Quantitative analysis of biological membrane lipids at the low picomole level by nano-electrospray ionization tandem mass spectrometry

Abstract

Nano-electrospray tandem mass spectrometry allows qualitative and quantitative analysis of complex membrane lipid mixtures at the subpicomole level. We have exploited this technique to selectively detect individual classes of phospholipids from unprocessed total cellular lipid extracts by either precursor ion or neutral loss scanning. This way phosphatidylcholine, sphingomyelin, phosphatidylinositol and -phosphates, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidic acid, and their plasmalogen analogues can be detected. The optimized ionization and fragmentation conditions described together with the principle of internal standardization by nonnatural analogues allow the rapid and quantitative determination of membrane lipid compositions down to sample amounts of 1000 cells.

Figures

Figure 1

ESI-MS from an unprocessed total lipid extract from CHO cells. (a) Negative ion mass spectrum. (b) Positive ion mass spectrum. The boxed numbers indicate full-scale intensity in arbitrary units.

Figure 2

Product ion spectra of negative ions of phospholipids. (a) Product ion spectrum of the [M-H]− ion of PI-(36:2) at 861.5 in Fig. 1a (collision offset, +40 V). (b) Product ion spectrum of the [M-H]− ion of PS-(36:1) at m/z 788.6 in Fig. 1a (collision offset, +25 V).

Figure 3

Collision offset plots for the loss of a phospholipid head group as ionic fragment [example: loss of inositolphosphate with m/z 241 from PI-(36:2)] and for the loss of a phospholipid head group as neutral fragment [example: loss of serine-H2O from PS-(36:1)].

Figure 6

Summary of the phospholipid class-specific scan modes available in positive and negative ion ESI-MS.

Figure 4

Specific detection of phospholipid classes in an unprocessed lipid extract from CHO cells by negative ion ESI tandem MS. (a) Detection of PI by precursor ion scanning for m/z 241 (offset, +45 V). (b) Detection of PS by scanning for neutral loss of 87 D (collision cell offset, +25 V). (c) Detection of PE by precursor ion scanning for m/z 196 (collision cell offset, +40 V). (d) Detection of SM by combined application of skimmer-CID and parent ion scanning for m/z 168 (sCID offset, +65 V; collision cell offset, +40 V).

Figure 5

Specific detection of phospholipid classes in an unprocessed total lipid extract of CHO cells by positive ion ESI-MS/MS. (a) Detection of [M+H]+ ions of PC and SM by precursor scanning for m/z 184 (collision cell offset, −35 V). (b) Detection of [M+H]+ ions of PE by scanning for neutral loss of 141 D (collision cell offset, −25 V). (c) Detection of [M+H]+ ions of PS by scanning for neutral loss of 185 D (collision cell offset, −22 V).

Figure 7

An unprocessed total lipid extract of 5000 CHO cells containing equimolar amounts of PC-(24:0), -(28:0), -(40:0), and -(44:0) was analyzed by parent ion scanning for m/z 184. (a) Uncorrected ion intensities. The signal intensities of the internal standards were used for generation of the calibration plot insert. (b) Corrected ion intensities of the PC signals so that the monoisotopic signals represent the true molar abundances of the corresponding PC molecular species.