First of all, commercial purified human histones H3 and H2B (EpiGex, Illkirch, France) were trypsinised and analysed using LC-MS/MS_DDA in a 5600 triple TOF (ABSciex, Framingham, MA, USA) to identify those unequivocal peptides with a good signal. From the different fragmentation spectra (MSMS), peptide precursors and fragment ion masses were selected for H3 and H2B to be analysed by MRM-MS. The MRM parameters were optimised in a 5500 QTRAP instrument (ABSciex) by the MRM-PILOT software (AB Sciex) to determine the declustering potential (DP) for each peptide and dwell time (DT) and collision energy for each transition (CE). Afterwards, histone-derived peptides with a large number of detectable transitions, high sensitivity and a wide linear dynamic range were chosen to perform the MRM-MS experiments. The selected peptide sequences (STELLIR for H3; LLLPGELAK for H2B) were used for absolute quantification purposes.
5500 qtrap instrument
Manufactured by AB Sciex
Sourced in United Kingdom
The 5500 QTrap instrument is a mass spectrometry system designed for quantitative and qualitative analysis. It features a triple quadrupole configuration with an enhanced linear ion trap, providing high-performance detection and sensitivity. The core function of the 5500 QTrap is to perform accurate and sensitive measurements of chemical compounds in complex samples.
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4 protocols using 5500 qtrap instrument
1
Quantitative Histone Peptide Analysis
2
Targeted Plasma Metabolomics by LC-MS/MS
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Tandem mass spectrometry was performed for targeted metabolomics of plasma samples using Biocrates MxP® Quant 500 Kit (Biocrates, Innsbruck, Austria) at the Fraunhofer Institute for Toxicology and Experimental Medicine (ITEM). FIA-MS/MS was utilized to measure lipids using a 5500 QTRAP® instrument with an ESI source (AB Sciex, Darmstadt, Germany). LC-MS/MS utilizing the same 5500 QTRAP® instrument was used for measuring small molecules as described previously (17 (link)). Appropriate MS software (Sciex Analyst®) was used to quantify data that was then imported into Biocrates MetIDQ™ software for calculation of analyte concentrations, assessment and compilation of data.
3
Quantification of Lipids and Oxysterol Sulfates
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Lipids from frozen tissues (10 mg) and CSF (100μL) were extracted by the Folch protocol [28 (link)]. Extraction was done in glass vials and repeated twice, the organic layer from each was combined and evaporated to dry under nitrogen stream in an ice bath. Phospholipids were quantified by spectrophotometry measurement of inorganic phosphorous as described before [29 (link)]. Lipid extracts were resuspended in chloroform: methanol (1:1, v/v) and normalized to a final concentration of 25 ng phospholipid per microliter in 100% methanol. Oxysterol sulfates were analyzed by mass spectrometry in a 5500 QTrap instrument (ABSciex, Warrington, UK) operating in the negative ion detection mode over the mass range of 350–1000 Da with direct infusion at a flow rate of 10μL min–1 as we described before [29 (link)]. Detection of oxysterol sulfates in lipid extracts was achieved by precursor ion scanning (PIS) at m/z 97.0 and confirmed by PIS at m/z 80 collected at 1000 Da/s scan speed with step size of 0.1 Da. Oxysterol PIS mass spectrum of samples at m/z 481.4 was used to calculate the levels of oxysterol sulfates.
4
Lipid Extraction and Mass Spectrometry
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Prior to MS analysis, lipid extracts were resuspended in CHCl3:MeOH (1:1, v/v) and normalized to a final concentration of 25ng phospholipid per microliter (See Optimization of Analysis Conditions Section). Screening of human fluid and cell lipids extracts was conducted by mass spectrometry in a 5500 QTrap instrument (ABSciex, Warrington, UK) operating in the negative ion detection mode were acquired for the ion of interest with Q1 set to low resolution at 10,000 Da/s with collision energy varying between -10 and -100eV. Dynamic fill time was used with a maximum fill time of 250 ms and all other parameters optimized to give maximum signal. Blank injections were included between samples.
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