# Blood Matrices and Sample Preparation Influence Blood Marker Discovery

## TL;DR

This study shows how different blood types and preparation methods affect the discovery of blood markers using mass spectrometry.

## Contribution

The paper systematically compares blood matrices and sample preparation methods for proteomic marker discovery.

## Key findings

- EDTA plasma and serum provided better protein identification than citrate plasma.
- SAX beads, ENRICH-iST, and perCA methods yielded high identification numbers but with increased variability.
- 181 protein groups were consistently identified across all samples using timsTOF HT.

## Abstract

While plasma and serum are widely used in high-throughput
proteomics,
the impact of different blood matrix types remains underexplored.
Routine diagnostics most commonly use serum or Li-heparin plasma,
while the proteomics community primarily focuses on advancing analytical
depth in EDTA plasma. Here, we systematically investigated the LC–MS/MS
proteomic profiles of pooled blood samples from three healthy, voluntary
probands including serum (with/without separation gel) and plasma
anticoagulated with EDTA, citrate, or Li-heparin. Sample preparation
was conducted with the commercially available iST and ENRICH-iST kits,
strong-anion exchange (SAX) beads, the TFA-based approach SPEED, and
perchloric acid (perCA) precipitation. Mass-spectrometric measurements
were performed on a Q Exactive HF-X and a timsTOF HT in data-independent
acquisition mode (DIA). Protein identifications varied considerably
across matrix types with EDTA plasma and serum outperforming citrate
plasma. Sample preparation methods with SAX beads, ENRICH-iST, and
perCA yielded the highest identification numbers but also showed increased
variability. Across all samples, 181 protein groups overlapped for
timsTOF HT data. Subsets of protein groups were specific for the matrix
and preparation. This study shows a systematic approach to determining
suitable sample preparation and matrix parameters for the robust identification
of individual body fluid marker proteins by mass spectrometry.