The serum proteins in each sample were separated into 12 fractions through pH 3–10 isoelectric points, using the OFFGEL fractionator (3100 OFFGEL Low Res Kit, pH 3–10; Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s instructions. Twelve fractions were loaded onto an Eksigent nanoLC 400 system and the cHiPLC® (AB Sciex, Concord, ON, Canada) and analyzed, and the proteins were identified using a TripleTOF 5600 mass spectrometer (AB Sciex). Thereafter, for relative analysis, SWATH acquisition was conducted. In each run, 100 μg/mL of samples was injected onto an Eksigent ChromXP nanoLC trap column (350 μm i.d. × 0.5 mm, ChromXP C18 3 μm) at a flow rate of 5000 nL/min. Samples were eluted from the Eksigent ChromXP nanoLC column (75 μm i.d. × 15 cm) at a flow rate of 300 nL/min for 120 min, and mobile phase B buffer was added gradually into the column (5–90%) over a 120-min total run time. The gradient of mobile phase B buffer was (time and % B) 0 min/mobile phase B 5%, 10.5 min/40%, 105.5 min/90%, 111.5 min/90%, 112 min/5%, and 120 min/5%. Mobile phase B and A buffer, and the search parameters are as described [12 (link)].
Nanolc 400 system
Manufactured by AB Sciex
Sourced in United States, Canada
The NanoLC 400 system is a liquid chromatography instrument designed for high-performance separation of complex samples. It features precise flow control and low dead volume to enable sensitive analysis of small sample volumes. The system is equipped with a nanoflow-to-capillary-flow fluidic pathway to accommodate a wide range of analytical column dimensions.
Automatically generated - may contain errors
Lab products found in correlation
Triple tof 5600 system, by AB Sciex (2 mentions)
3100 offgel low res kit, by Agilent Technologies (1 mentions)
Chromxp nanolc trap column, by AB Sciex (1 mentions)
Chromxp nanolc column, by AB Sciex (1 mentions)
Tripletof 5600 mass spectrometer, by AB Sciex (1 mentions)
Tripletof5600plus, by AB Sciex (1 mentions)
Proteinpilot software, by AB Sciex (1 mentions)
Qtrap 4500 system, by AB Sciex (1 mentions)
Tripletof 6600 system, by AB Sciex (1 mentions)
6 protocols using nanolc 400 system
1
Serum Protein Fractionation and Identification
2
Proteomic Analysis of FOXQ1 Interactome
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Liquid chromatography–mass spectrometry (LC-MS) analysis was conducted with a NanoLC 400 system, and a TripleTOF 5600-Plus (AB Sciex, Toronto, Canada) system was used for the mass spectrometry (MS) analysis. ProteinPilot software (AB Sciex, Toronto, Canada) was used to analyze data from the TripleTOF 5600-Plus and identified proteins bound to FOXQ1.
The potential interactions among identified proteins were evaluated with the STRING pathway database [16 ]. Specifically, we tried to identify proteins associated with the Wnt/β-catenin pathway, and the proteins that were identified through the use of the STRING database were then ranked by their percentage of coverage in the LC-MS/MS results.
The potential interactions among identified proteins were evaluated with the STRING pathway database [16 ]. Specifically, we tried to identify proteins associated with the Wnt/β-catenin pathway, and the proteins that were identified through the use of the STRING database were then ranked by their percentage of coverage in the LC-MS/MS results.
3
Targeted Protein Quantification by Scheduled MRM
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Samples, high- and low-concentration standards were analyzed using an ekspert nanoLC 400 system coupled to an AB SCIEX QTRAP® 4500 System operating in scheduled MRM mode (Fig. 3 ). Mass scans were performed in positive ionization mode with curtain gas flow at 30 psi, collisionally activated dissociation set on high, ion spray voltage at 2700 V, ion gas 1 at 20 psi, ion gas 2 at 0 psi, interface heater temperature at 150 °C, entrance potential at 10 V and collision cell exit potential at 15 V. Declustering potential and collision energy were optimized for each target transition (Table S1 ).
Skyline software was used to design experiments, and MultiQuant was used to analyze data. As described previously,29 (link) we selected 1–3 tryptic peptides per protein, and 1–3 y-serious fragment ions for each peptide to increase specificity and confidence (Table S1 ). Standard curves were constructed for each protein. Finally, protein abundance was determined by integrating the peak area of the transitions of each protein, and normalizing to corresponding 15N-labeled internal standards. As before, samples were analyzed in triplicate.
Skyline software was used to design experiments, and MultiQuant was used to analyze data. As described previously,29 (link) we selected 1–3 tryptic peptides per protein, and 1–3 y-serious fragment ions for each peptide to increase specificity and confidence (
4
MRM-HR Analysis of Peptide Samples
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Peptide samples were prepared in the same way as the previous proteomic section except no TMT labeling was performed. Eksigent NanoLC 400 System (Eksigent, Dublin, CA, USA) coupled with TripleTOF 6600 system (SCIEX, CA, USA) was applied for MRM-HR experiment. The peptide digests were separated at a 5 μL/min with a 10 min gradient (buffer B: 5%–10% for 1 min, 10%–40% for 6 min, 40%–80% for 0.1min, maintained 80% for 2.9 min, 80%–5% for 1 min) using an analytical column (3 μm, ChromXP C18CL, 120 Å, 150∗0.3 mm). IDA mode (rolling collision energy, +2 to +5 charge states with intensity criteria above 2,000,000 cps to guarantee all untargeted peptides will not be acquired) for time-scheduling was set up for 51 peptides including 10 iRT peptides (Escher et al., 2012 (link)) with a mass tolerance of 50 ppm. Accumulation time for TOF-MS scan (350-1250 m/z) and MS/MS scans (100-1500 m/z) was 250 ms and 50 ms, respectively. The data acquired by MRM-HR experiment were analyzed by Skyline (MacLean et al., 2010 (link)). The retention time was predicted by the iRT, and the isolation time window is 2 min. The mass analyzer for MS1 and MS/MS was set as “TOF” with the resolution power of 30,000.
5
Serum Proteome Analysis by SWATH-MS
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Briefly, serum proteome analysis was performed as previously described (Cai et al., 2023 (link)). The peptides were extracted from the serum samples and were then digested with a two‐step overnight tryptic digestion (Hualishi Tech. Ltd, Beijing, China) at 32°C for 4 h and 12 h, using an enzyme‐to‐substrate ratio of 1:60 (final ratio 1:30) for each digestion step. The SWATH‐MS analysis for the peptide samples were perform on an Eksigent NanoLC 400 System (Eksigent, Dublin, CA, USA) coupled with a TripleTOF 5600 system (SCIEX, CA, USA). The MS files were analyzed using DIA‐NN (1.8) against a plasma spectral library containing 5102 peptides and 819 unique proteins from the Swiss‐Prot database of Homo sapiens. Protein inference was set to the protein names (from the FASTA file), and the cross‐run normalization was set as “RT dependent”. A total of 413 proteins from ~20,000 proteomes were quantified. The detailed methods of LC–MS/MS were provided in Appendix S1 .
6
SWATH-MS Workflow for Quantitative Proteomics
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For the SWATH-MS experiment, Eksigent NanoLC 400 System (Eksigent, Dublin, CA, USA) and TripleTOF 5600 system (SCIEX, CA, USA) were used. Peptides were separated at a ow rate of 5 µL/min with a 20 min gradient (5%-30% buffer B) by an analytical column (3 µm, ChromXP C18CL, 120 A, 150*0.3 mm). The acquisition range for the TOF-MS scan was 350-1250 Th with an ion accumulation time of 250 ms, while the mass tolerance for MS/MS scans was 50 ppm with an ion accumulation time of 50 ms. SWATH-MS data were analyzed using OpenSWATH (version 2.4.0). The retention time was calibrated using common internal Retention Time standards (CiRT) peptides with m/z extraction of 50 ppm tolerance. Peptide precursors were identi ed by PyProphet with an FDR < 0.01 at both the peptide and protein levels using a self-built spectral library containing 3287 peptide precursors from 537 protein groups. The TMT method was described in our previous paper 3 . The German cohort protein data matrix has been described previously 16 .
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