Innovative approaches for in-depth quantitative analysis of the serum proteome in non-model organisms

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Blood serum is a rich source of protein biomarkers that plays a significant role in understanding the physiological responses of a body to adapt to different conditions. However, the serum proteome is highly complex due to the large dynamic range of protein concentration, only 22 proteins being responsible for ~99% of the whole protein content. Hence, thousands of low abundant proteins are easily masked during mass spectrometry analysis, preventing access to the full proteome. The conventional fractionation and immuno-depletion techniques are partial solutions to the problem, but not suitable for large cohort studies and the latter is less applicable to non-model organisms. In the light of this, two innovative commercial solutions - in-Stage Tip and ENRICH-iST (Preomics GmbH) were tested for the first time to analyze the serum proteome of two hibernating species: the brown bear and the Daurian ground squirrel. The combination of the novel workflows with the next generation dia-PASEF MS acquisition, enabled to identify nearly 1000 serum proteins with iST workflow, a result already outstanding, and this number exceeded 1700 with ENRICH technology. The preliminary data suggest that ENRICH-iST preserved the inherent abundance ratios for the majority of proteins, hence providing biologically unbiased enrichment. To further validate the quantitative performance of ENRICH technology and its application to other biological matrixes in non-model organisms, new experiments are planned.

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