Phospholipid class-specific profiling by LC/MS (Utrecht protocol)

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 quantification 3.1 Data handling 3.2 Isotope correction 4 Sample data 4.1 Contour Plot 4.2 Separation of isobaric species 5 Reference

Sample preparation

• Extraction according to Bligh and Dyer (with or without modifications to enhance recovery of acidic- and lyso-phospholipids and glyco(sphingo)lipids)
• sample solvent: Samples stored dry under nitrogen atmosphere at -20^oC

Instrumentation and method

Pump

• Type: Perkin Elmer series 200 micropump (2x)
• Mode: gradient and subsequent isocratic elution
  
• Solvent(s): A: MeOH/Acetonitrile/H₂O (45/30/25) B: MeOH/Acetonitrile (60/40). Both A and B contain 1 µM serine and 2.5 mM ammoniumacetate
  
• Gradient (including re-equilibration for subsequent run):
• Indication of operating pressure: around 120 bar

Time [min]	Flow [ml/min]	% Solvent A	% Solvent B
0	0.425	100	0
25	0.425	0	100
50	0.425	0	100
51	0.425	100	0
55	0.425	100	0

Column

• Type: Synergi 4 µm MAX-RP 18A column (250 × 3 mm; Phenomenex, CA, US)
• Temperature: ambient

Autosampler

• Type: HTC PAL
  
• Injection volume: 10 µl
  
• Wash solvent: MeOH
  

Mass spectrometer

• Type: 4000 QTRAP (Sciex)
• Ionization mode: either negative (PI, PA, PG, PS) or positive (GPCho, GPEtn, SM)
  
• Ionization voltage: -4500V or +4500V/5500V (optimize)
  
• Source temperature(s): 450^oC
  
• Collision gas: nitrogen
• Collision gas pressure: set to "medium"
  
• Operating mode: phospholipid class dependent

MS scan modes and settings

class	scan type	scan speed	DP (V)	CE (V)
PC/SM	precursor m/z +184	300 amu/s	110	+45
PtdEtn1	neutral loss 141 (pos)	300 amu/s	110	+35
PtdEtn1	precursor m/z -196	300 amu/s	-110	-45
PtdIns	precursor m/z -241	300 amu/s	-110	-30
PtdGro2	precursor m/z -153	300 amu/s	-110	-40
PtdSer	neutral loss 87 (neg)	300 amu/s	-110	-40

¹Positive mode analysis of diacyl PtdEtn has higher sensitivity than negative mode, but positive mode analysis is very insensitive towards ether-linked PtdEtn species.

²All glycerophospholipid species can generate the fragment ion of m/z -153 upon CID, but PtdGro and cardiolipin do so with much higher efficiency

Data analysis and quantification

Data handling

• Data are acquired with Analyst^TM (version 1.4.2). Using Analyst's "Translat.exe", data may be converted to open formats such as mzXML and netCDF.

Isotope correction

• Isotope correction is generally done by the data processing software (mzMine, XCMS).

Sample data

Contour Plot

Contour plot showing PtdCho profiling. Note the separation of lyso-species (5-15 min) from diradyl species (20-50 min), as well as the separation of sn-1 and sn-2 lyso species (double peaks between 5 min and 15 min).

Separation of isobaric species

Multiple examples of isobaric species that are separated by liquid chromatography

Reference

• PubMed
• Rapid Communications in Mass Spectrometryl

Please add categories as below for:

• analytical techniques applied
• lipid classes analyzed (see lipid class categories)