The GeLC-MS workflow, which combines low-cost, easy-to-use SDS-polyacrylamide gel electrophoresis (SDS-PAGE) with liquid chromatography-mass spectrometry (LC-MS), is very popular in current bottom-up proteomics. However, GeLC-MS requires that PAGE-separated proteins undergo overnight enzymatic digestion in a gel, resulting in more than 20 hours of sample preparation for LC-MS. In this study, we overcame the limitations of GeLC-MS by developing a rapid digestion workflow for PAGE separation proteins using N,N'-bis(acryloyl)cystamine (BAC) cross-linked gels that can be solubilized by reductive treatment. Making use of an established workflow called BAC-DROP (BAC-gel dissolution to digest PAGE-resolved objective proteins), crude proteome samples were fractionated based on molecular weight by BAC cross-linked PAGE. After fractionation, the gel fragments were reductively dissolved in under 5 minutes, and in-solution trypsin digestion of the protein released from the gel was completed in less than 1 hour at 70 °C, equivalent to a 90–95 % reduction in time compared to conventional in-gel trypsin digestion. The introduction of the BAC-DROP workflow to the MS assays for inflammatory biomarker CRP and viral marker HBsAg allowed for serum sample preparation to be completed in as little as 5 hours, demonstrating successful marker quantification from a 0.5 μL sample of human serum. Combining reduced proteome complexity via PAGE fractionation with rapid trypsin digestion provides a unique opportunity for conducting high-throughput analysis of in-depth proteome with a focus on specific molecular weight ranges.

The LC-SRM assay was performed using an Eksigent nanoLC 400 system (SCIEX) connected to a QTRAP 5500 mass spectrometer (SCIEX). The LC mobile phases consisted of 0.1% formic acid (solvent A) and 0.1 % (v/v) formic acid/80 % (v/v) acetonitrile (solvent B). Each peptide sample was injected onto a 200 µm i.d. × 0.5 mm cHiPLC trap column (SCIEX) and desalted at 5 μl/min for 10 minutes using 0.1 % (v/v) TFA. Concentrated peptides were then separated on a fused-silica capillary column packed with C18 resin (12.5 cm × 75 µm i.d.; Nikkyo Technos) at a flow rate of 300 nL/min according to the following gradient schedule: 0–15 minutes, 2–50 % B; 15–18 minutes, 50–90 % B; hold at 90 % B for 6 minutes; and re-equilibrate at 2% B for 20 minutes. Eluate was ionized using a NanoSpray III Ion Source (SCIEX). The QTRAP 5500 was operated in positive ion mode with the following parameters: ion spray voltage = 2300 V; curtain gas = 20; ion source gas1 = 20; collision gas = 12; interface heater temperature = 150; entrance potential, 10; collision cell exit potential = 9; and Q1/Q3 = low resolution. The SRM transitions for each target peptide are shown in Supplementary Table S4. The obtained SRM data was analyzed by Skyline software (University of Washington, Seattle, WA, USA).

For CRP quantification, human serum samples were collected from three healthy donors, one patient with rheumatoid arthritis (RA), four patients with antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), and two patients with bacterial pneumonia (BP), all treated at Ehime University Hospital, following the procedures approved by the human ethics committee of Ehime University. For HBsAg quantification, human serum samples were collected from five HBV infected patients and one uninfected healthy donor following the procedures approved by the human ethics committee of Hamamatsu University School of Medicine. For HBV inactivation, 180 μL of the collected serum sample were mixed with 20 μL of 10 % (w/v) SDS solution and subjected to UV irradiation. The samples were then mixed with 160 μL of 150 mM Tris-HCl (pH 8.8) and stored at -80 °C until use. BSA was purchased from Wako (Osaka, Japan).