Extending the Depth of Human Plasma Proteome Coverage Using Simple Fractionation Techniques

Breaking the barriers of plasma proteome discovery

Plasma is an easily accessible and minimally invasive tissue that affords a significant opportunity to learn about human biology. Blood samples such as serum and plasma can be considered a form of “liquid biopsy” for personalized medicine applications, and early detection of disease-specific biomarkers

Mass spectrometry has been widely used in quantification of proteomic expression changes, biomarker discovery in plasma, and disease mechanism investigations. However, the main analytical challenge when applying proteomics methods to plasma is the presence of a few extremely high abundant proteins that dominate the protein content. Roughly 55% of the total protein mass in plasma is made up of albumin alone and as few as seven proteins together make up 85% of the total protein mass.

Although proteomics technologies have advanced significantly in recent years, only a few biomarkers identified through proteomics have progressed to successful and reliable clinical use. This is partly due to challenges in identifying high-throughput, reproducible, and robust workflows to identify and validate biomarkers in large sample sets.

Also, highly-abundant proteins in plasma often obscure lower-abundant proteins from detection by mass spectrometry. Plasma prefractionation techniques can play an important role in overcoming the challenges of complex plasma samples, therefore providing an avenue to identify low-abundance proteins.

To overcome these challenges Australian scientists from the University of New South Wales: Gurjeet Kaur, Anne Poljak, Ling Zhong, Mark Raftery and Perminder Sachdev in collaboration with Syed Azmal Ali from the National Dairy Research Institute, India, evaluated multiple fractionation techniques, to identify methods that would yield extensive plasma proteome coverage, identifying tissue-specific proteins, while remaining compatible with the high sample throughput requirement of most clinical studies. Their research work is published in the Journal of Proteome Research.

The research team compared low-abundance plasma proteins enrichment and high-abundance protein depletion followed by low-abundance protein fractionation using electrophoretic and chromatographic techniques. They compared the performance of each of the proposed fractionation techniques in a bid to choose one that befits the requirements of large clinical studies and to combine superior plasma proteome coverage with practical sample  throughput.

The researchers found that, compared with serial chromatographic separation, 1-dimensional gel-based prefractionation allowed for parallel sample processing, accelerated analysis time, which is particularly needed for large clinical projects, and proteome coverage of greater than 2000 proteins. However, all of the eight methods of plasma low-abundance protein enrichment and high-abundance protein depletion evaluated in their study allowed for substantial enhancement of typical plasma proteome coverage.

The findings of the study provide a clear demonstration of reproducibility of the evaluated prefractionation techniques and that gel-based methods can become suitable alternatives to time-consuming and expensive chromatographic separation, thereby accelerating analysis time. It’s quite promising to report that the several prefractionation techniques evaluated in the study could yield relatively high proteome coverage, thereby providing choice flexibility depending on project-specific needs, for example, whether a researcher is seeking quantitative or qualitative information, project size, availability of lab resources, etc.

The study by Gurjeet Kaur and colleagues further boosts the efforts of accelerated plasma proteomics research highly desired to provide dependable, precise, and efficient diagnoses, clinical research studies, population-based health evaluation, among other clinical work.