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

Plasma and cerebrospinal fluid are complementary sources of biomarkers for neurodegenerative diseases. The wide dynamic range of protein abundances, particularly in plasma, hampers 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 for use in large-scale clinical proteomic studies. We developed a scalable sample preparation workflow for plasma and cerebrospinal fluid (CSF) that integrates depletion of high-abundance proteins, optimized digestion using Lys-C and trypsin, and compatibility with both label-free and tandem mass tag (TMTpro)-based quantification. Depletion was performed using a multi-affinity resin with immobilized antibodies targeting 14 high-abundance plasma proteins, which collectively constitute ≈ 95% of total plasma protein content. We systematically evaluated protein depletion and enzyme digestion conditions, and the effect of deoxycholate on digestion, monitoring the number of detectable proteins and the quantitation precision. A resin-to-plasma ratio of ≥ 75 and a mixing speed of 900 rpm ensured complete and reproducible depletion. Depletion resulted in an increase in the number of identified proteins by ~ 65% in CSF, and ~ 80% in plasma, tripling the number of brain-enriched proteins, with maintained quantitative precision (median coefficient of variation (CV) for relative protein abundances < 11%). A two-step digestion protocol using Lys-C/trypsin followed by trypsin yielded the highest reproducibility and detectability in plasma. Adding the detergent deoxycholate to the samples had little effect in CSF and only marginally improved proteome coverage for plasma but decreased quantification precision and throughput. Technical replicates from a 528-sample clinical amyotrophic lateral sclerosis (ALS) cohort showed high reproducibility, with intra-sample CVs substantially lower than inter-individual variation. The sample preparation workflow described here enabled deep and reproducible proteome profiling of plasma and CSF in high-throughput formats and was found to be suitable for biomarker discovery in large clinical studies.