3.2.1 Main Lipid Classes in Mammalian Samples
Direct infusion MS typically analyzes the crude lipid extracts. Therefore, it is impor- tant to understand which lipid classes and species are expected within the respec- tive sample material. In the following, we will focus on the lipid compositions of mammalian samples. Today, lipids are categorized according to the LIPID MAPS nomenclature introduced in 2005 by Fahy et al. [30]. This system defines eight lipid categories: fatty acyls (FAs), glycerolipids (GLs), glycerophospholipids (GPs), sphin- golipids (SPs), sterol lipids (STs), prenol lipids (PRs), saccharolipids (SLs), and pol- yketides (PKs). Each of the categories is subdivided into lipid classes, e.g.
phosphatidylcholine (PC) in the GP category.
The typical membranes of mammalian cells contain about two‐thirds of glycer- ophospholipid, about 25% cholesterol, and 10% sphingolipid molecules (Figure 3.1) [31]. The main glycerophospholipid is commonly PC, followed by phosphatidyle- thanolamine (PE), phosphatidylserine (PS), and phosphatidylinositol (PI) [32]. The major sphingolipid class is sphingomyelin (SM), which could contribute at about 10% of the membrane lipids in human blood cells [32]. Cholesterol has a high impact on membrane fluidity [33] and therefore shows a substantial variation among cell types with up to 50% for red blood cells [32].
Besides membrane lipids, mammalian cells contain a variable content of storage lipids deposited in lipid droplets. The most prominent lipid classes in lipid droplets are triacylglycerols (TGs) and cholesteryl ester (CE) (Figure 3.2). In adipocytes, TGs represent the main lipid class [34], as well as in adipose tissue, with the TG content up to 99% [35]. In blood plasma from (fasted) healthy human subjects, the content
3.2 Complexity CofCrul xmxu eiCrait 43
of CE is higher than that of TG. The transporters of blood lipids, the lipoproteins, comprise TG‐rich particles (chylomicrons and very low‐density lipoprotein [VLDL]) as well as CE‐rich lipoproteins (LDL and high‐density lipoprotein [HDL]) [36]. In contrast to mammalian cells or tissues, blood plasma contains a high fraction of lysophosphatidylcholine (LPC) in HDL and bound to albumin. These examples demonstrate that the lipid composition may vary substantially between different sample materials and therefore may require adaptation of the analysis strategy.
3.2.2 Bond Types as Structural Features
Their polar head groups differentiate glycerophospholipid classes (Figure 3.3), which mainly consist of diacyl species. However, besides an ester bond at the
Glycerophospholipids Sphingolipids Sterols
Polar head group
Glycerol O
HO
HO CH3
CH3 O
O
N HO
O N OO
O O O
Sphingosine
Fatty acid
Carbo- hydrate
PC, PE, PS, PI Sphingomyelin Glycosphingolipids Cholesterol
65 mol% 10 mol% 25 mol%
H
P P
Figure 3.1 Lipid composition of mammalian cell membranes. The main lipid categories of mammalian cells are depicted. The major glycerophospholipid classes are
phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylinositol (PI). Source: Adapted from van Meer and de Kroon [31].
Triacylglycerols
Cholesteryl ester
Fatty acid
Glycerol
Cholesterol O
O O
O
CH3 H3C
O
O
O
O
Figure 3.2 Main lipid classes stored in lipid droplets of mammalian cells.
Bond type
R1
R1
R1 O
O
O
O O
O
O
Typical in mammalians:
Number of carbon atoms 14 – 22 Number of double bonds 0 – 6
O O
O
O
O O O O O
H
X
N
Polar head group
P –O H R1
R2
O O
O O O
O
O–
O
O O Ester
phosphatidyl-
Phosphatidic acid
-ethanolamine
-choline
-glycerol
-serine
-inositol Ether
plasmanyl- Vinylether plasmenyl-
Nonpolar chains
+ N+ N+
Figure 3.3 Glycerophospholipid structures in mammalian cells. The lipid class of glycerophospholipid is defined by its polar head group. Subclasses are defined by the type of bond at the sn1-position. The nonpolar chains vary in their number of carbon atoms and double bonds (DBs).
sn1‐position, there also occur species with an ether bond and ethers with a double bond (DB) at position 1 termed as plasmanyl‐ or alkyl and plasmenyl‐ or plasmalo- gens, respectively. The occurrence of alkyl and plasmalogens is characteristic for cells or tissues. In particular, heart and brain [37, 38] contain high fractions of plas- malogens. Interestingly, heart contains a high fraction of PC plasmalogen, whereas brain almost exclusively contains PE plasmalogens. Typically, PE and PC are the only glycerophospolipid classes that contain relevant fractions of alkyl species, and the diacyl fraction is higher than the plasmalogen fraction. For example, granulo- cytes are the only human blood cells that contain a larger fraction of PE plasmalo- gens (PE P) than diacyl‐PE [32], and in human plasma, the HDL fraction contains the highest amount of PE P compared to other lipoprotein fractions [39].
3.2.3 Fatty Acids as the Major Building Blocks
Lipidomic analysis provides information not only about lipid class but also about lipid species, i.e. their fatty acyls/alkyl linked to the core structure (see Section 3.3.1 for annotation of lipids). In order to set up the analysis and evaluate the data appro- priately, it is necessary to have information about this elemental building block of the majority of lipid classes.
A convenient method to obtain fatty acid profiles is gas chromatography (GC) coupled to either flame ionization detection (FID) or GC‐MS, which typically applies derivatization to fatty acid methyl ester (FAME) [40]. The most abundant fatty acids in mammalian samples are palmitic acid with 16 carbon atoms and no DB (FA 16:0) and FAs with 18 carbon atoms, i.e. saturated stearic acid,
3.2 Complexity CofCrul xmxu eiCrait 45
monounsaturated oleic acid (cis DB at carbon 9; FA 18:1n‐9), and linoleic acid (FA 18:2) (Figure 3.4). The main FAs with more than two DBs are arachidonic acid (FA 20:4n‐6) and docosahexanoic acid (DHA, FA 22:6n‐3).
3.2.4 Lipid Species and Double-Bond Series
The FAs available in a biological system, i.e. a cell, are used to synthesize complex lipids. However, FAs are not incorporated into distinct lipid classes randomly but instead highly regulated in order to create a specific composition of membranes required for their respective biological function [2, 9, 10]. As shown in Figure 3.4 in
35
25
15
5 0 30
20
% mol of total FA 10
1.2
0.8
0.2 0.0 1.0
0.4 0.6
% mol of total FA
FA 14:0 FA 16:0 FA 18:0
FA 16:1 (n-7)
FA 18:1 (n-9)
FA 18:1 (n-7)
FA 18:2 (n-6)
FA 20:4 (n-6)
FA 22:6 (n-3)
FA 15:0 FA 17:0 FA 20:0 FA 22:0 FA 24:0
FA 18:3 (n-6)
FA 18:3 (n-3)
FA 20:1 (n-9)
FA 20:3 (n-6)
FA 22:1 (n-9)
FA 20:5 (n-3)
FA 22:4 (n-6)
FA 24:1 (n-9) Human plasma (low) Human plasma (high) Murine liver
Figure 3.4 Fatty acid profiles. The total fatty acid compositions were analyzed by GC–MS of human plasma with low and high total lipid content (n = 6 replicates. Source: Adapted from Ecker et al. [40] and murine liver (n = 6; unpublished data).
20
10
0 15
5
% of total PE
White Brite Brown
PE 32:2PE 32:1PE 34:2PE 34:1PE 36:5PE 36:4PE 36:3PE 36:2PE 36:1PE 38:6PE 38:5PE 38:4PE 38:3PE 38:2PE 40:7PE 40:6PE 40:5PE 40:4
Figure 3.5 Lipid species of PE. PE species profiles of murine white, brite, and brown adipocytes analyzed by FIA–ESI–MS/MS. Preadipocytes, isolated from inguinal (white), epididymal white (brite), and intrascapular (brown) adipose tissue, were lefvxvC differentiated to adipocytes (n = 3). Source: Schweizer et al. [34]/PLOS/CC BY-4.0.
mammals, FAs are dominated by even numbers of carbons, and the number of DBs increases with their length up to six DBs in DHA. The abundance of FAs represents the basis for species profiles observed for complex lipid classes. A basic lipidomic analysis provides data at the species level, i.e. sum of both acyl/alkyl chains (see details of annotation in 3.3.1). Thus, glycerophospholipids typically comprise a series of species with an increasing number of DBs for the same number of carbon atoms. For example, PE in adipocyte samples contains PE species with 38 carbons and two to six DBs (Figure 3.5). Advanced lipidomics analysis are eligible to differ- entiate acyl combinations, e.g. PE 18:0_20:4, PE 18:1_20:4, and PE 16:0_22:6.