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Analytical columns

Manufactured by Thermo Fisher Scientific
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Analytical columns are a type of scientific instrument used in various analytical techniques, such as liquid chromatography and gas chromatography. Their core function is to separate and analyze the components of a complex mixture by passing it through a column packed with a stationary phase material. This separation allows for the identification and quantification of the individual components within the sample.

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Lab products found in correlation

Rhcd10, by Abcam (1 mentions) Thermo scientific q exactive, by Thermo Fisher Scientific (1 mentions) Dionex ultimate 3000 rslcnano system, by Thermo Fisher Scientific (1 mentions) Proteomics grade trypsin, by Promega (1 mentions) Proteasemax surfactant, by Promega (1 mentions) Easy nlc 1200, by Thermo Fisher Scientific (1 mentions) Xcalibur software, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

Acclaim PepMap RSLC analytical column CarboPac-PA1 2 × 250 mm analytical column IonPac AS11 analytical column MAbPac SEC-1 analytical column PA200 analytical column HyPURITY® C18 analytical column EASY C18-A2 analytical column Accucore 150-C18 analytical column C18 reverse-phase analytical column Easy Spray PepMap C18 analytical column

4 protocols using analytical columns

1

CD10 Cleavage Sites on OGP

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OGP (4 µL) was incubated with or without 0.5 µL of rhCD10 (Cat# ab157051, Abcam) in a final volume of 200 µL of 50 × 10−3 m Tris‐HCl buffer (pH 7.5) for 1 h at 37 °C. To detect the cleavage sites of CD10 on OGP, enzymatic hydrolysis products of OGP after incubation with rhCD10 were used for HPLC‐MS/MS analysis.[111] Briefly, the peptides were first loaded onto peptide trap columns (300 µm × 5 mm, 5 µm, Cat# 160 454, Thermo Fisher Scientific) and then separated on analytical columns (75 µm × 150 mm, 3 µm, Cat# 160 321, Thermo Fisher Scientific). Peptides bound to the columns were eluted with ACN in water (4–72% (v/v)) containing 0.1% FA at a flow rate of 300 nL min−1 for 40 min. The MS analysis was performed online with an Orbitrap spectrometer (Thermo Scientific Q Exactive; Thermo Fisher Scientific). Full‐mass and MS/MS scans were acquired as previously described.[112]Peak list files were analyzed against an in‐house constructed database based on the sequence of OGP using Mascot software. Peptides with at least four amino acids were included in the analysis. The FDR for the peptides was set to 0.05.
Yu S., Lu Y., Su A., Chen J., Li J., Zhou B., Liu X., Xia Q., Li Y., Li J., Huang M., Ye Y., Zhao Q., Jiang S., Yan X., Wang X., Di C., Pan J, & Su S. (2021). A CD10‐OGP Membrane Peptolytic Signaling Axis in Fibroblasts Regulates Lipid Metabolism of Cancer Stem Cells via SCD1. Advanced Science, 8(19), 2101848.
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2

Nano-LC-MS/MS Analysis of Peptides

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Samples were analyzed in the Dionex UltiMate 3000 RSLC nano System combined with the Q-Exactive High-Field (HF) Hybrid Quadrupole Orbitrap MS (Thermo Fisher Scientific). Peptides were re-suspended in 3% ACN/0.1% Formic Acid (FA)/96.9% MilliQ, and 5 μL was used for LC-MS/MS analysis. Peptides were separated using a trap (300 μm ID × 5 mm packed with 5 μm 100 Å PepMap C18 medium) and the analytical columns (75 μm × 50 cm packed with 2 μm of 100 Å PepMap C18 medium) (Thermo Fisher Scientific) using a 120 min method at a flow rate of 300 nL/min. Mobile phase A consisted of 0.1% FA in water and mobile phase B consisted of 0.1% FA in 80% ACN. The linear gradient started at 5% B and reached 30% B in 80 min, 45% B in 91 min, and 100% B in 93 min. Next, the column was held at 100% B for the next 5 min before bringing back to 5% B and held for 20 min to equilibrate the column. The column temperature was maintained at 37 °C. MS data were acquired with a Top 20 data-dependent MS/MS scan method with a maximum injection time of 100 ms, a resolution of 120,000 at 200 m/z. Fragmentation of precursor ions was performed by high-energy C-trap dissociation (HCD) with the normalized collision energy of 27 eV. MS/MS scans were acquired at a resolution of 15,000 at m/z 200. The dynamic exclusion was set at 20 s to avoid repeated scanning of identical peptides.
Kim H., Yuk S.A., Dieterly A.M., Kwon S., Park J., Meng F., Gadalla H.H., Cadena M.J., Lyle L.T, & Yeo Y. (2021). Nanosac, a Non-Cationic and Soft Polyphenol Nanocapsule, Enables Systemic Delivery of siRNA to Solid Tumors. ACS nano, 15(3), 4576-4593.
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3

Mass Spectrometry Proteomics Workflow

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Proteomics-grade trypsin and ProteaseMax surfactant were purchased from Promega. Reverse-phase C18 spin columns, precolumns, and analytical columns were all obtained from Thermo Fisher Scientific. Solvents and ion-pairing agents were certified liquid chromatography-mass spectrometry grade, and all other chemicals were purchased from Sigma-Aldrich with the highest purity available.
Martins L., Knuesting J., Bariat L., Dard A., Freibert S.A., Marchand C.H., Young D., Dung N.H.T., Voth W., Debures A., Saez-Vasquez J., Lemaire S.D., Lill R., Messens J., Scheibe R., Reichheld J.P, & Riondet C. (2020). Redox Modification of the Iron-Sulfur Glutaredoxin GRXS17 Activates Holdase Activity and Protects Plants from Heat Stress. Plant physiology, 184(2).
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4

Peptide Analysis by Q‐Exactive Orbitrap MS

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The peptides were separately analyzed by using Q‐Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipped with an EASY‐nLC 1200 (Thermo Fisher Scientific, Bremen, Germany). Data dependent acquisition (DDA) mode was applied. Precolumn (2 cm, 100 μm inner diameter, 5 μm C18 filler; Thermo Fisher Scientific, Bellefonte, USA) and analytical columns (10 cm, 75 μm inner diameter, 3 μm C18 filler; Thermo Fisher Scientific, Bellefonte, USA) were used for analysis. Peptides were eluted with a 90 min HPLC gradient from 0% to 100% in buffer (80% acetonitrile, 1% formic acid) at a flowrate of 250 nL/min. The scan range was set to 400–1700 m/z, and 70,000 resolution (at m/z 200) was used. The ten most‐abundant MS1 features were selected for high‐energy and MS/MS scans. Raw data were processed with Xcalibur software (Thermo Fisher Scientific, Bremen, Germany). The mass spectrometry proteomics data were deposited in the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD029631 [17 (link)].
Liu J., Yang C., Zhang H., Hu W., Bergquist J., Wang H., Deng T., Yang X., Zhang C., Zhu Y., Chi X., Mi J, & Wang Y. (2022). Quantitative proteomics approach reveals novel biomarkers and pathological mechanism of keloid. Proteomics. Clinical Applications, 16(4), 2100127.
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