Poster Presentation 24th Annual Lorne Proteomics Symposium 2019

Optimizing red blood cell protein extraction for biomarker discovery (#82)

Stephan Klatt 1 , Anne Roberts 1 2 , Blaine Roberts 1 2
  1. The Florey Institute of Neuroscience and Mental Health, Melbourne/Parkville, VIC, Australia
  2. Cooperative Research Centre for Mental Health, Parkville, Victoria 3052, Australia

Red blood cells (RBCs) are the most common cell type found in the blood. However, not many biomarker assays have been conducted on RBCs. This can be attributed to the large dynamic range of proteins, high abundance of lipids and metabolites, and difficulties accompanying hemoglobin interference particularly with colorimetric and ELISA assays. In this study, we developed a semi-quantitative assay by comparing the efficiency of urea, sodium deoxycholate, acetonitrile and HemoVoidTM to extract the RBC proteome. Protein lysates were analyzed on an Agilent 6495 QQQ instrument, targeting 144 proteins. The results confirm that RBC protein extraction with HemoVoidTM leads to a tremendous reduction of hemoglobin and subsequently to a detection increase of most remaining proteins. In addition, peptide normalization to the RBC housekeeping proteins resulted in very stable coefficient of variation (CV) values. Hemoglobin interference after deoxycholate and urea extraction is still strong, but signal intensities even for low abundant proteins are in a satisfying range, and most CV values are strong. Extraction with Acetonitrile leads to an overall decreased protein abundance probably due to protein precipitation. High-throughput feasibility is most promising after urea extraction. We therefore recommend RBC protein extraction with either HemoVoidTM or urea.