How blood matrices and sample preparation can influence blood marker discovery

ABSTRACT: While plasma and serum are increasingly being studied by using high throughput proteomics, it remains largely unexplored how different types of blood materials collected in the routine clinical diagnostics laboratories influence protein marker discovery. We compared the LC-MS/MS proteomic profiles of pooled blood samples from three healthy, voluntary donors in the form of EDTA-, citrate- and heparin-anticoagulated blood as well as serum generated with and without separation gel. Sample preparation was conducted with five different methods. We systematically evaluated the commercially available iST and ENRICH iST kits from Preomics as well as strong-anion exchange (SAX) beads from MagReSyn, the TFA-based approach SPEED and a depletion-based method which uses perchloric acid (perCA) for precipitation of large proteins. Mass-spectrometric measurements were performed on a Q Exactive HF-X and a timsTOF HT in data independent acquisition (DIA) mode. Regarding all possible combinations of sample preparation and matrices, the Q Exactive HF-X MS instrument identified between 250 and 600 protein groups whereas the timsTOF HT identified between 275 and 1100 protein groups. Differences on protein group ID level in the applied sample preparation techniques were evident. Workflows using SAX-beads, ENRICH iST and the perchloric acid precipitation showed highest identification numbers but also highest variability between the different blood matrices. EDTA-plasma and serum exhibited the highest ID numbers while citrated plasma showed the lowest. 116 protein groups overlapped between all 25 sample sets for Q Exactive HF-X and 181 protein groups for timsTOF HT. Importantly, subsets of protein groups were unique for specific combinations of sample preparation and blood matrix. This study shows a systematic approach to determine suitable sample preparation and matrix parameters for the robust identification of individual body fluid marker proteins by mass spectrometry.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Plasma, Blood Serum

SUBMITTER: Thomas Gronauer  

LAB HEAD: Dr. Stefanie Hauck

PROVIDER: PXD066304 | Pride | 2026-01-12

REPOSITORIES: Pride