Characterization of alkylacyl, alk‐1‐enylacyl and lyso subclasses of glycerophosphocholine by tandem quadrupole mass spectrometry with electrospray ionization

Abstract

Positive ion tandem quadrupole mass spectrometric methods for structural characterization of the subclasses of sn-glycero-3-phosphocholine (PC), including alkylacyl- and alk-1-enylacylphosphocholine and lysophosphatidylcholine (LPC), are described. Following collisionally activated dissociation, the [M + Li](+) ions generated by electrospray ionization yield abundant informative fragment ions that permit structural determination, and distinction of regioisomers among lysophosphatidylcholine can be easily achieved. In contrast, structurally informative ions arising from [M + H](+) or [M + Na](+) ions are less prominent. The most abundant ion observed in the product-ion spectra of the [M + Li](+) ions of plasmenyl- and plasmanyl-PC and of LPC arises from loss of N(CH(3))(3) ([M + Li - 59](+)). This feature permits their distinction from a product-ion spectrum arising from a diacylphosphatidylcholine, in which the [M + Li - 183](+) ion reflecting loss of phosphocholine is the most prominent. Examples for identification of various subclasses of PC in biological extracts by tandem mass spectrometry applying various constant neutral loss scannings are also shown.

Algorithm for processing raw mass spectrometric data to identify and quantitate complex lipid molecular species in mixtures by data-dependent scanning and fragment ion database searching

“…First, peak list files of both MS and MS/MS spectra are loaded into memory and undergo data pretreatment that includes smoothing (5-point Triangular Smooth)/noise filtering. MS/MS data are searched against a fragment ion database from a library of reference spectra for complex lipids that we have acquired [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28] or constructed from established fragmentation rules [29] to identify lipid molecular structures, and deduced chemical formulae are used to calculate theoretical isotope distributions subsequently used in the deisotoping algorithm. The m/z values of those ions in the MS spectra that do not correspond to a recorded MS/MS spectrum are searched against a lipid chemical formula database, and identified formulae are used to calculate theoretical isotope distributions for deisotoping, as described further below.…”

“…For positive ion analyses, LiOH (final concentration 5 mol/100 L) was added to sample solutions to permit formation of [M ϩ Li] ϩ adducts, which simplifies ESI/MS spectra by minimizing contributions of other metal ion adducts and facilitates informative fragmentation [11,19,20]. For negative ion analyses, NH 4 OH (final concentration 0.3%) was added to facilitate formation of [M Ϫ H] Ϫ ions.…”

“…Rules to explain fragmentation patterns of a wide variety of glycerolipids comprising hundreds of molecular species from different head-group classes have been established [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29], and we have constructed a lipid fragment ion database that includes species with fatty acid side-chain lengths of 10 -30 carbon atoms and 0 -6 double bonds. Searching the MS 2 spectra of different lipid species against this database permits deduction of their structures and chemical formulae.…”

“…Searching the MS 2 spectra of different lipid species against this database permits deduction of their structures and chemical formulae. Table 1 lists the fragment ions (Column 1) and their relative intensities for tandem spectra of Li ϩ adducts of glycerophosphocholine (GPC) lipids [11,19,20]. The ions…”

“…Unfractionated lipid extracts are directly infused and analyzed by ESI/MS with data-dependent switching to MS/MS mode. MS/MS data are searched against a library of reference spectra for complex lipids constructed from studies of standard glycerophospholipids of all major head-group classes [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28], which we used to establish fragmentation rules [29] that facilitate identification of glycerophospholipids in biological mixtures [30,31]. Structure assignment permits determination of an elemental formula, and a theoretical isotope profile is then calculated and used in a deisotoping algorithm.…”