Tanuja Koppal

Early detection and accurate diagnosis of a disease is a critical step and one that often determines the outcome of the disease. Hence the discovery of biomarkers that can predict and/or monitor the progression of a disease is an area of research that holds a lot of promise.

Identification and quantification of proteins using the direct tissue proteomics (DTP) method. Click to enlarge.

Proteins play an important role in determining cellular function and large-scale protein profiling under various physiological conditions is paving the way for protein biomarker discovery. However, the nature and quality of the data obtained is often restricted by the limitations of the technology used. Routinely used proteomic techniques such as surface-enhanced laser desorption ionization mass spectrometry (SELDI-MS) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI–TOF-MS) that identify protein expression patterns, fail to provide the exact chemical identity of the protein associated with a certain phenotype.

“With current mass spectrometers and existing methodologies we are able to detect only about half the proteins that get translated,” says David Han Ph.D., associate professor in the department of cell biology and director of the proteomics and biological mass spectrometry facility at the University of Connecticut, School of Medicine. In a recently published paper in Molecular and Cellular Proteomics, Han and his team have outlined a shotgun proteomics approach combined with a new protein extraction procedure to disrupt the cross-linked proteins from formaldehyde-fixed, paraffin-embedded tissue samples. This methodology, which they refer to as direct tissue proteomics (DTP), was shown to identify individual proteins from minute quantities of archived clinical tissue samples.

This research provided, for the very first time, a large-scale proteomic map of human coronary atherosclerotic plaques from paraformaldehyde-fixed, paraffin embedded and frozen coronary arteries. The study identified a total of 806 proteins, many of which had previously been implicated to play a role in atherosclerosis. However, there were other regulatory proteins such as PEDF, periostin, MFG-E8 and annexin I, that had not been previously identified in human coronary atherosclerotic lesions. Many low abundance proteins, such as growth factors and cytokines, were also detected and quantified.

Han and his colleagues had previously used DTP to identify proteins in paraformaldehyde-fixed, punch biopsy tissue samples from prostate cancer patients. The study published last year in Oncogene helped identify more than 400 proteins from the cancer tissue biopsies, including picogram levels of prostate-specific antigen (PSA), an important serum biomarker used to detect prostate cancer.

Serum biomarkers like PSA are preferred in the clinic because the sample extraction protocol is quick, fairly inexpensive and largely non-invasive. However, the limitation with working with serum is that it contains high abundance proteins such as albumin. “With human proteomic techniques, fishing for important biomarkers in the serum is not as productive as going directly to the source tissue,” says Han. “With serum, I am not sure you can routinely measure very low levels of important proteins using current technologies.”

These two studies act as a proof of principle for using DTP for conclusive identification of proteins directly from minute amounts of archived clinical tissue samples. However, Han does acknowledge that additional studies need to be done before specific biomarkers can be identified and validated. “This study is not a comprehensive study where we engage clinical questions,” says Han. “Larger patient samples and detailed analysis are needed to gain further insight.”

Limitations to the technique were also brought forth by this study. For instance, certain cytokines and growth factors known to be found in plaques were not detected. “One possibility is that they are rapidly consumed or they are present only in certain stages of the plaque,” says Han. “A second possibility is that they are present in very low abundance.”

Han is hopeful that in the next 3 to 5 years mass spectrometry will permit the detection of very low copy numbers of protein in complex samples. “Clearly there are people who are developing mass spectrometers that are a thousand times faster and more sensitive [than those available today],” says Han. “That’s when you can go after the post-translational modifications, splice isoforms and low abundant enzymes that have a huge impact on the biology of the disease. At that point I have no doubt in my mind that MS and tissue proteomics will be used in a clinical setting for diagnosis or biomarker detection. It will then be a true bench-side to bedside kind of application.”

Exploiting interdisciplinary skills

David Han (center, white shirt) and colleagues from the University of Connecticut, School of Medicine. Exploiting interdisciplinary skills Trained as a molecular biologist, David Han received his Ph.D. in genetics from the Department of Molecular Biology at George Washington University. As a graduate student, he worked on vascular smooth muscle maturation, and continued to build his knowledge of smooth muscle cell biology as a Cardiovascular Research Fellow, with Steve Schwartz and Leroy Hood in the Department of Pathology at the University of Washington (UW), School of Medicine. His second post-doctoral stint, in the UW Department of Molecular Biotechnology, offered the opportunity to work under Reudi Abersold and learn about proteomic technologies like mass spectrometry.