Blood Matrices and Sample Preparation Influence Blood Marker Discovery

Area of Science:

• Proteomics
• Biomarker Discovery
• Mass Spectrometry

Background:

• Plasma and serum are common in high-throughput proteomics, but matrix effects are understudied.
• Routine diagnostics use serum or Li-heparin plasma, while research often uses EDTA plasma.
• Understanding matrix and preparation impacts is crucial for robust proteomic analysis.

Purpose of the Study:

• To systematically investigate LC-MS/MS proteomic profiles across different blood matrices and sample preparation methods.
• To identify optimal parameters for robust identification of body fluid marker proteins.
• To compare the performance of various anticoagulants (EDTA, citrate, Li-heparin) and preparation techniques.

Main Methods:

• Pooled blood samples (serum, EDTA plasma, citrate plasma, Li-heparin plasma) from three healthy individuals were analyzed.
• Sample preparation involved iST, ENRICH-iST kits, strong-anion exchange (SAX) beads, SPEED, and perchloric acid (perCA) precipitation.
• Mass spectrometry was performed using Q Exactive HF-X and timsTOF HT instruments in data-independent acquisition (DIA) mode.

Main Results:

• Protein identification numbers varied significantly across matrices; EDTA plasma and serum performed better than citrate plasma.
• SAX beads, ENRICH-iST, and perCA methods yielded high protein identification but also increased variability.
• 181 protein groups overlapped across all timsTOF HT analyzed samples, with matrix- and preparation-specific protein subsets identified.

Conclusions:

• EDTA plasma and serum are suitable matrices for mass spectrometry-based proteomics.
• Specific sample preparation methods (SAX, ENRICH-iST, perCA) can enhance protein identification but require careful optimization.
• This systematic comparison provides guidance for selecting appropriate blood matrices and preparation protocols for biomarker discovery.