Proteomic analysis were performed by data-dependent acquisition (DDA) mass spectrometry assay. Sample preparation and fractionation were performed as previously described (Wiśniewski et al., 2009 (link)). Equal aliquots of the samples in this experiment were pooled into one sample for DDA library generation and quality control. All fractions for DDA library generation were injected into a Thermo Scientific Q Exactive HF mass spectrometer connected to an Easy-nLC 1200 chromatography system (Thermo Scientific). Each sample peptides were analyzed in a data-independent acquisition (DIA) mode. The DIA cycle contained a full MS–selected ion monitoring (SIM) scan, and 30 DIA scans were performed covering a mass range of 350–1650 m/z with the following settings: SIM full scan resolution, 60,000 at 200 m/z; automatic gain control (AGC), 3e6; maximum ion trap (IT) time, 50 ms; profile mode; DIA scan resolution, 30,000; AGC target, 3e6; maximum IT, auto; and normalized collision energy, 30 eV. The runtime was 120 min with a linear gradient of buffer B (80% acetonitrile and 0.1% formic acid) at a flow rate of 250 nL/min. Quality control samples (pools of equal aliquots of all the samples in the experiment) were injected in DIA mode at the beginning of the MS study and after every 5 injections throughout the experiment and were used to monitor MS performance.
Easy nlc 1200 chromatography system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Easy nLC 1200 is a chromatography system designed for high-performance liquid chromatography (HPLC) applications. It features a compact design and automated sample handling capabilities. The system is capable of performing nano-scale liquid chromatography separations.
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Lab products found in correlation
Easy spray c18 trap column, by Thermo Fisher Scientific (5 mentions)
Easy spray c18 lc analytical column, by Thermo Fisher Scientific (5 mentions)
Q exactive hf x mass spectrometer, by Thermo Fisher Scientific (3 mentions)
Q exactive hf mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Trypsin, by Promega (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Q exactive hf, by Thermo Fisher Scientific (1 mentions)
Proteome discoverer software, by Thermo Fisher Scientific (1 mentions)
Q executive mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Q exactive hf instrument, by Thermo Fisher Scientific (1 mentions)
Q exactive plus instrument, by Thermo Fisher Scientific (1 mentions)
Nexera x2 lc 30ad, by Shimadzu (1 mentions)
Qtrap 5500 mass spectrometer, by AB Sciex (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Q exactive plus, by Thermo Fisher Scientific (1 mentions)
Q exactive hf x, by Thermo Fisher Scientific (1 mentions)
Acetonitrile, by Merck Group (1 mentions)
Orbitrap fusion lumos tribrid mass spectrometer, by Thermo Fisher Scientific (1 mentions)
14 protocols using easy nlc 1200 chromatography system
1
Quantitative Proteomics by DIA-MS
2
DDA Proteomics Analysis on Q Exactive HF
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All fractions for DDA library generation were injected on a Thermo Scientific Q Exactive HF mass spectrometer connected to an Easy nLC 1200 chromatography system (Thermo Scientific). Peptides (2 μg) were first loaded onto an EASY-SprayTM C18 Trap column (Thermo Scientific, P/N 164946, 3 μm, 75 μm * 2 cm), and then separated on an EASYSprayTM C18 LC Analytical Column (Thermo Scientific, ES803, 2 μm, 75 μm * 50 cm) with a linear gradient of buffer B (80% acetonitrile and 0.1% formic acid) at a flow rate of 250 nL/min over 120 min. MS detection method was positive ion, the scan range was 300–1650 m/z, and resolution for MS1 scan was 60000 at 200 m/z, target of automatic gain control (AGC) was 3e6, maximum IT was 25 ms, and dynamic exclusion was 30.0 s. Each full MS–SIM scan followed 20 ddMS2 scans. Resolution for MS2 scan was 15000, AGC target was 5e4, maximum IT was 25 ms, and normalized collision energy was 27 eV.
3
Peptide Separation and Identification
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The appropriate amounts of peptides for each sample were separated by chromatography using the Easy nLC 1200 chromatography system (Thermo Scientific). The peptides were separated by DDA (data-dependent acquisition) mass spectrometry with a Q-Exactive HF-X mass spectrometer (Thermo Scientific). The resulting LC-MS/MS raw file was imported into the Sequest HT search engine in Proteome Discoverer software (version 2.4; Thermo Scientific) for database retrieval.
4
LC-MS/MS Proteomic Analysis of Vibrio cholerae
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The LC-MS/MS analysis was carried out at HooGen Biotech, Shanghai, China using Q Executive Mass Spectrometer (Thermo Fisher Scientific (TFS), Waltham, MA, USA) coupled with Easy nLC 1200 Chromatography System with the same parameters described previously (Zhu et al., 2020 (link)). The automated peptide identification using UniProt V. cholerae 89344 20210707 databases (download in July 2021) in Mascot version 2.2 server (Matrix Science, London, United Kingdom), as described previously (Zhu et al., 2020 (link); Shan et al., 2021 (link)).
5
Detailed DDA Proteomic Workflow
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All fractions for DDA library generation were analysed by a Thermo Scientific QExactive HF X mass spectrometer connected to an Easy nLC 1200 chromatography system (Thermo Scientific). The peptide (1.5 μg) was first loaded onto an EASY-SprayTMC18 Trapcolumn (Thermo Scientific, P/N 164946, 3 µm, 75 um*2 cm), then separated on an EASY-SprayTM C18 LC Analytical Column (Thermo Scientific, ES802, 2 µm, 75 um*25 cm) with a linear gradient of buffer B (84% acetonitrile and 0.1% Formic acid) at a flow rate of 250 nl/min over 120 min. MS detection method was positive ion, the scan range was 300–1800 m/z, the resolution for the MS1 scan was 60,000 at 200 m/z, the target automatic gain control (AGC) was 3e6, the maximum IT was 25 ms, and the dynamic exclusion parameter was 30.0 s. Each full MS–SIM scan followed 20 ddMS2 scans. The resolution for the MS2 scan was 15,000, the AGC target was 5e4, the maximum IT was 25 ms, and the normalized collision energy was 30 eV.
6
Proteomic Analysis of Phosphorylated Peptides
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Tryptic peptides from in-gel digestion were analyzed on a Q Exactive plus instrument (Thermo Fisher Scientific) and phosphoenrichment peptides on a Q Exactive HF instrument (Thermo Fisher Scientific), both instruments coupled with an EASY nLC 1200 chromatography system (Thermo Fisher Scientific). Sample was loaded on an in-house packed 25-cm (for in-gel digestion) and 53-cm (phosphoenrichment) nano-HPLC column with C18 resin (1.9-μm particles, 100-Å pore size; Reprosil-Pur Basic C18-HD resin; Maisch GmbH, Ammerbuch-Entringen, Germany) after an equilibration step in 100% solvent A (H2O, 0.1% FA). Peptides were first eluted using a 2% to 5% gradient of solvent B (ACN, 0.1% FA) during 5 min, a 5% to 10% gradient during 20 min, a 10% to 30% gradient during 70 min, and finally a 30% to 60% gradient during 20 min, all at 300 nl·min−1 flow rates. The instrument method was set up in the data-dependent acquisition mode. After a survey scan in the Orbitrap (resolution, 70,000 and 60,000), the 10 most intense precursor ions were selected for HCD fragmentation with normalized collision energy set to 27 and 28. Charge state screening was enabled, and precursors with unknown charge state or a charge state of 1, 7, 8, and >8 were excluded. Dynamic exclusion was enabled for 20 s and 30 s.
7
Proteomics and Metabolomics Analytical Workflow
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In proteomics assays, the EasynLC1200 chromatography system (Thermo Scientific) was used to perform chromatographic separations. Buffer consists of two liquid aqueous solutions: A is 0.1% formic acid, and B is 0.1% formic acid acetonitrile (acetonitrile is 85%). DDA (data-dependent acquisition) mass spectrometry was conducted using a Q-Exactive HF-X mass spectrometer (Thermo Scientific). All mass spectrometry data were merged by Proteome Discoverer 2.4 software to analyze the search library and build a spectral database. In targeted metabolomics assays, separation was performed by high-performance liquid chromatography using Shimadzu NexeraX2LC-30AD. Mobile phase: Liquid A was 5% aqueous acetonitrile, 10 mM ammonium acetate, pH 9; Liquid B was 95% aqueous acetonitrile, 10 mM ammonium acetate, pH 9. The sample was injected into the autosampler at a column temperature of 40°C, a flow rate of 300 µL/min, and a sample volume of 8 µL. Mass spectrometry was conducted utilizing a QTRAP 5500 mass spectrometer (ABSCIEX) in positive/negative ion mode. To determine the ion pair that needed to be monitored, MRM mode was employed.
8
Proteomic Analysis of Gemcitabine and DTLL
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The proteomic analysis of the cell samples was conducted using the combination of DIA and a data-dependent acquisition (DDA)-based ion library. After treatment with 2 μM gemcitabine, 0.1 nM DTLL and both at 37 °C for 4 h, cells were harvested and lysed with lysis buffer (8 M urea, 100 mM Tris-HCl pH 7.6, 1 mM PMSF), and then centrifuged at 18,000 × g for 15 min at 4 °C. The extracted protein in the supernatant was quantified using a BCA protein assay kit (Beyotime Biotechnology, China) and digested in trypsin (Promega, Madison, WI) after reduction and alkylation using the FASP (filter aided sample preparation) method. The concentration of digested peptides was determined by measuring the absorbance at 280 nm using a NanoDrop 2000 instrument (Thermo Fisher Scientific, Waltham, MA, USA). An EASY-nLC 1200 chromatography system and an Q Exactive HF mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) were used for mass spectrometry acquisition and analysis. The DIA analysis was performed according to a previous study71 (link). All results were filtered by a Q-value cutoff of 0.01 (corresponding to an FDR of 1%). The P-value estimator was performed by the Kermel Density Estimator. The area was used for protein quantification. Every peptide was validated with at least three fragments.
9
Venom Protein Characterization by LC-MS
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Reduced/alkylated venom filtrate was digested overnight at a temperature of 37 °C with 0.1 μg of trypsin (Promega, Madison, WI, USA). Tryptic digests were analyzed on a Q‐Exactive Plus instrument (Thermo Fisher Scientific, Bremen, Germany) coupled to an EASY‐nLC 1200 chromatography system (Thermo Fisher Scientific). Two micrograms was loaded on an in‐house packed 50‐cm nano‐HPLC column (75 μm inner diameter) filled with C18 resin (1.9 μm particles, 100 Å pore size, Reprosil‐Pur Basic C18‐HD resin; Maisch GmbH, Ammerbuch‐Entringen, Germany) and equilibrated in 97% solvent A and 3% solvent B (ACN, 0.1% (v/v) FA).
Peptides were eluted at 250 nL·min−1, using 3–22% gradient of solvent B for 112 min, then 22–38% gradient of solvent B for 35 min, and finally 38–60% gradient of solvent B for 15 min. The instrument method for the Q‐Exactive Plus was set up in the data‐dependent acquisition mode. MS and MS‐MS spectra were acquired at a resolution of 60 000, 10 of the most abundant precursor ions were selected for HCD fragmentation with collision energy adjusted to 27. Mono‐charged precursors and those with a charge state of > 7 were excluded.
Peptides were eluted at 250 nL·min−1, using 3–22% gradient of solvent B for 112 min, then 22–38% gradient of solvent B for 35 min, and finally 38–60% gradient of solvent B for 15 min. The instrument method for the Q‐Exactive Plus was set up in the data‐dependent acquisition mode. MS and MS‐MS spectra were acquired at a resolution of 60 000, 10 of the most abundant precursor ions were selected for HCD fragmentation with collision energy adjusted to 27. Mono‐charged precursors and those with a charge state of > 7 were excluded.
10
Peptide Fractionation for DDA Library
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Peptide fractions for DDA library generation were analysed using a Q Exactive HF X mass spectrometer (Thermo Scientific Corp) coupled to an Easy nLC 1200 chromatography system (Thermo Scientific Corp). Peptide samples (1.5 μg) were first loaded onto an EASY-Spray C18 Trap column (P/N 164946, 3 μm, 75 μm × 2 cm; Thermo Scientific Corp), then separated on an EASY-Spray C18 LC Analytical Column (ES802, 2 μm, 75 μm × 25 cm; Thermo Scientific Corp) over a 120 min gradient from buffer B (0.1% formic acid and 84% acetonitrile). The column flow rate was maintained at 250 nL/min. MS detection was performed in positive ion mode, full scans were performed between 300 and 1800 m/z, the resolution for the MS1 scan was 60,000 at 200 m/z, the automatic gain control (AGC) target for the MS scan was set to 3e6, and the maximum injection time (IT) was 25 ms. The dynamic exclusion was set to 30.0s. Each full mass spectrometry (MS)-selected ion monitoring (SIM) scan followed 20 ddMS2 scans. The MS2 scan was performed at 15,000 resolution, the AGC target was 5e4, the maximum IT was 25 ms, and the collision energy was 30 eV.
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