Plasma proteomics has advanced considerably, yet the field faces a critical challenge: the vast dynamic range of plasma proteins makes it difficult to detect low-abundance biomarkers that may hold diagnostic value. While recent innovations in mass spectrometry and sample preparation workflows have enabled detection of thousands of proteins, there remains uncertainty about which workflows perform optimally across different biological contexts, species, and sample types.

This exploratory study by Emery-Corbin et al. aimed to benchmark eight plasma sample preparation workflows across human serum, human plasma, and rat plasma to determine how workflow selection influences proteome coverage and composition.

Samples were analysed using an Orbitrap Astral connected to a Vanquish Neo with direct injection onto Aurora® Ultimate™ 25×75 C18 UHPLC columns (65-minute gradient, ‘Discovery’ method) and Aurora® Elite™ 15×75 C18 UHPLC columns (30-minute gradient, ‘Throughput’ method), both operated at 50°C.

The researchers identified up to 2,726 human proteins and 3,767 rat proteins across all workflows. Corona enrichment methods, particularly MagNet, achieved the deepest coverage with over 1,700 proteins in human biofluids and 3,000 in rat plasma.

These findings demonstrate that plasma proteomic workflows must be selected based on the specific biological questions and disease context, as different methods illuminate different subsections of the plasma proteome.

Publication
bioRxiv

Authors

Samantha J Emery-Corbin, Joel R Steele, Dylan H Multari,Erwin Tanuwidjaya, Iresha Hanchapola, Komagal Kannan Sivaraman, Han-Chung Lee, Scott A Blundell, Idrish Ali, Terence J O’Brien, Pouya Faridi, & Ralf B Schittenhelm;

Title

‘If the shoe fits?’ – technical nuances of plasma proteomic workflows in clinical and preclinical contexts