Katharina Limm (Planegg / DE), Zuzana Demianova (Planegg / DE), Katrin Hartinger (Planegg / DE), Nils Kulak (Planegg / DE)
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.
We use cookies on our website. Cookies are small (text) files that are created and stored on your device (e.g., smartphone, notebook, tablet, PC). Some of these cookies are technically necessary to operate the website, other cookies are used to extend the functionality of the website or for marketing purposes. Apart from the technically necessary cookies, you are free to allow or not allow cookies when visiting our website.