Poster Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

Small-protein enrichment assay enables the rapid, unbiased quantification of over 100 low abundance factors from human plasma (#559)

Dylan J Harney 1 , Amy T Hutchison 2 , Zhiduan Su 1 , Luke Hatchwell 1 , Leonie K Heilbronn 2 , Samantha Hocking 3 , David E James 1 , Mark Larance 1
  1. School of Life and Environmental Sciences, University of Sydney, Camperdown, NSW, Australia
  2. Discipline of Medicine, University of Adelaide, Adelaide, South Australia, Australia
  3. Central Clinical School, Faculty of Medicine and Health, University of Sydney, Camperdown, NSW, Australia

Unbiased and sensitive quantification of low abundance small proteins in human plasma (e.g. hormones, immune factors, metabolic regulators) remains an unmet need. These small protein factors are typically analysed individually and using antibodies that can lack specificity. Mass spectrometry (MS)-based proteomics has the potential to address these problems, however the analysis of plasma by MS is plagued by the extremely large dynamic range of this body fluid, with protein abundances spanning at least 13 orders of magnitude. Here we describe an enrichment assay (SPEA), that greatly simplifies the plasma dynamic range problem by enriching small-proteins of 2-10 kDa, enabling the rapid, specific and sensitive quantification of >100 small-protein factors in a single untargeted LC-MS/MS acquisition. Applying this method to perform deep-proteome profiling of human plasma we identify C5ORF46 as a previously uncharacterized human plasma protein. C5ORF46 is expressed in either major blood vessels, or the heart, and whose role may be positively correlated with HDL particle abundance and reverse cholesterol transport. We further demonstrate the reproducibility of our workflow for low abundance protein analysis using a stable-isotope labelled protein standard of insulin spiked into human plasma. We applied this method to study the intermittent fasting response in an overweight human female cohort and observed several unexpected changes including; decreased plasma abundance of the iron homeostasis regulator hepcidin, decreased VLDL-associated protein APOC4 and increased plasma osteopontin. Thus, SPEA provides the ability to study numerous important, low-abundance hormones in a single rapid assay by overcoming much of the dynamic range problem in plasma proteomics, which makes it a promising tool for plasma protein discovery and quantitative assay development.