Clinprot purification reagent sets

We used magnetic-bead-based weak cation - exchange chromatography (MB- WCX) (ClinProt purification reagent sets; Bruker Daltonics, Bremen, Germany) for peptidome separation of all serum samples. The 96 serum samples were fractionated according to the standard protocol. With the magnet lowered, 5 μ L serum samples were diluted in 10 μ L binding solution in a standard thin wall PCR tube, added to 10 μ L of MB-WCX beads and then carefully mixed using the mixing feature of the robot. After thorough stirring, samples were incubated at room temperature for 5 min, then the tubes were placed into the magnetic separator to collect the beads on the wall of the tube until the supernatant was clear (~1 min). The supernatant was then removed and the magnet was lowered again. Following the stepwise application of sample and MB-WCX separation, we eluted the peptide fraction from the magnetic beads with 5 μ L of elution solution and 5 μ L of stabilization buffer. The eluted peptides were spotted onto the MALDI AnchorChip with 1 μ L α - cyano - 4 -hydroxy -cinnamic acid (Bruker) in 50% acetonitrile, and 0.5% trifluoroacetic acid was added twice to the MALDI AnchorChip surface. Samples were spotted in triplicate to evaluate the reproducibility of this method.

The procedure of serum peptides enrichment and MALDI-TOF data acquisition were described previously [14 (link)]. The serum peptides were fractionated using weak cation exchange magnetic-beads (MB-WCX) (ClinProt purification reagent sets; Bruker Daltonics, Bremen, Germany) with a magnetic separator. Mixture with the eluted sample and matrix was spot onto a MALDI-TOF mass spectrometry target (AnchorChip™, Bruker Daltonics) for peptide profiling acquisition in Microflex mass spectrometer (Bruker Daltonics). Cilnplot standard was used for mass calibration. The scan range was 0.7–10 KD. Peptide profiling by different mass to charge ratio was obtained through FlexControl2.2 software. The coefficient of variability less than 30% indicated that the system was stable and reliable.