TL and SL aliquots were resuspended in 250 μl chloroform/methanol (1:1 v/v) (LC–MS/HPLC grade) and sonicated for 5 min. The samples were pipetted in a 96-well plate (final volume = 100 μl). The TL were diluted 1:4 in negative-mode solvent (chloroform/methanol (1:2) + 5 mM ammonium acetate) and 1:10 in positive-mode solvent (chloroform/methanol/water (2:7:1 v/v) + 5 mM ammonium acetate). The SL were diluted 1:10 in positive-mode solvent and infused onto the mass spectrometer. Tandem mass spectrometry for the identification and quantification of SL molecular species was performed using Multiple Reaction Monitoring (MRM) with a TSQ Vantage Triple Stage Quadrupole Mass Spectrometer (Thermofisher Scientific, Waltham, MA) equipped with a robotic nanoflow ion source, Nanomate HD (Advion Biosciences, Ithaca, NY). The collision energy was optimized for each lipid class. The detection conditions for each lipid class are listed below (Table 2 ). Cer species were also quantified with a loss of water in the first quadrupole. Each biological replica was read in 2 technical replicas (TR). Each TR comprised 3 measurements for each transition. Lipid concentrations were calculated relative to the relevant internal standards and then normalized to the total lipid content of each lipid extract (mol %).
Nanomate hd
Manufactured by Advion
Sourced in United States
The Nanomate HD is a high-resolution nanoelectrospray infusion system designed for direct sample introduction into mass spectrometers. It provides precise control of sample flow rates and ensures stable spray formation for sensitive and reproducible mass spectrometry analysis.
Automatically generated - may contain errors
Lab products found in correlation
Tsq vantage triple stage quadrupole mass spectrometer, by Thermo Fisher Scientific (3 mentions)
Uflc xr, by Shimadzu (1 mentions)
Ltq orbitrap xl mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Qtrap 5500, by AB Sciex (1 mentions)
Tsq quantum ultra, by Thermo Fisher Scientific (1 mentions)
Apex 396, by AAPPTec (1 mentions)
6 protocols using nanomate hd
1
Quantification of Sphingolipid Species
2
Comprehensive Lipidomics Analysis of Human Myotubes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The lipidomics analysis was performed as described in Loizides-Mangold et al. (2017 (link)). Briefly, human primary myotubes were harvested from one confluent 10 cm dish (~1.5×106 cells) and lipid extracts were prepared using the MTBE protocol (Matyash et al., 2008 (link)). Total phosphorus was determined as described in (Loizides-Mangold et al., 2017 (link)). Phospho- and sphingolipid were analyzed by mass spectrometry using on a TSQ Vantage Triple Stage Quadrupole Mass Spectrometer (Thermo Fisher Scientific) equipped with a robotic nanoflow ion source (Nanomate HD, Advion Biosciences, Ithaca, NY), using multiple reaction monitoring (MRM). Lipid concentrations were calculated relative to the relevant internal standards and then normalized to the total phosphate content of each total lipid extract. Triacylglyceride analysis was performed by mass spectrometry analysis on a hybrid ion trap LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific) equipped with a micro LC binary pump UFLC-XR (Shimadzu, Kyoto, Japan). Lipid identification was carried out with the Lipid Data Analyzer II (LDA v. 2.5.1, IGB-TUG Graz University) (Hartler et al., 2011 (link)).
3
Shotgun Lipidomic Analysis of Phospholipids
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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
Mass Spectrometric Analysis of Derivatized Lipids
5
Peptide Synthesis and Characterization
6
Quantification of Sphingolipid Molecular Species
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
TL and SL aliquots were resuspended in 250 μL chloroform/methanol (1:1 v/v) (LC-MS/HPLC grade) and sonicated for 5 min. The samples were pipetted in a 96-well plate (final volume = 100 μL). The TL were diluted 1:4 in negative-mode solvent (chloroform/methanol (1:2) + 5mM ammonium acetate) and 1:10 in positive-mode solvent (chloroform/methanol/water (2:7:1 v/v) + 5mM ammonium acetate). The SL were diluted 1:10 in positive-mode solvent and infused into the mass spectrometer. Tandem mass spectrometry for the identification and quantification of sphingolipid molecular species was performed using Multiple Reaction Monitoring (MRM) with a TSQ Vantage Triple Stage Quadrupole Mass Spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a robotic nanoflow ion source, Nanomate HD (Advion Biosciences, Ithaca, NY, USA). The collision energy was optimized for each lipid class. The detection conditions for each lipid class are listed below (Table 1 ). Ceramide species were also quantified with a loss of water in the first quadrupole. Each biological replica was read in 2 technical replicas. Each of these replicas comprised 3 measurements for each transition. Lipid concentrations were calculated relative to the relevant internal standards and then normalized to the total lipid content of each lipid extract (mol%).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!