Mass spectrometry measures the mass of molecules that have been converted to ions, and can be used to determine the structure and properties of those molecules. It can also be used to identify unknown substances. Ionization techniques include electrospray and matrix-assisted laser desorption/ionization (MALDI).A New Phase
David Clemmer, a professor in the Department of Chemistry at Indiana University Bloomington (Bloomington, IN) and his group developed ion mobility mass spectrometry as a new way to make the ions required for all mass spectrometry analysis. A gas phase step is used to separate ions by shape and mass. The conformation of a protein affects its mobility through the high-pressure buffer gas and can be used to obtain structural information. Although the structures of proteins in the gas phase may not be exactly the same as their structure in solution, the information obtained is still useful. His group is looking at how denatured proteins fold into their native state by looking at the structures of naked proteins in the gas phase.
“This technology is our claim to fame in the field,” Clemmer says. There are many applications, such as differentiating proteins from tissues in different developmental or disease states. “These days,” Clemmer observes, “there isn't a disease state where biomarkers aren't being looked at. It's valuable to distinguish disease states, for example, liver cancer from hepatitis caused by a viral infection from normal tissue.” He says that one way to do this is to look at the shape of glycans or branched polysaccharides on the surface proteins; their location suggests that they may be involved in signaling. “Changes in glycans may be associated with changes in protein structure,” he continues. “Currently, on a limited scale, we can distinguish these, so glycans appear to be a very good way to distinguish disease states.”
“If we don't apply technology, no one uses it,” Clemmer says, noting that one of his group's interests is mapping the human plasma proteome. Determining how a protein folds involves looking at individual proteins, whereas proteomics requires looking at samples containing complex mixtures of many proteins. “All of the proteins the body produces end up in the plasma,” he observes, and he estimates there are 100,000–1,000,000 proteins in the plasma. “Two-dimensional gel electrophoresis can see about 60 of these,” he says. “At this time, state-of-the-art ion mobility mass spectrometry can identify thousands of molecules, or at least thousands of peaks in milliseconds.” To identify all of these molecules and understand what is normal will require the ability to look at more than one molecule in a systems-biology type of approach. Clemmer notes that it “certainly was the case, and a little optimistic, to let the mainstream think we could interpret the genome” from the human genome sequence.
At the Georgia Institute of Technology in Atlanta, GA, the Center for Bio-Imaging Mass Spectrometry (BIMS) was created in an attempt to study complex biologic samples at the systems level. The center was created in 2007 to harness the potential of mass spectrometry imaging tools and the expertise of scientists at Georgia Tech, according to Al Merrill, who is Professor and Smithgall Institute Chair in Molecular Biology at the School of Biology and the Petit Institute for Bioengineering and Bioscience. In addition to the types of protein-containing specimens mentioned by Clemmer, other potential targets of work at BIMS include histologic sections of tissues, and communities of organisms in biofilms, which masquerade as shower curtain soap scum, occur as dental plaque, or exist on implanted medical devices where their structure may make them resistant to antibiotic treatment.
MALDI-mass spectrometry has been widely used for studying biologic samples. It requires the uniform deposition of a matrix compound on the sample. Merrill and colleagues—including Cameron Sullards, director of BIMS, and Yanfeng Chen, research scientist at the School of Chemistry—have shown that an oscillating capillary nebulizer used to spray matrix aerosol onto the surface of histologic tissue slides could improve homogeneity of the matrix.
They used tissue samples from mouse models of Tay-Sachs disease and Sandhoff disease, two lysosomal storage diseases (LSDs) that were provided by colleague Timothy Cox, Professor of Medicine at the University of Cambridge in Cambridge, UK. LSDs are a group of rare, inherited disorders caused by the deficiency or absence of a lysosomal enzyme. This causes the accumulation of substrate—normally processed or degraded by the enzyme—which damages organs and tissues. In Tay-Sachs disease and Sandhoff disease, β-hexosaminidase deficiency can result in the accumulation of the sphingolipid GM2 ganglioside and related glycolipids, damages organs, including the central nervous system, and causes death in childhood. The group's technique allowed improved localization of sphingolipids. They suggest imaging MALDI-mass spectrometry could be used for “lipomic” studies.
Other projects at Georgia Tech include the use of desorption electrospray ionization (DESI), which, unlike MALDI, allows samples to be analyzed outside of a vacuum. Facundo Fernandez, assistant professor at the School of Chemistry and Biochemistry at Georgia Tech, has used DESI to detect counterfeit anti-malarial drugs. Other applications of DESI include analysis of tumor tissue and environmental samples.
Johannes Drach is a professor of medicine and Program Director for Multiple Myeloma and Malignant Lymphoma at the Clinical Division of Oncology at Medical University Vienna, Vienna, Austria. Multiple myeloma, the second most common hematologic malignancy, is not a single disease, Drach points out. This is reflected by the ability to divide patients by those with standard-risk disease and those with high-risk disease, he says, based on cytogenetic changes such as chromosomal deletions, gains, and translocations. The role of the bone marrow microenvironment in supporting myeloma cell growth has been shown to be crucial, involving interactions between bone marrow stromal cells and myeloma cells that are mediated by cytokines, chemokines, and adhesion molecules that are secreted and modulate cell-cell interactions.
Drach and colleagues are using shotgun proteomics to investigate the myeloma “secretome.” Protein mixtures from the cytoplasmic fraction of bone marrow cells from patients with myeloma, as well as that of plasma cells from patients with monoclonal gammopathy of undetermined significance (MGUS) are being analyzed. MGUC sometimes progresses to myeloma, although this does not occur in every patient with MGUS, and the factors that cause progression are unknown. Protein mixtures from patients with MGUS are digested into peptides, separated by chromatography, and identified by tandem mass spectrometry. The spectra are translated into the amino-acid sequence to identify these proteins to create a non-quantitative clinical multiple myeloma proteomics database. Additionally, protein quantification via two-dimensional PAGE helps identify up-or downregulated proteins and modifications to them.
Drach's group is currently characterizing five myeloma cell-specific proteins. They have identified adhesion molecules that are upregulated in myeloma cells, and have created a secretion profile of bone marrow fibroblasts from healthy donors. “MGUS cells are already different from normal,” Drach says. They specifically express proteins associated with inflammation, chitinase-3–like proteins associated with neutrophil defense, and insulin-like growth factor-2 (IGF-2) in short-term co-culture with stromal cells. Survival factors appear to be upregulated in myeloma cells. Myeloma cells express the same proteins as MGUS cells plus others (e.g., stem cell growth factor and proteins involved in calcium and phosphate homeostasis). One goal is to look at how different therapeutic agents affect protein expression by these cells over time. Another goal is to analyze a larger series of samples that will allow identification of novel markers that might be used in diagnosis or determining prognosis.Non-routine Analysis
Leeder Consulting in Brisbane, Australia, is using mass spectrometry to identify the chemical makeup of odorants and link them to human perceptions of these compounds. They have coupled the separation of individual components of odorous substances by mass spectrometry in real time with people's descriptions of these components: their observations are recorded by voice activation software and transferred to the chromatogram, producing what Leeder calls “high-resolution gas chromatography mass spectrometry olfactometry.” This may allow better odor investigation, management, and reduction strategies, and might be useful in environmental investigations.