Overcoming the dynamic range difficulties in CSF samples through a novel enrichment step for biomarker discovery studies

Introduction

Cerebrospinal fluid (CSF) is an essential body fluid that is particularly important for investigating brain disorders. However, CSF not only exhibits a very high dynamic range, but also has a very low protein concentration, which poses a particular challenge for proteome analysis. ENRICH-iST is a novel high-throughput workflow that addresses the dynamic range challenge in plasma/serum samples by providing a fast and straightforward sample preparation solution for LC-MS-based proteomic studies. It is based on the enrichment of low-abundance proteins, resulting in a significant increase in protein identifications and improved proteome coverage. Thus, we evaluated and optimized the ENRICH-iST workflow for CSF samples to provide an easy-to-use and standardized workflow for in-depth proteome analysis.

Methods

The ENRICH-iST workflow is based on the novel ENRICH technology that provides efficient dynamic range compression by enriching low-abundance proteins in biofluid samples onto non-functionalized paramagnetic microbeads. To address the challenge of low protein concentration in CSF samples, the ENRICH step was optimized by adjusting the starting volumes. For subsequent LC-MS sample preparation, the iST-BCT protocol optimized for biofluids (PreOmics) was employed for on-bead denaturation, reduction, alkylation, digestion, and peptide cleanup. Peptides were analyzed by nanoLC coupled to a timsTOF instrument (Bruker) using dia-PASEF^® acquisition mode.

Results

Considering the low concentration of CSF samples (~0.1 mg/mL) compared to plasma/serum samples (~60-80 mg/mL), the ENRICH step must be adapted to ensure successful dynamic range compression. Therefore, different input volumes of CSF were compared, and the workflow was optimized accordingly. Of note, the ENRICH-iST workflow has been successfully applied to CSF samples with input volumes ranging from 20 µl to 200 µl, resulting in improved proteome coverage. The workflow is therefore suitable for a wide range of starting volumes and can be scaled to the available CSF volume, providing maximum flexibility. Processing 200 µL of rat CSF with ENRICH-iST resulted in the identification of ~2300 protein groups; this corresponds to a 1.8-fold increase in protein identifications compared to the iST-BCT workflow without enrichment.

Furthermore, as an antibody-free approach, the ENRICH-iST workflow is not specific to human samples and can be applied to other mammalian species, making it particularly interesting for preclinical studies.

Conclusion

The optimized ENRICH-iST workflow enables in-depth proteome analysis for large CSF cohorts, simplifying and improving biomarker discovery studies.