Zorbax 300 extend c18 column

Manufactured by Agilent Technologies

Sourced in United States

The ZORBAX 300 Extend-C18 column is a high-performance liquid chromatography (HPLC) column designed for the separation and analysis of a wide range of organic compounds. It features a silica-based stationary phase with a C18 bonded ligand, which provides effective reversed-phase separation. The column is suitable for a variety of applications, including pharmaceutical, environmental, and industrial analyses.

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C18 column (380 citations) Zorbax Extend-C18 column (94 citations) Zorbax C18 column (93 citations) Zorbax Extend C18 (51 citations) Extend-C18 column (51 citations) Extend-C18 (17 citations) C18 ZORBAX (2 citations)

52 protocols using zorbax 300 extend c18 column

Peptide Separation and Identification

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The peptides were separated on a Thermo Scientific UltiMate™ 3000 Binary Rapid Separation System (Thermo Fisher Scientific, San Jose, CA, USA) with a 3.5 μm 4.6 × 150 mm Agilent ZORBAX 300Extend-C18 column for liquid phase separation of the samples. The eluted peaks were monitored at 214 nm and one component was collected per minute. The samples were combined with the chromatographic eluted peaks to obtain 10 components, which were then lyophilized. The lyophilized peptides were re-dissolved, centrifuged at 20,000× g for 10 min, and the supernatant was sampled. Subsequently, the samples were separated with the Thermo Scientific EASY-nLC™ 1200 system (Thermo Fisher Scientific, San Jose, CA, USA). The peptide fragments separated in the liquid phase were ionized with the nanoESI source and then entered the mass spectrometer Orbitrap Exploris 480 (Thermo Fisher Scientific, San Jose, CA, USA) for DDA (data-dependent acquisition) mode detection. Two QC samples were inserted before, during, and after the whole experiment to evaluate the stability and repeatability of the experiment.

Wang L., Zheng X., Ma J., Gu J, & Sha W. (2023). Comparative Proteomic Analysis of Exosomes Derived from Patients Infected with Non-Tuberculous Mycobacterium and Mycobacterium tuberculosis. Microorganisms, 11(9), 2334.

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TMT-Based Proteomic Profiling Workflow

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Peptides were aliquoted for TMT labeling and dried in a vacuum centrifuge, with 250 μg per channel used for TMT16 multiplexes. Peptides were reconstituted in 500 mM HEPES, pH 8.5, to a concentration of 5 μg/μL. TMT reagents were dissolved in anhydrous acetonitrile (ACN) at a concentration of 20 μg/μL and added to peptide samples at a 1:1 peptide:label ratio (by mass). Labeling was allowed to proceed for 1 hour in a thermomixer set to 25°C and 400 rpm, and reactions were quenched with 5% hydroxylamine. Samples from each multiplex were combined, concentrated in a vacuum centrifuge, and cleaned with C18 SPE cartridges. Each combined multiplex sample was fractionated into 96 fractions by high-pH reversed phase separation using a 3.5 μm Agilent Zorbax 300 Extend-C18 column (4.6 mm ID x 250 mm length). Peptides were loaded onto the column in buffer A (4.5 mM ammonium formate, pH 10, in 2% v/v ACN) and eluted off the column using a gradient of buffer B (4.5 mM ammonium formate, pH 10, in 90% v/v ACN) for 96 minutes at a flow rate of 1 mL/minute. After fractionation, samples were concatenated to 12 fractions (Wang et al. 2011 (link)).

Feng S., Sanford J.A., Weber T., Hutchinson-Bunch C.M., Dakup P.P., Paurus V.L., Attah K., Sauro H.M., Qian W.J, & Wiley H.S. (2023). A Phosphoproteomics Data Resource for Systems-level Modeling of Kinase Signaling Networks. bioRxiv.

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Quantification of Free Amino Acids in Fermented Milk

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Free amino acids in fermented milk samples were determined by reversed-phase liquid chromatography (RP-HPLC) (Agilent Technologies 1260 Infinity, Germany) [21 (link)]. Briefly, aqueous extracts (<3 kDa) from fermented milk samples were derivatized with fluoraldehyde OPA reagent solution. After derivatization, 10 μL of each sample was injected on a Zorbax 300Extend C18 column, 5 μm, 250 × 4.6 mm (Agilent Technologies, Germany), with detection at 254 nm. Mobile phase A was 100 mM C₂H₃NaO₂, adjusted to pH 7.2 with 1 M HCl, while mobile phase B was methanol (100%). Separation was carried out at a flow rate of 0.75 mL/min with a gradient of 0–5 min with 80% A; followed by 5–8 min with 70% A and 8–15 min with 50% A; and finally, 15–25 min with 20% A. Quantification of free amino acids was calculated with a standard curve constructed with a pool of amino acid standards (0–50 mg/mL).

Beltrán-Barrientos L.M., Garcia H.S., Reyes-Díaz R., Estrada-Montoya M.C., Torres-Llanez M.J., Hernández-Mendoza A., González-Córdova A.F, & Vallejo-Cordoba B. (2019). Cooperation between Lactococcus lactis NRRL B-50571 and NRRL B-50572 for Aroma Formation in Fermented Milk. Foods, 8(12), 645.

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High pH Reversed-Phase Fractionation

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The TMT labeled samples were fractionated using offline high pH reversed-phase chromatography (bRP) as previously described^{70 (link)}. Samples were fractionated using Zorbax 300 Extend C18 column (4.6 × 250 mm, 300 Å, 5 μm, Agilent) on an Agilent 1100 series high-pressure liquid chromatography (HPLC) system. Samples were reconstituted in 900 µL of 4.5 mM ammonium formate (pH 10) in 2% (vol/vol) acetonitrile (MeCN) (bRP solvent A). Samples were injected with Solvent A at a flow rate of 1 ml/min and separated using a 96 min gradient. The gradient consisted of an initial increase to 16% solvent B (90% MeCN, 5 mM ammonium formate, pH 10), followed by 60 min linear gradient from 16% solvent B to 40% B and successive ramps to 44% and 60% at a flow rate of 1 ml/min. Fractions were collected in a 96-deep well plate (GE Healthcare) and pooled in a non-contiguous manner into final 24 proteome fractions. Pooled fractions were dried to completeness using a SpeedVac concentrator.

Bernasocchi T., El Tekle G., Bolis M., Mutti A., Vallerga A., Brandt L.P., Spriano F., Svinkina T., Zoma M., Ceserani V., Rinaldi A., Janouskova H., Bossi D., Cavalli M., Mosole S., Geiger R., Dong Z., Yang C.G., Albino D., Rinaldi A., Schraml P., Linder S., Carbone G.M., Alimonti A., Bertoni F., Moch H., Carr S.A., Zwart W., Kruithof-de Julio M., Rubin M.A., Udeshi N.D, & Theurillat J.P. (2021). Dual functions of SPOP and ERG dictate androgen therapy responses in prostate cancer. Nature Communications, 12, 734.

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Quantitative Proteomics of Mouse Brain

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After termination, the SN and striatum (STR) of WT and HET mice (n = 3 per group) were dissected and snap‐frozen in liquid nitrogen. After the sample was lysed by using ultrasound, it was digested with trypsin. The digested peptides were desalted with Strata‐X C18 (Phenomenex, Inc., Torrance, CA, USA), freeze‐dried in vacuo, and labelled with tandem mass tags. The peptides were fractionated by using high‐pH reversed‐phase HPLC and a ZORBAX 300Extend C18 column (5‐μm particle size, 4.6‐mm inner diameter, 250‐mm length; Agilent Technologies Inc., Santa Clara, CA, USA). After dissolving the peptides with mobile phase A during liquid chromatography, an EASY‐nLC 1000 liquid chromatograph (Thermo Fisher Scientific, USA) was used for separation. The peptides were separated by using ultra‐high‐performance liquid chromatography, injected into a nanospray ionisation source (Thermo Fisher Scientific, USA), and analysed by using a Q Exactive Plus Hybrid Quadrupole‐Orbitrap mass spectrometer (Thermo Fisher Scientific, USA). Data were acquired with a data‐dependent acquisition scan program. Secondary mass spectrometry data were retrieved by using the MaxQuant software package version 1.5.2.8.S.

Fan L., Zhang S., Li X., Hu Z., Yang J., Zhang S., Zheng H., Su Y., Luo H., Liu X., Fan Y., Sun H., Zhang Z., Miao J., Song B., Xia Z., Shi C., Mao C, & Xu Y. (2022). CHCHD2 p.Thr61Ile knock‐in mice exhibit motor defects and neuropathological features of Parkinson's disease. Brain Pathology, 33(3), e13124.

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Radioiodination of PEN peptides

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Two variants of human PENs were utilized for radioiodination in this study: one with a tyrosine added at the aminoterminal end (Tyr-PEN: YAADHDVGSELPPEGVLGALLRV-NH₂), and one with an additional tyrosine attached to the carboxyterminal end (PEN-Tyr: AADHDVGSELPPEGVLGALLRVY-NH₂). Both were labelled with ¹²⁵I using the chloramine-T method. Chloramine-T and sodium metabisulfite solutions were freshly prepared in water. For labelling, 10 µL of peptide (stock 1 mM) with 15 µL of sodium phosphate buffer (0.5 M, pH 7.6) was mixed with 37 MBq carrier-free Na¹²⁵I (NEZ033L010MC, Perkin Elmer, Waltham, MA, USA). A 4 μL volume of chloramine T (1 mg/mL) was added to start the reaction, and after 30 s, 4 μL sodium metabisulfite (2 mg/mL) was added to stop the iodination. The labeled radioactive peptide was separated from unlabeled peptide with HPLC purification (Agilent ZORBAX 300 Extend-C18 column) using a gradient from 20–50% acetonitrile (+0.1% TFA) against water (+0.1% TFA) for 20 min. To determine the retention time of the radioactive peptide, 1–2 µL of the reaction mixture was analyzed before the purification run. The fraction containing the radiolabeled peptide peak was then collected, diluted with binding buffer to prevent radioautolysis, aliquoted, and stored at −80 °C.

Giesecke Y., Asimi V., Stulberg V., Kleinau G., Scheerer P., Koksch B, & Grötzinger C. (2023). Is the Neuropeptide PEN a Ligand of GPR83?. International Journal of Molecular Sciences, 24(20), 15117.

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Radioactive Labeling of Chemerin-9

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Radioactive iodination of the nonapeptide chemerin-9 (Tyr-Phe-Pro-Gly-Glu-Phe-Ala-Phe-Ser, representing amino acids 149-157 of full-length chemerin) was performed by the chloramine T method 23 (link). For labeling, 10 nmol of chemerin-9 in 25 μL iodination buffer (0.5 M sodium phosphate, pH 7.4) were mixed with 1 mCi carrier-free Na[¹²⁵I] (NEZ033L010MC, PerkinElmer, Waltham, USA) in an HPLC glass vial with microvolume insert. The reaction was started by adding 4 μL chloramine T (1 mg/mL in water). After 20-30 seconds, 4 μL of sodium metabisulfite (2 mg/mL in water) were added to stop the iodination. HPLC purification was performed to separate unlabeled from labeled radioactive peptide on an Agilent ZORBAX 300 Extend-C18 column using a gradient from 20 to 50% acetonitrile (+0.1% TFA) against water (+0.1% TFA) for 20 minutes. First, 1-2 μL of the reaction mixture were analyzed to determine the retention time of the radioactive peptide. This fraction was then collected during the main run, diluted with radioactive binding buffer to prevent radiolysis, aliquoted and stored at -20 °C.

Erdmann S., Niederstadt L., Koziolek E.J., Gómez J.D., Prasad S., Wagener A., von Hacht J.L., Reinicke S., Exner S., Bandholtz S., Beindorff N., Brenner W, & Grötzinger C. (2019). CMKLR1-targeting peptide tracers for PET/MR imaging of breast cancer. Theranostics, 9(22), 6719-6733.

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Quantitative Proteomic Analysis via TMT Labeling

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Lyophilized samples obtained from transferred 100 µl of the eluted peptide solution were resuspended in 100 µl of TEAB, and 20 µl of such peptide solution were labeled using a TMT 6-plex isobaric label reagent (Thermo Scientific, 90406). Each samples were labeled with 185 µg of TMTs according to the scheme presented in Fig. 2a and incubated overnight at room temperature before to be quenched by addition of 1-M Tris-HCL pH 7.5.
The labeled samples were combined, acidified, and desalted on Sep-Pak C18 cartridges (Waters, WAT051910). The desalted samples were dried, and resuspended in 5% acetonitrile, 5% ammonium hydroxide solution (pH 10.0), and then pre-fractionated into 80 fractions using a high pH reverse-phase Zorbax 300 Extend-C18 column (5 um, 4.6 × 250 mm, Agilent) on an ÄKTAmicro system (GE Healthcare). The pre-fractionated samples were concatenated into 20 injection fractions for each sample.

Bisteau X., Lee J., Srinivas V., Lee J.H., Niska-Blakie J., Tan G., Yap S.Y., Hom K.W., Wong C.K., Chae J., Wang L.C., Kim J., Rancati G., Sobota R.M., Tan C.S, & Kaldis P. (2020). The Greatwall kinase safeguards the genome integrity by affecting the kinome activity in mitosis. Oncogene, 39(44), 6816-6840.

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High-pH Fractionation for Deep CSF Proteomics

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To enhance the depth of the discovery CSF proteome, these samples were subjected to high-pH fractionation as previously described (71 (link)). TMT-labeled peptides (160 μg) from each discovery sample were dissolved in 100 μl of loading buffer [1 mM ammonium formate in 2% (v/v) ACN], injected completely with an autosampler, and fractionated using a ZORBAX 300Extend-C18 column (2.1 mm by 150 mm, 3.5 μm; Agilent Technologies) on an Agilent 1100 HPLC (high-performance liquid chromatography) system monitored at 280 nm. A total of 96 fractions were collected over a 60-min gradient of 100% mobile phase A [4.5 mM ammonium formate (pH 10) in 2% (v/v) ACN] from 0 to 2 min, 0 to 12% mobile phase B [4.5 mM ammonium formate (pH 10) in 90% (v/v) ACN] from 2 to 8 min, 12 to 40% mobile phase B from 8 to 36 min, 40 to 44% mobile phase B from 36 to 40 min, 44 to 60% mobile phase B from 40 to 45 min, and 60% mobile phase B until completion with a flow rate of 0.4 ml/min. The 96 fractions were collected with an even time distribution and pooled into 30 fractions.

Higginbotham L., Ping L., Dammer E.B., Duong D.M., Zhou M., Gearing M., Hurst C., Glass J.D., Factor S.A., Johnson E.C., Hajjar I., Lah J.J., Levey A.I, & Seyfried N.T. (2020). Integrated proteomics reveals brain-based cerebrospinal fluid biomarkers in asymptomatic and symptomatic Alzheimer’s disease. Science Advances, 6(43), eaaz9360.

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TMT Labeling and Phosphopeptide Enrichment

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Approximately 3.5 mg of 10-plex TMT labeled sample was separated on a reversed phase Agilent Zorbax 300 Extend-C18 column (250 mm x 4.6 mm column containing 3.5-mm particles) using the Agilent 1200 HPLC System. Solvent A was 4.5 mM ammonium formate, pH 10,2% acetonitrile and solvent B was 4.5 mM ammonium formate, pH 10, 90% acetonitrile. The flow rate was 1 mL/min and the injection volume was 900 mL. The LC gradient started with a linear increase of solvent B to 16% in 6 min, then linearly increased to 40% B in 60 min, 4 min to 44% B, 5 min to 60% B and another 14 of 60% solvent B. A total of 96 fractions were collected into a 96 well plate throughout the LC gradient. These fractions were concatenated into 24 fractions by combining 4 fractions that are fractions apart (i.e., combining fractions #1, #25, #49, and #73; #2, #26, #50, and #74; and so on). For proteome analysis, 5% of each concatenated fraction was dried down and re-suspended in 2% acetonitrile, 0.1% formic acid to a peptide concentration of 0.1 mg/mL for LC-MS/MS analysis. The rest of the fractions (95%) were further concatenated into 12 fractions (i.e., by combining fractions #1 and #13; #3 and #15; and so on), dried down, and subjected to immobilized metal affinity chromatography (IMAC) for phosphopeptide enrichment.

Dou Y., Kawaler E.A., Zhou D.C., Gritsenko M.A., Huang C., Blumenberg L., Karpova A., Petyuk V.A., Savage S.R., Satpathy S., LiU W., WU Y., Tsai C.F., Wen B., Li Z., Cao S., Moon J., Shi Z., Cornwell M., Wyczalkowski M.A., Chu R.K., Vasaikar S., Zhou H., Gao Q., Moore R.J., Li K., Sethuraman S., Monroe M.E., Zhao R., Heiman D., Krug K., Clauser K., Kothadia R., Maruvka Y., Pico A.R., Oliphant A.E., Hoskins E.L., Pugh S.L., Beecroft S.J., Adams D.W., Jarman J.C., Kong A., Chang H.Y., Reva B., Liao Y., Rykunov D., Colaprico A., Chen X.S., Czekański A., Jędryka M., Matkowski R., Wiznerowicz M., Hiltke T., Boja E., Kinsinger C.R., Mesri M., Robles A.I., Rodriguez H., Mutch D., Fuh K., Ellis M.J., DeLair D., Thiagarajan M., Mani D.R., Getz G., Noble M., Nesvizhskii A.I., Wang P., Anderson M.L., Levine D.A., Smith R.D., Payne S.H., Ruggles K.V., Rodland K.D., Ding L., Zhang B., Liu T, & Fenyö D. (2020). Proteogenomic Characterization of Endometrial Carcinoma. Cell, 180(4), 729-748.e26.

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