Dataset Information

Shake and bake: a robust and cost-effective sample preparation recipe for plasma and cerebrospinal fluid

ABSTRACT: Background Plasma and cerebrospinal fluid CSF are complementary sources of biomarkers for neurodegenerative diseases. However, the wide dynamic range of protein abundances, particularly in plasma, hinders detection of low-abundance proteins. Depletion of high-abundance proteins and efficient enzymatic digestion can improve proteome coverage but must be carefully optimized for reproducibility, throughput, and cost-efficiency in large-scale clinical studies. Results We developed a scalable sample preparation workflow for plasma and CSF that integrates high-abundance protein depletion, optimized digestion using Lys-C and trypsin, and compatibility with both label-free and TMT-based quantification. We systematically evaluated depletion efficiency, mixing speed, enzyme digestion strategy, and the use of sodium deoxycholate, monitoring the number of detectable proteins and quantitative precision. A resin to plasma ratio of more than 75 and a mixing speed of 900 rpm ensured complete and reproducible depletion. Depletion increased protein group identifications by about 80percent in plasma and 65percent in CSF, while maintaining quantitative precision median coefficient of variation CV less than 11 percent. A two-step digestion protocol using Lys-C trypsin followed by trypsin yielded the highest reproducibility. DOC marginally improved proteome coverage but reduced quantification precision and throughput. In depleted plasma, detection of brain-enriched proteins tripled compared to non-depleted plasma. Technical replicates from a 528-sample clinical ALS cohort showed high reproducibility, with intra-sample CVs substantially lower than inter-individual variation. Conclusions This depletion-based sample preparation workflow enables deep and reproducible proteome profiling of plasma and CSF in high-throughput formats and is well suited for biomarker discovery in large clinical studies.

INSTRUMENT(S): timsTOF HT

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Johan Gobom