Four weeks after MCT injection, left lungs of Cirp‐KO and WT rats were digested into peptides and dissolved in triethylammonium bicarbonate (TEAB). Subsequently, the peptides were incubated with Tandem Mass Tag (TMT) 6‐plex mass tagging reagents, separated on 1100 HPLC System (Agilent) using an Agilent Zorbax Extend RP column (5 μm, 150 mm × 2.1 mm). ProteomeDiscoverer (v.2.2) was used to search all of the Q Exactive raw data thoroughly against Uniport rat database. A global false discovery rate (FDR) was set to 0.01. 1.5‐fold change was defined as the threshold for a significant change in expression.
Zorbax extend rp column
Manufactured by Agilent Technologies
Sourced in United States
The Zorbax Extend RP column is a reversed-phase high-performance liquid chromatography (HPLC) column manufactured by Agilent Technologies. It is designed for the separation and analysis of a wide range of compounds, including polar, nonpolar, and ionic substances. The column features a proprietary stationary phase that provides excellent peak shape and resolution.
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Lab products found in correlation
1100 hplc system, by Agilent Technologies (15 mentions)
Nanospray flex source, by Thermo Fisher Scientific (4 mentions)
Q exactive mass spectrometer, by Thermo Fisher Scientific (3 mentions)
Q exactive hf mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Nanospray flex, by Thermo Fisher Scientific (2 mentions)
Q exactive ms, by Thermo Fisher Scientific (1 mentions)
Hplc system, by Agilent Technologies (1 mentions)
Agilent 1100 hplc system, by Agilent Technologies (1 mentions)
Proteome discover software, by Thermo Fisher Scientific (1 mentions)
Tripletof 5600 mass spectrometer, by AB Sciex (1 mentions)
Nanospray 3 source, by AB Sciex (1 mentions)
19 protocols using zorbax extend rp column
1
Quantitative Lung Proteomics in Cirp-KO Rats
2
HPLC-based Proteomics Analysis of Mycobacterium tuberculosis
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A 1100 HPLC system (Agilent) was used for reversed-phase (RP) chromatographic separation with an Agilent Zorbax Extend RP column (5 µm, 150 × 2.1 mm). Mobile phases A (2% acetonitrile in HPLC water) and B (98% acetonitrile in HPLC water) were used for the RP gradient. Trypsin peptides were separated at a steady flow rate of 300 µl/min, monitored at 210 and 280 nm. Then elution buffer at every minute was collected, lasting for 8–50 min. The separated peptides were lyophilized for mass spectrometer (MS) detection as follows.
All analyses were performed by a Q-Exactive MS (Thermo, United States) equipped with a nanospray flex source (Thermo, United States). The peptides were separated on a C18 analytical RP column (75 µm × 15 cm) at a flow rate of 300 nL/min and a linear gradient of 70 min. A full MS scan was obtained in the mass range of 300 to 1600 m/z. Ten strongest peaks in the MS were fragmented by high-energy collision dissociation. The resolution of the obtained MS/MS spectrum is 17,500 with the maximum injection time of 50 ms. Q-E dynamic exclusion was set to 15 s.
All of the Q exactive MS/MS raw data were analyzed by using proteome discoverer v.2.2 (Thermo Company, United States) software and the uniprot-M.tb database. The false-positive rate of peptide identification was controlled below 1%.
All analyses were performed by a Q-Exactive MS (Thermo, United States) equipped with a nanospray flex source (Thermo, United States). The peptides were separated on a C18 analytical RP column (75 µm × 15 cm) at a flow rate of 300 nL/min and a linear gradient of 70 min. A full MS scan was obtained in the mass range of 300 to 1600 m/z. Ten strongest peaks in the MS were fragmented by high-energy collision dissociation. The resolution of the obtained MS/MS spectrum is 17,500 with the maximum injection time of 50 ms. Q-E dynamic exclusion was set to 15 s.
All of the Q exactive MS/MS raw data were analyzed by using proteome discoverer v.2.2 (Thermo Company, United States) software and the uniprot-M.tb database. The false-positive rate of peptide identification was controlled below 1%.
3
Reversed-phase HPLC Peptide Separation
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Reversed-phase (RP) separation was performed on an 1100 high-performance liquid chromatography (HPLC) system (Agilent) using an Agilent Zorbax Extend RP column (5 μm, 150 mm × 2.1 mm). Mobile phases A (2 % acetonitrile in HPLC water) and B (90 % acetonitrile in HPLC water) were used for RP gradient. The solvent gradient was set as follows: 0–8 min, 98 % A; 8–8.01 min, 98 %–95 % A; 8.01–48 min, 95 %–75 % A; 48–60 min, 75–60 % A; 60–60.01 min, 60–10 % A; 60.01–70 min, 10 % A; 70–70.01 min, 10–98 % A; 70.01–75 min, 98 % A. Tryptic peptides were separated at a fluent flow rate of 300 μL/min and monitored at 210 nm and 280 nm. Samples were collected for 8–60 min, and eluent was collected in centrifugal tubes 1–15 every minute in turn. Samples were recycled in this order until the end of gradient. The separated peptides were lyophilized for mass spectrometry.
4
Reversed-Phase Peptide Separation and Fractionation
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Reversed-phase (RP) separation was performed using an Agilent Zorbax Extend RP column (5 μm, 150 mm × 2.1 mm) on a 1100 HPLC System (high-pH separation liquid chromatograph, Aglient, Beijing, China). RP gradient chromatography separation was achieved using mobile phases A (containing 2% acetonitrile (ACN, Thermo Fisher, Waltham, MA, USA)) and B (containing 90% acetonitrile). In this step, the mixed sample was chromatographically separated into 15 components for subsequent testing. The gradient elution conditions were 0~8 min, 98% A; 8~8.01 min, 98%~95% A; 8.01~48 min, 95~75% A; 48~60 min, 75~60% A; 60~60.01 min, 60~10% A; 60.01~70 min, 10% A; 70~70.01 min, 10~98% A; 70.01 to 75 min, 98% A. The tryptic peptides were separated at a flow rate of 300 µL/min and monitored at 210 nm. The samples were collected after 8–60 min, the eluent was collected into centrifuge tubes 1–15 at 1-minute intervals in sequence, and then the samples were collected repeatedly according to the sequence 1 → 15. The samples were collected, vacuum freeze-dried, and evacuated before being frozen and kept ready for mass spectrometry analyses.
5
Reversed-phase HPLC Peptide Separation
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Reversed-phase (RP) separation was performed on an Agilent 1100 HPLC System (Agilent Technologies Inc., CA, USA) using an Agilent Zorbax Extend RP column (5 μm, 150 mm × 2.1 mm). The RP separation was conducted with mobile phases A (2% acetonitrile in high-performance liquid chromatography (HPLC) water) and B (98% acetonitrile in HPLC water) as follows: 0−8 min, 98% A; 8.00−8.01 min, 98−95% A; 8.01−38 min, 95−75% A; 38−50 min, 75−60% A; 50−50.01 min, 60−10% A; 50.01−60 min, 10% A; 60−60.01 min, 10−98% A; and 60.01−65 min, 98% A. Tryptic peptides were separated at a fluent flow rate of 300 μL·min−1 and were monitored at 210 and 280 nm. Dried samples were harvested for 8−50 min, and the elution buffer was collected once per minute and numbered from 1 to 10 with pipeline. The separated peptides were lyophilized for MS detection.
6
HPLC Fractionation and MS Detection
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Reversed-phase liquid chromatography was performed on an 1100 HPLC System (Agilent) using an Agilent Zorbax Extend RP column (5 μm, 150 mm × 2.1 mm). The elution buffer was collected every 1 min and placed in turn into a 1–15 centrifuge tube (Thermo Fisher Scientific, Waltham, MA, USA); samples were harvested from 8 min to 60 min. After collection, the samples were vacuum freeze-dried and cryopreserved for MS detection.
7
Peptide Separation and Identification
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The iTRAQ-labeled peptide mixtures were separated using an Agilent Zorbax Extend RP column (C18, 5 μm, 150 mm × 2.1 mm). Mobile phases A (2% ACN in water) and B (98% ACN in water) were used for gradient. The solvent gradient was set as described previously (Hu et al., 2018 (link)). Peptides were separated at a fluent flow rate of 300 μL/min and monitored at 210 nm and 280 nm. Dried samples were harvested from 8 to 50 min and elution buffer were collected in every minute and numbered from 1 to 10 with pipeline. The separated peptides were lyophilized for MS detection.
8
Serum Proteomics Analysis Pipeline
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40 ul serum for each sample was used for proteomics analysis. Briefly, the total protein was extracted and removed albumin/IgG. Protein concentration was determined by the BCA method. 10 μg protein of each sample was separated by 12% SDS-PAGE. The digested peptides were desalted by the C18-Reverse-Phase SPE Column. RP separation was performed on an 1100 HPLC System (Agilent) using an Agilent Zorbax Extend RP column (5 μm, 150 mm × 2.1 mm). The separated peptides were lyophilized for mass spectrometry. All analyses were performed by a Q-Exactive HF mass spectrometer (Thermo, USA) equipped with a Nanospray Flex source (Thermo, USA). The machine signal is transformed into peptide and protein sequence information by matching the mass spectrum output with the theoretical spectrum generated by fasta library, and then the spectrum DDA (Data-dependent Acquisition) library is established by combining the sequence information, peptide retention time, and fragment ion information, to facilitate the subsequent Data Independent Acquisition (DIA) analysis. The original LC-MS/MS files are imported into Spectronaut Pulsar software to search and build the database. The original data of DIA is processed by Spectronaut Pulsar software.
9
Serum Proteomics: Comprehensive Workflow
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For each sample, 40 ul serum was used for the proteomics analysis. Briefly, total protein was extracted, and albumin/IgG was removed. The protein concentration was determined by the BCA method. Moreover, 10 μg protein of each sample was separated by 12% SDS-PAGE. The digested peptides were desalted by C18-Reverse-Phase SPE Column. RP separation was performed on an 1100 HPLC System (Agilent) using an Agilent Zorbax Extend RP column (5 μm, 150 mm × 2.1 mm). The separated peptides were lyophilized for mass spectrometry. All analyses were performed by a Q-Exactive HF mass spectrometer (Thermo, USA) equipped with a Nanospray Flex source (Thermo, USA). The machine signal is transformed into peptide and protein sequence information by matching the mass spectrum output with the theoretical spectrum generated by fasta library, and then the spectrum Data-Dependent Acquisition library is established by combining the sequence information, peptide retention time, and fragment ion information so as to facilitate the subsequent Data Independent Acquisition (DIA) analysis. The original LC–MS/MS files are imported into the Spectronaut Pulsar software to search and build the database. The original data of DIA is processed by the Spectronaut Pulsar software.
10
Reversed-phase HPLC Separation of Tryptic Peptides
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Reversed-phase separation was conducted on an 1100 HPLC System (Agilent, Santa Clara, California, USA), and an Agilent Zorbax Extend RP column (5 μm, 150 mm × 2.1 mm) was applied. Mobile phases A (2% acetonitrile in HPLC water) and B (90% acetonitrile in HPLC water) were applied for RP gradient. The solvent gradient was set as follows: 0–8 min, 98% A; 8.00 min, 98–95% A; 8–48 min, 95–75% A; 48–60 min, 75–60% A; 60 min, 60–10% A; 60–70 min, 10% A; 70 min, 10–98% A; 70–75 min, 98% A. Tryptic peptides were separated at a velocity of 300 μL/min. Monitoring was at 210 nm and 280 nm. Samples were collected after 8 min and up to 60 min, and elution buffer was harvested every 1 min into centrifuge tubes. The separated peptides were lyophilized in a vacuum.
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