Some biomarkers are easier to find than others. Once a class of molecules has been noticed, and the assay methodology to measure their levels has been optimized, data rapidly accumulates. Related molecules frequently pop up (often as a result of artifacts appearing in the assays under certain conditions or when particular samples are analysed). Its rather like unearthing an ancient pyramid – if the first dig identifies the tip of the pyramid, the rest follows quite quickly.
But imagine what it would be like trying to rebuild the pyramid if the blocks had been scattered over a wide area. Finding one block wouldn’t necessarily help you find the next one. That seems to be the case with the ever-growing superfamily of peptide modifications. A trickle of discoveries of naturally occurring modifications of peptides is turning into a flood. And the molecules that are being discovered seem to be associated with fascinating biology, and offer great promise as biomarkers now and in the future.
Modifications such as phosphorylation, sulphation, glycosylation and more recently glycation have been so extensively studied that they are taken for granted as part of the molecular landscape. But the molecular diversity they generate is still under-appreciated. Total Scientific have comprehensively analysed the unexpected array of natural antibodies against the oligosaccharides that decorate many extracellular proteins and peptides – and extended initial observations by others that changes in these anti-carbohydrate antibodies are useful biomarkers for the early stages of cancer development in man. But even these studies, using multiplexed assays to profile the portfolio of anti-carbohydrate antibodies, hardly scratch the surface of the molecular diversity that exists in this domain.
Over the last decade the range of covalent tags on peptides and proteins has expanded much further. The ubiquitin family of small peptide tags now numbers at least 46, and these can be added to proteins in a staggering variety of chains, ranging from a single ubiquitin tag to branched chains of different ubiquitin family members. These modifications play central roles in diverse biological pathways, from cell division and organelle biogenesis to protein turnover and antigen presentation. Our understanding of the importance of ubiquitinylation is progressing rapidly, but in the absence of good methodology to differentiate the vast diversity of tag structures the possibilities that proteins and peptides modified in this way may be valuable biomarkers is all but unexplored.
Covalent tags, such as phosphorylation, ubiquitination or nitrosylation, are not the only natural modifications of peptides now known. More surprisingly, mechanisms exist to modify the amino acids composing the peptide chain itself. Some seem highly specific for a single metabolic pathway (such as the formation of S-adenosylmethionine in the folate cycle controlling methyl group transfer); others at least seem limited to a single class of protein targets (such as lysine acetylation in histones to regulate the strength of DNA binding); but more recently it has become clear that enzymes exist to modify peptidyl amino acid side chains in a wide range of different substrates. The best-studied example is the enzyme peptidyl arginine deiminase (PAD), which converts arginine in peptides and proteins into citrulline. This unusual reaction only came to light because of the misregulation of PAD that occurs in almost all cases of rheumatoid arthritis (RA). Dysregulated PAD activity in the extracellular space results in the generation of hundreds of different citrulline-containing proteins and peptides, many of which are immunogenic. This, in turn, results in the formation of antibodies against citrulline-containing protein antigens (called ACPAs or anti-CCPs). Diagnostic kits measuring anti-CCP levels have revolutionized the clinical diagnosis of RA, almost completely supplanting the use of rheumatoid factor, which has poorer sensitivity and specificity. Today, the presence of anti-CCP antibodies is almost pathomnemonic for classical RA, and sales of the proprietary kits for measuring this biomarker are generating millions annually for their discoverers.
Conversion to citrulline is not the only fate for arginine residues in peptides and proteins. In bacteria, conversion of arginine to ornithine is a key step in the generation of self-cleaving peptides called inteins. Intriguingly, one of Total Scientific’s clients has recently discovered an analogous pathway in eukaryotes (including humans) that generates naturally occurring lactam-containing peptides, and we are helping them generate new assay methodology for this novel and exciting new class of potential biomarkers.
Even simpler than covalent tagging and metabolic transformation of the amino acid side chains is simple cleavage of the peptide or protein. Removal of a handful of amino acids from the N-terminus (by dipeptidyl peptidases) or the C-terminus (by carboxypeptidases) of peptides can already generate hundreds of different sequences from a single substrate peptide. Endoproteolytic cleavage at specific internal sites generates further diversity. The problem here is that both the product and the substrate contain the same sequence, making the generation of antibodies specific for a particular cleavage product very difficult to generate. Total Scientific are developing generally-applicable proprietary methods for successfully raising antibodies specific for particular cleavage products, and these tools should greatly accelerate the growing field of biomarkers that are specific cleavage products (such as the use of N-terminally processed B-type Naturetic Peptide, or ntBNP for the diagnosis of heart failure).
If the detection of different, closely related, cleavage products from a single substrate is a challenging analytical conundrum, then the specific detection of particular non-covalent aggregates of a single peptide or protein is surely the ultimate badge of honour for any assay developer. Recent data suggests that some peptide hormones, such as adiponectin, may signal differently when aggregated in higher molecular weight complexes compared to when present in lower molecular weight forms.
Frustratingly, none of this wealth of diversity in the potential biomarker landscape is captured in the genome. The glittering insights of the vast space beyond this post-genomic biomarker frontier have mostly come from fortuitous stumbling across a particular example. But the sheer frequency with which such discoveries are now being made suggests there is a substantial horde of buried treasure out there waiting for us to develop the appropriate analytical tools to find it. Total Scientific have built up an impressive toolkit, capable of shining a flashlight into the darkest corners of the post-genomic biomarker space and we relish any opportunity to turn this expertise into exciting new biomarker discoveries for our clients.
Dr. David Grainger
CBO, Total Scientific Ltd.
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