High select top14 abundant protein depletion resin

Eight microliters of each plasma sample were processed with the High-Select™ Top14 Abundant Protein Depletion Resin according to the manufacturer’s instruction (A36372, Thermo). Following depletion, proteins were subjected to LC–MS/MS with the Easy-nLC 1200 system coupled with a Q Exactive HF-X hybrid Quadrupole-Orbitrap (both Thermo Fisher Scientific, San Jose, USA) [11 (link)]. Purified peptides were separated on 25-cm HPLC columns with an inner diameter of 75 μm packed in-house with ReproSil-Pur C18-AQ 1.9 μm resin (Dr. Maisch GmbH). And about 1-μg peptides were loaded for each LC–MS/MS analysis. Raw data were acquired with a Data-Independent Acquisition method and analyzed by Spectronaut 15.0 (Biognosys AG, Switzerland). Q value (FDR) cutoff was set to 1% at the peptide and protein level. The average top 3 filtered peptides which passed the 1% Q value cutoff were used to calculate the major group quantities.

To increase the depth of proteome coverage, immunodepletion of highly abundant proteins was performed as previously described [7 ]. For CSF samples, 130 μl was incubated with equal volume (130 μl) of High Select Top14 Abundant Protein Depletion Resin (Thermo Fisher Scientific, A36372) at room temperature in centrifuge columns (Thermo Fisher Scientific, A89868). After 15 min of mixing with gentle rotation, the samples were centrifuged at 1000×g for 2 min. Sample flow-through was concentrated with a 3K Ultra Centrifugal Filter Device (Millipore, UFC500396) by centrifugation at 14,000×g for 30 min, and then the immunodepleted samples were diluted to equal volumes of 75 μl with phosphate-buffered saline. Immunodepleted CSF (60 μl) was then digested with LysC and trypsin. Briefly, the samples were reduced and alkylated with 1.2 μl of 0.5 M TCEP and 3 μl of 0.8 M CAA at 90°C for 10 min, followed by water bath sonication for 15 min. Samples were diluted with 193 μl of 8 M urea buffer [8 M urea in 10 mM Tris, 100 mM NaH₂PO₄ (pH 8.5)] to a final concentration of 6 M urea. LysC (4.5 μg) was used for overnight digestion at room temperature. Samples were then diluted to 1 M urea with 50 mM ABC. Trypsin (4.5 μg) was then added, and the samples were subsequently incubated for 12 h. The digestion was then stopped by adding final concentration of 1% FA and 0.1% TFA.