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hardwired into the structures of these proteins as dictated by their gene sequences. But, they are also tightly controlled by
reversible modifications after they are initially manufactured, which are added on by regulatory proteins that operate
within cellular intelligence systems.
While gene sequences can provide some clues as to the potential functions and interactions of proteins with each other
and other molecules, this information is extremely limited. Even now, we do not have a real sense of what over a third of
these diverse proteins do, and less than 20% of these proteins have received any real serious attention in research labs.
The disconnect between genetic information and the actual occurrence of disease is due to the high impact of
environmental factors such as diet, life style and exposure to agents in the environment that can affect the proteome.
Proteomes are immensely complex and dynamic. For example, blood plasma may contain as many as 40,000 different
protein products, and their individual concentrations can range over a trillion-fold. Consequently, tracking proteins offers
much better insights into the occurrence of diseases than genetic profiling, and importantly the opportunity for more
rational therapeutic intervention.
While about 21,300 genes encode proteins in the human genome, the actual number of distinct protein entities in the
proteome may actually exceed several million, largely due to the range and degree of added modifications and other
processing. More than 50 types of modifications have been documented in proteins, with phosphorylation as the
predominant reversible regulatory mechanism. Over 85% of the proteome is known to be phosphorylatable at over
250,000 sites, but the actual number of phosphosites appears to be closer to a million. The occurrence of these and other
modifications in proteins represent a rich source of biomarkers that may correlate better with the development of
pathologies.
Most sites of known protein modification were originally revealed by mass spectrometry (MS). However, apart from
being very expensive, MS requires milligram amount of biological sample material and is finicky for reliable detection
of desired target proteins. For example, out of some 3000 phosphosites in proteins that have been well documented to be
functionally important in the scientific literature, about 22% have not been reported in any MS studies, whereas another
16% were documented in only one of thousands of MS analyses that had been performed.
Antibodies have been well proven to be reliable and effective probes for the detection and quantification of specific
proteins for their present and modification states. Over a million different antibodies against diverse proteins are
presently commercially available. Furthermore, the printing of antibodies as individual microdots on microscope slide-
sized chips with densities exceeding 5000 spots per chip has paved the way for biomarker discovery that is easily
translatable into the development of routine diagnostic tests. Biomarker antibodies can readily be re-deployed into other
tried and true platforms such as immunoblotting, ELISA, and immunohistochemistry.
Problems with sample preparation, high background issues, and low sensitivity of detection initially hampered the wide-
spread adoption of antibody microarrays. However, recent breakthroughs on all of these fronts have poised antibody
microarrays to become the most versatile, reproducible, and cost-effective tools in the foreseeable future for biomarker
discovery, using as little as 25 microgram amounts of protein samples from crude, unfractionated lysates from cells,
tissues, and bio fluids. High content antibody microarrays can identify the most appropriate and robust panel of
biomarkers. When used to probe lysate microarrays printed instead with hundreds of patient specimen samples on each
slide, these biomarker antibodies can provide accurate, comprehensive and economical diagnoses for diseases and for the
monitoring of the effectiveness of therapeutic treatments.
36 | December 2018