Profiling of Neutral Lipids (Utrecht protocol)

From LipidomicsWiki

A manuscript describing this method and its properties is in preparation.

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 MS2 of TAG(54:6) [M+H]+ 4.2 Contour plot (MZmine output) 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: Acetonitrile B: Acetone/chloroform (85/15). A may be replaced bij MeOH, which leads to better ionisation efficiency but somewhat decreased chromatographic quality. In some countries, chloroform may not be allowed as mobile phase constituent (carcinogenic). It can be omitted, which leads to slightly increased retention times.
• Gradient (including re-equilibration for subsequent run):
• Indication of operating pressure: around 100 bar

Time [min]	Flow [ml/min]	% Solvent A	% Solvent B
0	1.0	100	0
15.0	1.0	0	100
35.0	1.0	0	100
36.0	1.0	100	0
40.0	1.0	100	0

Column

• Lichrospher RP18e (endcapped) 250x4.6 mm (Merck)
  
• Temperature: ambient

Autosampler

• Type: HTC PAL
  
• Injection volume: 10 µl
  
• Wash solvent: 2-propanol
  

Mass spectrometer

• Type: 4000 QTrap or other
• Ionization mode: APCI positive ionisation
  
• Ionization current: typically 2 µA, may be optimized
  
• Source temperature(s): 450^oC
• Collision gas: UHP nitrogen (5.0)
  
• Collision gas pressure: medium
• Operating mode: Enhanced Mass Spectrometry (full scans using linear ion trap)
  
• MS/MS-conditions: suitable information dependent acquisition (IDA) settings to record product spectra are: DP 110V; CE 30V; CES 10V; m/z range: 200-1050; IDA tresh: 1e6; Fill time 5 ms; scan speed 4000 amu/s (sum 2 spectra).
  

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. Peak recognition, integration and alignment can be performed in XCMS using this basic script (in R language)

library(xcms)

# get the files

wpath <- "/media/WD Passport/Phago/ExpX"
setwd(wpath)
CDFiles <- list.files(wpath,pattern="(.*.mzXML$)",full.names = T,recursive = T)
CDFiles

xraw <- xcmsRaw(CDFiles[])
xset <- xcmsSet(files=CDFiles[], profmethod = "binlinbase", method="matchedFilter", max = 25, fwhm=5,sigma=10, snthresh = 6, step = 0.28, steps = 1, mzdiff = 0.6, sleep=0.01)
xset <- group(xset, max= 10, bw=5, minfrac=0.5)

# Do retetiontime correction and plot
xset2 <- retcor(xset, method="loess", family = "gaussian", plottype = "mdevden",span=.41)

#re-group results, fill missed peaks, and store peaklist in variable for further processing
xset2 <- group(xset2, max= 10, bw=3, minfrac=0.5)
xset3 <- fillPeaks(xset2)
peaks <- xset3@peaks

Isotope correction

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

Sample data

MS2 of TAG(54:6) [M+H]⁺

Contour plot (MZmine output)

Reference

• Manuscript in preparation

Please add categories as below for:

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