Ceramide Profiling LC-MS/MS

From LipidomicsWiki

Contents 1 Sample preparation 2 Instrumentation and method 2.1 Pump 2.2 Column 2.3 Autosampler 2.4 Mass spectrometer 3 Data analysis and quantitation 3.1 Data handling 3.2 Calibration and quantitation 4 Reference

Sample preparation

• Lipid extracts of tissues according to Bligh and Dyer are spiked with internal standards, and get diluted 1:100 in acetonitrile/2-propanol 5:2, 1% ammonia acetate, 0.1% formic acid.

Material	Material used	Internal Standard	Internal Standard added
Cell extracts	5 - 100 mg tissue	17:0 Ceramide	10ng each

Instrumentation and method

Pump

• Type: Thermo Accela
• Mode: Gradient
• Solvent A: Water with 1% ammonia acetate and 0.1% formic acid
• Solvent B: Acetonitrile/2-propanol 5:2 with 1% ammonia acetate and 0.1% formic acid
• Gradient:

Time [min]	Flow [ml/min]	% Solvent A	% Solvent B
0	0.25	65	35
4.0	0.25	30	70
20.0	0.25	0	100
30.0	0.25	0	100

Column

• Type: Thermo Hypersil GOLD C18, 100x1 mm, 1.9µm
• Temperature: 50°C

Autosampler

• Type: Accela
• Injection volume: 5ul
• Wash solvent: Solvent B

Mass spectrometer

• Type: Thermo TSQ Quantum Ultra
• Ionization mode: positive ESI
• Ionization voltage: 4500V
• Capillary temperature: 220°C
• HESI temperature: 300°C
• Capillary voltage: 35V
• Collision gas: Argon
• Collision gas pressure: 0.7mTorr
• MS/MS-conditions:
  • Precursor ion scan

Analyte	Precursor [m/z]	Scan range [m/z]	Collision energy [eV]
Ceramides	264	400 - 700	30

Data analysis and quantitation

Data handling

• Peak areas are calculated by QuanBrowser and identification is done by precursor ion scan and retention time.

Calibration and quantitation

• calibration type: relative quantitation 
• The internal standard is set to 100% and all other peaks are expressed as % relative to internal standards.

Reference

• PubMed
• electronic journal