Phosphoinositide quantification was performed as described previously (Haag et al., 2012 (link)). Briefly, MDCK cells were plated in 6-well plates and grown for 5 days in Matrigel. Cells from two 6-well plates were combined and subjected to an acidic–neutral extraction of trichloroacetic acid (TCA)-washed cell pellets using PI(4)P 17:0–20:4 and PI(4,5)P2 17:0–20:4 from Avanti Polar Lipids as internal standards for quantification. Mass spectrometry was performed on a QTRAP 5500 instrument (AB Sciex) equipped with a Triversa NanoMate system (Advion Biosciences). Phosphoinositides were measured by scanning for neutral losses of m/z 357 (phosphatidylinositol) and m/z 437 (PIP2) on an AB Sciex QTRAP 5500 instrument at collision energies of 25 eV and 35 eV, respectively. Mass spectra were evaluated using LipidView software (AB Sciex). PIP and PIP2 amounts were normalized to total phospholipids, which was determined in neutral and acidic phases, as described previously (Özbalci et al., 2013 (link)).
QTRAP 5500
Manufactured by AB Sciex
Sourced in United States, Japan, Germany, Canada, United Kingdom, Denmark, Austria, France
The QTRAP 5500 is a high-performance hybrid triple quadrupole linear ion trap mass spectrometer. It combines the quantitative capabilities of triple quadrupole technology with the qualitative power of a linear ion trap, enabling both quantitative and qualitative analysis in a single platform.
Automatically generated - may contain errors
Lab products found in correlation
Analyst 1.6.3 software, AB Sciex (23 mentions)
1290 HPLC, Agilent (15 mentions)
LC-20AD HPLC, Shimadzu (12 mentions)
Nexera X2, Shimadzu (11 mentions)
Acquity UPLC BEH C18 column, Waters Corporation (10 mentions)
Analyst software, AB Sciex (10 mentions)
MultiQuant, AB Sciex (9 mentions)
MultiQuant software, AB Sciex (8 mentions)
MultiQuant 3.0.2 software, AB Sciex (8 mentions)
UFLC XR, Shimadzu (7 mentions)
Agilent 1290 HPLC system, Agilent (6 mentions)
1290 Infinity LC, Agilent (6 mentions)
TriVersa NanoMate, Advion (6 mentions)
SIL-20AC autoinjector, Shimadzu (5 mentions)
Eclipse Plus C18 column, Agilent (5 mentions)
UPLC, Waters Corporation (5 mentions)
ACQUITY UPLC BEH Amide column, Waters Corporation (5 mentions)
LipidView software, AB Sciex (5 mentions)
C18 HPLC column, Agilent (5 mentions)
LC-30 ultrahigh performance liquid chromatography system, Shimadzu (5 mentions)
504 protocols using QTRAP 5500
1
Quantifying Phosphoinositide Levels in MDCK Cells
2
Comprehensive Metabolite Profiling Protocol
The methods for measuring blood metabolites, including amino acids, hormones, vitamins, microelements and heavy metals, were described in detail by Jie et al.67 (link) Briefly, we detected the amino acids from 40 μL plasma using ultra-high pressure liquid chromatography (UHPLC) coupled to an AB Sciex Qtrap 5,500 mass spectrometer (MS) (AB Sciex, US) with an electrospray ionization (ESI) source; to measure hormones, 250 μL plasma was used and detected via UHPLC-MS (AB Sciex Qtrap 5,500) with an atmospheric pressure chemical ionization (APCI) source; water-soluble vitamins were detected from 200 μL plasma via UPLC-MS (Waters Xevo TQ-S Triple Quad, Waters, US) with an ESI source; and fat-soluble vitamins were detected from 250 μL plasma via UPLC-MS (AB Sciex Qtrap 4,500, AB Sciex, US) with an APCI source; we detected the microelements and heavy metals in 200 μL whole blood via an Agilent 7,700x ICP-MS (Agilent Technologies, Japan) equipped with an octupole reaction system (ORS). UPLC-MS was used in positive ion mode. All measured items are shown in Table S1 .
3
Shotgun Lipidomic Analysis of Phospholipids
The species of all phospholipids, SM, DAG and CE were analyzed by shotgun analysis on a hybrid triple quadrupole/linear ion trap mass spectrometer (QTRAP 5500, AB SCIEX) equipped with a robotic nanoflow ion source (NanoMate HD, Advion Biosciences) [59 ]. These analyses were performed using both positive and negative ion modes using multiple precursor ion scanning (MPIS) and neutral loss (NL) based methods [56 (link), 60 (link)], whereas CEs were analyzed in positive ion mode [61 (link)]. Sphingolipids were analyzed by reverse phase ultra-high pressure liquid chromatography (UHPLC) as previously described [62 (link)] using an Acquity BEH C18, 2.1×50 mm column with a particle size of 1.7 μm (Waters, Milford) coupled to a hybrid triple quadrupole/linear ion trap mass spectrometer (QTRAP 5500, AB SCIEX). A 25 min gradient using 10 mM ammonium acetate in water with 0.1% (v/v) formic acid (mobile phase A) and 10 mM ammonium acetate in 4:3 (v/v) acetonitrile:2-propanol containing 0.1% (v/v) formic acid (mobile phase B) was used. Quantification of sphingolipids was performed using multiple reaction monitoring. Data from one such experiment are shown; similar results were obtained in another independent experiment.
4
Quantitative AP-MALDI Mass Spectrometry Analysis
The quantitative experiments were performed on an AB SCIEX QTRAP 5500 (SCIEX, Framingham, MA, USA) mass spectrometer coupled to an AP-MALDI ion source (MassTech, Inc., Columbia, MD, USA) equipped with a 355 nm Nd: YAG laser source.
The AB SCIEX QTRAP 5500 system was operated in SRM with positive ion polarity mode. Analyst software 1.7.1 (SCIEX) was used for data acquisition and parameter optimization for SRM. For SRM analysis, ion spray voltage was optimized to 4000 V, interface heater temperature was set at 220 C, and declustering potential (DP) was set to 91 V for best signals. The collision energy (CE) was optimized to 33 eV for 273.040 u and 45 eV for 229.007 u to achieve fragmentation. The laser energy of the AP-MALDI ion source was optimized at 80% for the analysis with the Target (MassTech, Inc., Columbia, MD) software. AP-MALDI quantitation sequence was run automatically with the Target software. The obtained AP-MALDI SRM data were qualitatively interpreted with PeakView 2.2 (SCIEX). PeakView 2.2 uses the Analyst-generated raw data as an input file and gives a text file as an output. The output text files contain the intensities of the chosen SRM transitions of a particular AP-MALDI spot(s)/well(s). These text files were imported in Microsoft Excel, and the quantitative graphs were plotted with Microsoft Excel.
The AB SCIEX QTRAP 5500 system was operated in SRM with positive ion polarity mode. Analyst software 1.7.1 (SCIEX) was used for data acquisition and parameter optimization for SRM. For SRM analysis, ion spray voltage was optimized to 4000 V, interface heater temperature was set at 220 C, and declustering potential (DP) was set to 91 V for best signals. The collision energy (CE) was optimized to 33 eV for 273.040 u and 45 eV for 229.007 u to achieve fragmentation. The laser energy of the AP-MALDI ion source was optimized at 80% for the analysis with the Target (MassTech, Inc., Columbia, MD) software. AP-MALDI quantitation sequence was run automatically with the Target software. The obtained AP-MALDI SRM data were qualitatively interpreted with PeakView 2.2 (SCIEX). PeakView 2.2 uses the Analyst-generated raw data as an input file and gives a text file as an output. The output text files contain the intensities of the chosen SRM transitions of a particular AP-MALDI spot(s)/well(s). These text files were imported in Microsoft Excel, and the quantitative graphs were plotted with Microsoft Excel.
5
NAD+ Metabolites Analysis by LCMS
Liquid chromatography and mass spectrometry were carried out on an Agilent 1260 Infinity pump coupled to a QTRAP5500 (AbSCIEX). The chromatographic separation of NAD+ metabolites was achieved using an XBridge Beh Amide column (130Å, 3.5 µm, 201 mm × 100 mm; Waters, Australia) at a flow rate of 0.2 mL/min with a total run time of 30 min. The mobile phase consisted of ACN with 20 mM ammonium acetate and 20 mM NH4OH (A) and 100% ACN (B). The initial composition of the mobile phase was 15% A and 85% B, which was ramped up to 30% A after 10 min and held for 3 min before being increased again to 70% A for 5 min and 30 s before returning to initial levels and being maintained for the duration of the run. An injection volume of 2.5 µL was used for all samples. The ion source voltage was set at 4500 V, the capillary temperature was set at 350 °C and the pressure was maintained at 45 bar. Data were acquired and analysed with Analyst 1.6.2 and MultiQuant software, respectively.
6
Metabolic Profiling of Cell Lysates
Except glucose, pyruvate and lactate were measured using specified reagent kits (Nanjing Jiancheng Biotech Co. Ltd) and HPLC‐MS for their extracellular and intracellular content; other metabolites were directly determined by HPLC‐MS. Cell and tissue lysates were extracted in the extraction buffer (containing methanol and water (1:1, v/v), 5 mmol/L ammonium acetate) in the cold room for 15 minutes, followed by scraping and centrifuging at 17 000 g for 10 minutes. Metabolic flux analysis was performed by liquid chromatography (Prominence LC‐20A, Shimadzu, JPN) with a tandem quadrupole mass spectrometry (QTRAP®5500, AB Sciex, MA, USA). Samples were injected into a 4.6 mm × 150 mm StableBond column (ZORBAX SB‐AQ 5 µm; Agilent). The chromatography was run started with 98% solution B (0.1% formic acid in H2O) and 2% solution A (0.1% formic acid in methanol), followed by 4 minutes going down to 80% solution B, 4.5 minutes coming back to 98% solution B, and then holding up until stopped. Data were collected and analysed by Analyst Software (AB Sciex).23
7
Quantitative Proteomics Using MRM
A spectral library of MS/MS data was generated on a TripleTOF5600 (AB SCIEX, Foster City, CA) and searched using Mascot v2.3 (Matrix Science, UK) against a human database that was downloaded from the Uniprot database and consisted of 127497 sequences. All 19 proteins had MS/MS spectra and unique peptides. Then we used a QTRAP 5500 (AB SCIEX) to verify whether the peptide had a co-elution chromatogram and correct retention time. Finally, we developed an MRM method for the 19 proteins. The data file was imported into Skyline software, where a library was built. The peptides were selected for MRM method development according to the following criteria: (1) the peptides had a unique sequence in the database, (2) a maximum m/z of peptide <1250 (limination of quadrupole scan), with a peptide length range of 5–40 aa, (3) the peptides lacked methionine, (4) had a carbamidomethyl group attached to residues and was without variable modification in the peptides, and (5) no missed cleavage by trypsin. We initially monitored 6 transitions per peptide to ensure specificity with the criteria that >5 y-ions had the same elution profile and were in the same ratios as the spectral library. The predicted retention time of targeted peptides was observed using an iRT strategy.
8
Quantification of Metabolites in Mouse Liver
We extracted 5‐10 mg mouse liver tissue in 80% high‐performance liquid chromatography‐grade methanol, as described, then lyophilized and resuspended the extract in 20 µL of liquid chromatography–mass spectrometry (LC‐MS)‐grade water. Next, 5‐10 µL of each sample was autoinjected into the QTRAP 5500 tandem LC‐MS (LC‐MS/MS) system (AB Sciex, LLC, Framingham, MA). Multiquant 2.0 software (AB Sciex, LLC) was used to quantify chromatographic peak areas. The Linear Models for Microarray Analysis package27 from the Bioconductor project was used to identify differentially expressed molecules among different groups compared to controls. Technical replicates were averaged, and the biologically distinct sample groups were compared by fitting a linear model for each variable (normalized expression values) and applying empirical Bayes smoothing to identify differentially expressed molecules. For pathway analysis, the differentially expressed molecules (P ≤ 0.05 and fold change > 2) were grouped into pathways using Metaboanalyst 3.0.28
9
Quantitative Lipid Mediator Profiling
10
Quantitative UPLC-MS/MS Analysis of STG
The UPLC System was coupled to a tandem mass spectrometry QTRAP 5500 (AB SCIEX; Toronto, ON, Canada) with electrospray ionization (ESI) in positive mode. This was equipped with a ACQUITY UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm; Waters) maintained at 40 °C, and the injection volume was 2 μL. The mobile phase included A: water (0.1% formic acid, 2 mM ammonium formate) and B: acetonitrile. The flow rate was 0.3 mL/min, and the gradient elution is given in Table S1 . The mass detection parameters for STG were optimized, including declustering potential (DP), entrance potential (EP), collision energy (CE), and collision cell exit potential (CXP) (Table S1 ).
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