Introduction

Protein corona-based proteomic workflows that utilise beads are being more popular as sample preparation tools to help mitigate the dynamic ranges issues of plasma. There are a number of options available in the market that all work, using similar mechanisms, by enriching for the ‘sample’ on the bead corona and thus depleting the problematic high abundance proteins. Although effective, these have a high cost per sample. We have developed novel methods that utilise volumetric absorptive microsampling devices (VAMS) to collect samples and to deplete high abundance proteins from blood samples at a much lower per sample cost. In this study we sought to compare a representative bead technology with our VAMS methods to assess differences and benefits of each technology.

Methods

Whole blood and plasma (single-spun and double-spun) from healthy volunteers was collected and processed using 2 workflows, (1) our standard workflow using VAMS and (2) the PreOmics ENRICH-iST workflow.

1. Samples were applied to 30µL Mitra VAMS tips and dried. Samples were incubated in an extraction solution (whole blood: 24 hours, plasma: no incubation), tips were then washed and proteins were digested in tip with trypsin (1 µg/µL in lysis buffer).

2. Samples were combined with prepared EN-BEADS and incubated for 30 minutes. The sample-bound beads were then lysed, alkylated, and digested with the proprietary provided reagents and methods.

For both workflows, peptide concentration of all samples were quantified and normalised to 0.2 µg/µL and were analysed by LCMS.

Preliminary data

Each workflow is compatible with a 96-well plate system and for plasma samples, both protocols can be completed within 24 hours. A cost comparison of the methods determined that the VAMS methods are 10-fold more affordable than the ENRICH-iST kit per sample.

ENRICH-iST is only validated for use with plasma samples, as such this was the primary comparison we used to determine reproducibility and validity of each method. Plasma was single-spun or double-spun before sample enrichment, processing, and analysis for both workflows. There were fewer missed cleavages in the ENRICH-iST processed samples compared to the VAMS samples (23% vs 33%), indicating greater trypsin digestion for this protocol.

The lowest mean %CV for plasma was observed for double-spun plasma on the ENRICH-iST beads with the double-spun plasma on the VAMS as a close second. VAMS produced a higher protein and peptide count (1,218 vs 1,030 for proteins and 11,306 vs 8,246 for peptides) and a lower overall %CV. Single-spun plasma produced more protein identifications than the double-spun for all samples (2634 vs 1664 for identifications with VAMS and ENRICH-iST respectively) but was significantly more variable for the ENRICH-iST beads.

Although the ENRICH-iST beads are not recommended for whole blood, they did produce a similar number of protein identifications to VAMS devices, with 3019 proteins detected. However, there was significant variability in the data in that 46.5% of the proteins had a %CV greater than 20%. The VAMS whole blood samples were highly reproducible (mean %CV of <10%) and produced the highest number of identifications of all samples with 3614 proteins detected.

Novel aspect

Novel blood sample enrichment methods providing better affordability and reproducibility and producing more protein identifications than corona-based workflows.