Nanoscale Real-Time Proteomics
Stanford University School of Medicine researchers, working with Cell BioSciences, have developed a nanofluidic proteomic immunoassay that measures protein charge, similar to immunoblots, mass spectrometry, or flow cytometry. But unlike these platforms, this approach can measure the amount of individual isoforms, specifically, phosphorylated molecules.
“We have developed a nanoscale device for protein measurement, which I believe could be useful for clinical analysis,” says Dean W. Felsher, M.D., Ph.D., associate professor at Stanford University School of Medicine.
Critical oncogenic transformations involving the activation of the signal-related kinases ERK-1 and ERK-2 can now be followed with ease. “The fact that we measure nanoquantities with accuracy means that we can interrogate proteomic profiles in clinical patients, by drawing tiny needle aspirates from tumors over the course of time,” he explains.
“This allows us to observe the evolution of tumor cells and their response to therapy from a baseline of the normal tissue as a standard of comparison.”
According to Dr. Felsher, 20 cells is a large enough sample to obtain a detailed description. The technology is easy to automate, which allows the inclusion of hundreds of assays. Contrasting this technology platform with proteomic analysis using microarrays, Dr. Felsher notes that the latter is not yet workable for revealing reliable markers.
“Microarray studies are not always consistent, and what works in one person’s hands doesn’t always appear to work for another investigator,” he argues. “This means that reliable clinical assays may not be as easily developed using this approach.”
Dr. Felsher and his group published a description of this technology in Nature Medicine. “We demonstrated that we could take a set of human lymphomas and distinguish them from both normal tissue and other tumor types. We can quantify changes in total protein, protein activation, and relative abundance of specific phospho-isoforms from leukemia and lymphoma patients receiving targeted therapy. Even with very small numbers of cells, we are able to show that the results are consistent, and our sample is a random profile of the tumor.”
Splice Variant Peptides
“Aberrations in alternative splicing may generate much of the variation we see in cancer cells,” says Gilbert Omenn, Ph.D., director of the center for computational medicine and bioinformatics at the University of Michigan School of Medicine. Dr. Omenn and his colleague, Rajasree Menon, are using this variability as a key to new biomarker identification.
It is becoming evident that splice variants play a significant role in the properties of cancer cells, including initiation, progression, cell motility, invasiveness, and metastasis. “Of course, one could spend a lifetime fully characterizing the consequences of variation in a single protein by classical biochemical methods,” says Dr. Omenn.
Alternative splicing occurs through multiple mechanisms when the exons or coding regions of the DNA transcribe mRNA, generating initiation sites and connecting exons in protein products. Their translation into protein can result in numerous protein isoforms, and these isoforms may reflect a diseased or cancerous state.
Regulatory elements within the DNA are responsible for selecting different alternatives; thus the splice variants are tempting targets for exploitation as biomarkers.
Despite the many questions raised by these observations, splice variation in tumor material has not been widely studied. Cancer cells are known for their tremendous variability, which allows them to grow rapidly, metastasize, and develop resistance to anticancer drugs.
Dr. Omenn and his collaborators used mass spec data to interrogate a custom-built database of all potential mRNA sequences to find alternative splice variants. When they compared normal and malignant mammary gland tissue from a mouse model of Her2/Neu human breast cancers, they identified a vast number (608) of splice variant proteins, of which peptides from 216 were found only in the tumor sample.
“These novel and known alternative splice isoforms are detectable both in tumor specimens and in plasma and represent potential biomarker candidates,” Dr. Omenn adds.
Dr. Omenn’s observations and those of his colleague Lewis Cantley, Ph.D., have also shed light on the origins of the classic Warburg effect, the shift to anaerobic glycolysis in tumor cells. The novel splice variant M2, of muscle pyruvate kinase, is observed in embryonic and tumor tissue. It is associated with this shift, the result of the expression of a peptide splice variant sequence.
It is remarkable how many different areas of the life sciences are tied into the phenomenon of splice variation. The changes in the genetic material can be much greater than point mutations, which have been traditionally considered to be the prime source of genetic variability.
“We now have powerful methods available to uncover a whole new category of variation,” Dr. Omenn says. “High-throughput RNA sequencing and proteomics will be complementary in discovery studies of splice variants.”
Splice variation may play an important role in rapid evolutionary changes, of the sort discussed by Susumu Ohno and Stephen J. Gould decades ago. They, and other evolutionary biologists, argued that gene duplication, combined with rapid variability, could fuel major evolutionary jumps.
At the time, the molecular mechanisms of variation were poorly understood, but today the tools are available to rigorously evaluate the role of splice variation and other contributors to evolutionary change.
“Biomarkers derived from studies of splice variants, could, in the future, be exploited both for diagnosis and prognosis and for drug targeting of biological networks, in situations such as the Her-2/Neu breast cancers,” Dr. Omenn says.