry and MS techniques, describing MS as "the future of molecular medicine."
One can imagine being able to take a thin slice of a diseased tissue sample and expose it to laser energy, causing the proteins to fly out of the sample and into a mass spectrometer, which would then identify and analyze the proteins, looking for specific biomarkers to guide diagnosis.
Eleftherioss Diamandis, M.D., Ph.D., head of clinical biochemistry at Mount Sinai Hospital in Toronto, explained the mechanics behind mass analyzers and the use of electric or magnetic fields, generated by ion traps and quadrupoles, for example, to manipulate ionized particles, allowing only one ion to enter the detector at a time.
Mass spectrometers analyze gas-phase ions based on their mass-to-charge ratio. Second-generation mass analyzers combine elements of earlier systems to generate hybrid instruments, such as dual time-of-flight, triple quadrupole, quadrupole-TOF, and triple quadrupole-ion trap instruments.
Like existing techniques, novel strategies in development to break down proteins and separate them for better resolution each have their own advantages and disadvantages in terms of accuracy, resolution, and throughput.
Dr. Diamondis described the advantages of top-down proteomics, analysis of whole proteins, for qualitative applications such as identifying proteins in a complex mixture and studying post-translational modifications (PTMs), versus the advantages of bottom-up proteomics, in which the proteins are first broken down into peptides, which are then analyzed.
The latter approach is useful more for quantitative proteomic applications such as biomarker studies, comparing normal versus disease tissue samples, for assessing changes in protein levels before or after drug treatment, or for time-course studies.
Reagents developed for quantitative proteomics, which label proteins with different colored tags, enable monitoring of multiple parameters in one experiment.
State-of-the-art MS-based protein analysis is far from a perfect science. Dr. Diamondis provided the example of a single sample that was analyzed by six different laboratories. In total, 1,109 proteins were identified, but only 52 proteins were identified by all six labs, and 63% of proteins were identified by only one group.
Nevertheless, he sees MS and protein microarrays as the future "workhorses of high-throughput proteomics." Describing the protein microarray as the "compact disk of the future," he envisioned it playing a central role in protein identification and functional studies aimed at evaluating protein-protein interactions.
During a panel discussion, participants identified the most critical issue for clinical proteomics going forward as the reproducibility of results from lab to lab. Whereas peak intensity may vary depending on the MS instrument used, mass accuracy should not.
The panelists also discussed the challenges inherent in working with biological specimens, including storage issues. Whatever is done to a sample once it leaves the patient will modify it, and the potential effects of freezing a sample, breaking open cells, and isolating the proteins, or digesting a protein sample into its peptide components at the bedside, for example, must all be characterized and understood.