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Sep 1, 2011 (Vol. 31, No. 15)

Mass Spec Dominating Protein Profiling

Latest Enhancements Have Allowed Technique to Become a Rising Star in Proteomics Studies

  • Global Proteome Quantification

    Rob Beynon, Ph.D., chair of proteomics at the University of Liverpool, put forth what he refers to as “a rather modest ‘grand challenge’. For a simple proteome, first quantify, in copies per cell, the abundances of all proteins. Secondly, determine the rate at which those proteins are turned over in the cell. Finally, determine both sets of parameters with high confidence and quantify the inherent biological variance in the data.”

    Dr. Beynon described his experiences with the Waters ExpressionE system, which employs a high bandwidth UPLC/MSE data-acquisition strategy to consistently oversample complex protein digests, thereby delivering datasets containing evidence for all peptides above the limits of detection.

    He discussed his research group's experience with label-free proteomics as a solution to quantification problems. This approach has some difficult issues; isoform resolution and quantification remain significant problems, as do post-translational variants. However, label-free methods, particularly those based on summed peptide intensities, are remarkably valuable for many proteomics studies.

    “Identification is not the same as quantification,” Dr. Beynon cautioned. “Label-free approaches start to struggle for peptides or ions at about 0.1–1 fmol on column on the better instruments—at 1 fmol, this equates to about 3,000 copies per cell in yeast.” The situation is more pronounced in mammalian cells, as a typical load of a digest of HeLa cells, for example, equates to only 4,000 cells, or 150,000 copies per cell.

    To get a handle on the problem, Dr. Beynon noted that the sensitivity required to meet the “grand challenge” would be a detection limit in yeast of around 10 copies of a given protein molecule per cell.

    “On current instruments, we can routinely apply a digest derived from 200,000 cells, and in principle we can therefore reach between 30 and 300 copies per cell,” Dr. Beynon stated. “We anticipate instrument and informatics developments that should bring such methods to the required depth.”

    He described his invention of the artificial QconCAT proteins, concatamers of tryptic peptides for several proteins, which when expressed heterologously in bacteria create stoichiometric equivalent sets of standard peptides. “The QconCAT approach is robust and the limiting factors are quantotypic peptide nomination and the development of appropriate selected reaction monitoring assays.”

  • Tissue Imaging

    “Ion-mobility spectrometry is capable of separating molecules on the basis of their size or shape, whereas imaging mass spectrometry is an effective tool to measure the molecular weight and spatial distribution of molecules,” said Ron Heeren, Ph.D., research scientist at the Institute AMOLF, a section of the Foundation for Fundamental Research on Matter of the Dutch National Science Foundation. He discussed his lab's investigations combining these approaches in biomedical tissue imaging.

    “Histological imaging with mass spectrometry has a number of advantages. No labeling is required, thus biomolecules are unaltered; it is possible to image biomolecular modification including post-translational modifications and distribution of metabolites; detailed structural information can be provided; and mass spectrometry imaging can generate a molecular picture of the pathology of the case.”

    Dr. Heeren discussed the application of ion-mobility mass spectrometry imaging. The technology is based on the use of generated ions that travel through a drift tube that has an applied electric field and a carrier buffer gas that opposes the ion motion. The larger the ion size, the more area is available for the buffer gas to collide and impede the ion's drift—the ion then requires a longer time to migrate through the drift tube. This added separation provided increased resolution and structural information.

    Dr. Heeren and his colleagues have applied ion-mobility mass spectrometry to histological analysis. One series of experiments demonstrated how application of this technology can produce a clearer image of cartilage development in normal and diseased states. The protocol used joints in animal models and in humans to map tissues, combining conventional histological staining with mass spectrometry analysis.

    One of the markers the team followed was fibronectin, which undergoes dramatic changes in conditions involving joint deterioration. Other markers that change in response to inflammatory conditions include p53 and Il-17, putative breast cancer, cell adhesion, and angiogenesis markers.

    Dr. Heeren strongly supports imaging mass spectrometry technology as a game changer in the field of molecular histology. With its ability to visualize the location and quantitative levels of critical proteins, it is a far more specific technology than classic staining used traditionally in the pathology laboratory. “The ‘new glasses’ help us visualize and understand more molecular details of different diseases,” he concluded.

    There are numerous mass spectrometry options available for characterization of biological molecules, and the list continues to expand. Mass spectrometry companies are understandably reticent about the nature of their forthcoming offerings, but consumers can anticipate that the mantra of “faster, better, easier, cheaper” will continue to drive the industry forward.


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