Identification of Lipids by Mass Spectrometry

Identification of the individual glycerophospholipids present in the total lipid extracts (both basal and AIG-stimulated) was accomplished by tandem mass spectrometry (ESI-MS/MS). Resolution and characterization of glycerophospholipids in an unprocessed total lipid extract are based on the predisposition of each lipid class to acquire positive or negative charges under the source energy. A single molecular ion is present with a mass-to-charge ratio (m/z) that refers to the monoisotopic molecular weight. Collision-induced dissociation (CID) of the peaks of interest yielded fragmentation patterns, which were used to unambiguously identify the lipid(s) present at a particular m/z value (Fig. 1 provides an illustration of this procedure) (11-23).

Fig. 1. Fragmentation and identification of lipid species.  Individual lipid species from the total cell extract were isolated and fragmented using ESI-MS/MS. Positive mode analysis was utilized in the identification of three phospholipid classes. Negative mode analysis was used to assign five classes and to determine fatty acid compositions

For tandem mass spectrometry, both positive and negative mode ionization were utilized. Traditionally, degree of structural information obtained as a result of this analysis varies by the type of instrumentation used. In negative ionization mode, triple quadrupole instruments tend to yield sn-1 and sn-2 fatty acid residue fragments, whereas ion traps form more lyso-lipid fragments (19). Positive ion ESI-MS/MS spectra from ion trap instruments are more likely to create lyso-PC fragmentation products, which reveal the fatty acid composition of the lipid. However, under our triple quadrupole MS experimental conditions, only glycerophospholipid head group information was routinely obtainable from positive mode fragmentation.

Three lipid classes were analyzed in positive ESI mode: phosphatidylcholines (PCs), phosphatidylethanolamines (PEs), and sphingomyelins (SMs). The choline containing species, PCs and SMs, both show a characteristic m/z 184 phosphocholine head group peak, as well as an [M+H-59]⁺ peak corresponding to the neutral loss of (CH₃)₃N. In addition to the diacyl PC compounds, a large number of plasmanyl and plasmenyl phosphocholines were also identified. All together, over 100 choline containing lipids were identified. Fragmentation of phosphatidylethanolamines exclusively yielded one peak, an [M+H-141]⁺ ion from the neutral loss of the phosphoethanolamine head group. Again, plasmanyl and plasmenyl lipids were a large proportion of the over 40 PE species identified.

Five lipid classes were detected in negative ESI mode: phosphatidylinositiols (PIs), phosphatidylserines (PSs), phosphatidylglycerols (PGs), glycerophosphatidic acids (PAs), and PEs. Negative mode fragmentation of these species yielded a wealth of structural information. In each case, head group fragmentation, lyso-lipid formation, and fatty acid fragments aided in the lipid identification process. Phosphatidylinositol fragmentation generated a wide variety of product ("daughter") ions. Four types of lysophosphatidic acid and lysophosphatidylinositols, phosphatidic acid, and five characteristic head group fragments were used in identifying the 27 observed PI and lyso-PI species. In a similar fashion, 33 distinct species of PS and lyso-PS compounds were identified from their phosphatidic (PA) and lysophosphatidic acid (LPA) fragments. A negative mode fragmentation library of the phosphatidylserines is provided as an example in Table 1. Fragmentation tables for the remaining phospholipid classes (for both fragmentation modes) can be viewed from the Supplemental Tables page. Phosphatidylcholine compounds were not identified during the routine negative mode scans. However, it was found that these compounds were detectable after the addition of ammonium acetate (15, 18, and 23). Two important categories of signaling lipids were not included in this analysis. Diacylglycerol (DAG) was not routinely detected under the optimized conditions for triple quadrupole MS described here; however, DAG species can be detected using a Fourier transform ion cyclotron resonance (FT-ICR) instrument (16). We have also found that DAG can be detected using a triple quadrupole MS but requires formation of a sodium adduct. In the current study, well over 200 glycerophospholipids have been detected and unambiguously identified in WEHI-231 total lipid extracts. A tabular listing of all identified lipids for both positive and negative MS modes is shown in Table 2.

Table 1. Fragmentation table for phosphatidylserines. Using negative mode ESI-MS/MS, 33 PS and lyso-PS species were identified. The numbers in parentheses following fragment ions (FA:D) refer to the total number of fatty acid carbons (FA) and fatty acid carbon-carbon double bonds (D). GP= glycerophosphate.
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Table 2. Library of identified glycerophospholipid species. All of the glycerophospholipids identified by ESI-MS/MS fragmentation in positive and negative modes are summarized. PC and PE compounds with lower case e or p refer to plasmanyl and plasmenyl (alkyl ether and plasmalogen) subspecies, respectively. When plasmanyl and plasmenyl PE or PC species are separated by and/or (a/o), this indicates that one or both species were detected at that m/z.

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