Magnetic bead-based weak cation exchange chromatography (MB-WCX) (Bruker Daltonic, Bremen, Germany) was used to separate the peptides in the serum samples. The experiment was conducted using a standard protocol according to the manufacturer's instructions: 5 µl of serum was mixed with 10 µl of binding buffer and 10 µl of MB-WCX beads. The tube was placed into the magnetic separator and the supernatant was subsequently removed. After adding 100 µl of washing buffer to the tube, and the tube was shaken back and forth ten times in the magnetic separator to wash the beads. Subsequently, the supernatant was removed, and the washing step was repeated twice. Next, 5 µl of elution buffer was added to dissolve the beads. The tubes were placed in the magnetic separator, and the clear supernatant was transferred to a fresh tube. Subsequently, 5 µl of stabilizing buffer was added to the tubes. One microliter of the eluted sample was spotted onto a steel target, air-dried, and subsequently 1 μL of the matrix (0.3 mg/ml HCCA, 50% ACN, 2% TFA) was spotted onto the MALDI-TOF steel target, followed by air-drying. The samples were detected using ultrafleXtreme MALDI-TOF/TOF (Bruker Daltonics Corp.) after calibrating the instrument according to the ClinProt standard.
Mb wcx
Manufactured by Bruker
Sourced in Germany, United States
The MB-WCX is a weak cation exchange chromatography column designed for the purification and separation of proteins and other biomolecules. It features a silica-based stationary phase with carboxyl-functionalized copolymer ligands, providing a versatile platform for ion exchange separations.
Automatically generated - may contain errors
Lab products found in correlation
Ultraflextreme maldi tof tof, by Bruker (1 mentions)
Clinprot purification reagent sets, by Bruker (1 mentions)
Alpha cyano 4 hydroxycinnamic acid, by Bruker (1 mentions)
Ca19 9, by Roche (1 mentions)
Maldi tof ms system, by Bruker (1 mentions)
Maldi tof ms, by Bruker (1 mentions)
Sinapinic acid, by Merck Group (1 mentions)
N 2 hydroxyethylpiperazine n 2 ethanesulfonic acid hepes, by Merck Group (1 mentions)
Tris hcl, by Merck Group (1 mentions)
Au chip, by Bio-Rad (1 mentions)
Acetonitrile, by Merck Group (1 mentions)
Urea, by Merck Group (1 mentions)
Sodium acetate, by Merck Group (1 mentions)
Trifluoroacetic acid, by Merck Group (1 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
9 protocols using mb wcx
1
Serum Peptide Separation by MB-WCX
2
Serum Peptidome Separation by MB-WCX
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Magnetic beads-based weak cation exchange chromatography (MB-WCX) (Bruker Daltonics, Germany) was used for peptidome separation of samples. We prepared a 200-μL sample tube of thoroughly mixed weak cation magnetic suspension by adding 10 μL magnetic beads binding buffer, 10 μL magnetic beads suspension, and 5 μL serum, mixing at least five times using the sampling gun, and standing at room temperature for 5 min. We put the sample tube into the magnetic separator, maintained the magnetic field for 1 min, used the sampling gun to absorb the liquid after separation of the magnetic beads and liquid, and added to the sample tube 100 μL magnetic beads cleaning buffer. We then moved the sample tube ten times between the two adjacent holes of the magnetic separator, left to stand, and used the sample gun to absorb the supernatant again after magnetic beads adherence. We repeated the cleaning process twice. We then took down the sample tube from the magnetic separator, added 5 μL magnetic beads elution buffer to the sample tube, and repeated the pipetting. We put the sample tube into the magnetic separator and let it stand for 2 min, transferred the supernatant to a clean 0.5-mL sample tube when the magnetic beads were completely adhered, then added 5 μL magnetic beads stabilizing buffer. The eluate was then ready for spotting onto MALDI-TOF MS targets and measured.
3
Serum Peptide Profiling using MB-WCX
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Serum samples were separated using MB-WCX (ClinProt purification reagent sets; Bruker Daltonics, Bremen, Germany) and a magnetic separator, with the magnet lowered, 5 μL serum samples were diluted in 10 μL binding solution in a standard thin wall PCR tube, added to 10 μL 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 μL of elution solution and 5 μL of stabilization buffer [6 (link)]. Eluted peptides were spotted onto the MALDI AnchorChip with 1 μL alpha-cyano-4-hydroxycinnamic acid (Bruker Daltonics) in 50% acetonitrile, and 0.5% trifluoroacetic acid was added twice to the MALDI AnchorChip surface. Each sample was spotted in triplicate in order to evaluate reproducibility.
4
Quantitative Determination of CEA and CA19-9
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The Quantitative determination kit for CEA and CA19-9 were from Roche Company (US), MB-WCX was from Bruker Company (Germany), Standard proteins and peptides were from Bruker Company (Germany), and MALDI-TOF MS system was from Bruker Company (Germany).
5
MALDI-TOF MS Bead Fractionation Protocol
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All samples were fractionated using weak cation exchange magnetic beads (MB-WCX) according to the manufacturer's recommendations (Bruker Daltonics, Bremen, Germany). Briefly, the suspension in the magnetic bead was mixed by shaking. After eluting and beating, the MB-WCX were separated from the protein, and the eluted peptide samples were transferred to a 0.5-mL clean sample tube for further MS analysis. Five microliters of alpha-cyano-4-hydroxycinnamic acid substrate solution (0.4 g/L, dissolved in acetone and ethanol) and 0.8–1.2 μL of elution were mixed. Next, 0.8–1.2 μL of this mixture was applied to a metal target plate and dried at room temperature. Finally, the prepared sample was analyzed by MALDI-TOF MS (Bruker Daltonics). A range of 1000–10,000 Da peptide molecular weights was collected, and 400 shots of laser energy were used.
To evaluate the precision of the assay, within-run and between-run variations were determined using multiple analyses of the bead fractionation and MS for 2 plasma samples. To assess within-run variability, each plasma sample was loaded onto 3 spots. To assess between-run variability, identical samples were assayed 3 times independently. In each profile, 3 peaks with different molecular masses were selected to evaluate the within-run and between-run coefficients of variance (CV).
To evaluate the precision of the assay, within-run and between-run variations were determined using multiple analyses of the bead fractionation and MS for 2 plasma samples. To assess within-run variability, each plasma sample was loaded onto 3 spots. To assess between-run variability, identical samples were assayed 3 times independently. In each profile, 3 peaks with different molecular masses were selected to evaluate the within-run and between-run coefficients of variance (CV).
6
MALDI-TOF MS Protein Profiling Protocol
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MALDI-TOF MS (ProteinChip Biology System II-c, PBS II-c) was purchased from Ciphergen Biosystems company (Austin, TX, USA), and Au chip was purchased from BioRad Laboratories, Inc. (Hercules, CA, USA). MB-WCX were purchased from Bruker Daltonics company (USA). Sodium acetate, urea, dithiothreitol (DTT), acetonitrile (ACN), Tris-HCl (pH9.0), Trifluoroacetic acid (TFA), sinapinic acid (SPA), water (HPLC grade), 3-[(3-choleamidopropyl) dimethylamino]-1-propanesulfonate (CHAPS), and N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES)were purchased from Sigma Aldrich (USA).
7
Peptide/Protein Separation from Serum
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According to the manufacturer's standard procedure (Bruker Daltonics), MB‐WCX (Bruker Daltonics,) was used to separate peptides/proteins from all serum samples. First, combine the magnetic beads with the protein/peptide: 10 μl MB‐WCX with 10 μl binding buffer, and 5 μl serum sample was added and mixed to be tested. Then, the Eppendorf tube was put into the magnetic separator for 1 min, and the supernatant was discarded. The second step was to wash the high‐abundance protein bound on the magnetic beads. Added 100 μl buffer solution to the Eppendorf tube and mixed it evenly, the magnetic separator was allowed to stand for 1 min, then pipette absorbed the supernatant and discarded it, and the washing step was repeated for 3 times. The final step was to elute the protein bound to the magnetic beads: 5 μl elution buffer was added to the Eppendorf tube, put it in a magnetic separator and stood for 2 min. The clear supernatant was collected in a fresh tube and mixed it with 5 μl of stabilization buffer.
8
Serum Protein Fractionation for Mass Spectrometry
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Serum samples were fractionated by MB-WCX (Bruker Daltonics, Bremen, Germany) according to a standard protocol (manufacturer’s instruction). Briefly, each 5-μL serum sample was mixed with 10 μL of MBs and 10 μL of buffer in a 200-μL tube. After mixing, the sample tubes were placed into a magnetic separator, and 1 min later the supernatant in each tube was carefully removed. After the samples were bound to the MBs and washed 3 times with 100 μL of MB cleaning buffer, 5 μL of MB elution buffer was added to the samples bound to the magnetic beads. The suspended fluid samples were then transferred into new 10-μL tubes after complete separation of the MBs from the supernatants was achieved. Five μL of stable buffer was added to each tube before they were stored at −20°C for MS analyses.
9
Serum Peptide Extraction Protocol
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The serum samples were collected in a 5 mL vacuum blood collection tube without anticoagulant, then centrifuged for 5 min at 12,000 g at room temperature. The serum samples were distributed into 1.5 mL aliquots and stored at −80°C. The frozen samples should be thawed at room temperature for 15 min before use. MB-WCX (Bruker Daltonik, Germany) kits were used according to the manufacturer’s protocol. We added 10 μL of MB-WCX beads and 10 μL of binding solution (BB) in 200 μL PCR tubes. Then we added 5 μL of serum samples and mixed thoroughly by pipetting up and down. Subsequently, samples were incubated at room temperature for 5 min, and then we used a magnetic separator to collect the beads. The supernatant was removed, and the magnetic beads were washed three times using washing solution (WB). Finally, we added 5 μL of elution solution (EB) and 4 μL of stabilization buffer (SB) to elute the peptide fraction from the magnetic beads.
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