Oasis hlb c18 cartridges

Proteomics procedures are scheduled in Supplementary Fig. S3 and fully described in Supplementary Information. The peptide pools were isobarically labelled (iTRAQ, Sigma-Aldrich), equally mixed and the peptide mixture was desalted onto Oasis HLB C18 cartridges (Waters). One-fifth of the tagged peptides were dried-down, and the remaining four-fifths were separated into 8 fractions using MCX Oasis cartridges (Waters). The peptide fractions were desalted using MicroSpin Colums C18 (The Next Group) and vacuum-dried.

5 µL of individual human serum from GC patients, healthy controls, or QC samples were diluted ten times with 8 M urea/0.1 M NH₄HCO₃, and then denatured by reduction (with 20 mM DTT at 37 °C for 2 h) followed by alkylation (with 40 mM IAA at 25 °C for 40 min). After urea concentration of the mixture was reduced to below 2 M with 0.1 M NH₄HCO₃, protein digestion was carried out with trypsin at an enzyme‐to‐protein ratio of 1:50 (w/w) at 37 °C for 16 h. Tryptic peptides were subsequently desalted using Oasis HLB C18 cartridges (Waters) and then lyophilized. Glycopeptide enrichment and MS analysis of intra‐batch samples were performed randomly to avoid bias. Intact N‐glycopeptides were enriched by the automated N‐glycopeptide enrichment method, according to previously reported.^[23 (link)
^] In brief, the lyophilized tryptic peptides were redissolved in 0.1% TFA/80% ACN and injected automatically onto a HILIC column at a flow rate of 200 µL min⁻¹. The glycopeptide fraction was collected after the non‐glycopeptide fraction was previously washed away. The following sample could be injected after system washing and re‐equilibration. The whole enrichment cycle for each sample needs 20 min.

Each sample was digested using the filter-aided sample preparation method (FASP) (Wiśniewski et al., 2009 (link)). Protein samples were denatured with 20 mM DTT at 37 °C for 1 h and alkylated with 50 mM IAA in darkness for 45 min. Then, the samples were loaded onto filter devices with a cut-off of 10 kD (Pall, Port Washington, NY, USA) and centrifuged at 14,000g at 18 °C. After washing twice with UA (8 M urea in 0.1 M Tris-HCl, pH 8.5) and twice with 25 mM NH₄HCO₃, the samples were redissolved in 25 mM NH4HCO3 and digested with trypsin (enzyme to protein ratio of 1:50) at 37 °C overnight. The digested peptides were collected as a filtrate and desalted using Oasis HLB C18 cartridges (Waters, Milford, MA). The IgAN 1-3 and pMN samples were individually labelled with 114, 115, 116, and 117 4-plex iTRAQ chemistry according to the manufacturer’s protocol (ABsciex, Framingham, MA, USA).