# Ion Mobility-Mass Spectrometry Approach for the Comparison of Sheep and Goat Milk Lipidomes
## 3 Results
### TAG Regiochemistry and FA at sn-2 Position  
Considering the nutritional importance of FA positions in the glycerol backbone of TAGs [20], we investigated and compared the regiochemistry of lipid species found in goat and sheep milk samples. With the aim to study the three isomeric forms of TAGs and the position of each FA in the glycerol backbone at sn-1, sn-2 and sn-3, we proceeded to perform an in-depth study of TAG mass spectra. An in-depth study of high-resolution MS/MS spectra of chromatography resolved TAG molecules allows an easy understanding of the FA composition [11]. It is commonly assumed that for collision-induced dissociation-based MS/MS acquisition, compared to sn-1/sn-3 FA dissociation, the sn-2 position requires more energy [21]. Thus, DAG+ ions generated after the loss of the sn-2 FA are supposed to be of lower abundance [22]. This procedure was used to annotate the sn-2 FAs of TAGs in both milk samples in this study. The results of a structural analysis of TAGs with their mass characteristics and CCS values are reported in Table S3. For the same TAGs, we can notice the inversion of the FA position in sheep and goat milk samples, e.g., the TAG (36:1) at a retention time of 10.802 min and m/z 654.5699, where butyric acid (FA 4:0) is positioned at sn-2 in sheep milk but at sn-3/1 in goat milk samples. As theoretically expected, the different positions of butyric acid provided two different CCS values, 271.49 vs. 272.57 A2. Furthermore, by analyzing the relative abundance of FAs at the TAG sn-2 position, we were able to report their distribution, along with their chain length (Figure 2) and saturation degree (Figure 3).  
Figure 1: Ion mobility capability to discriminate isomers.  
Considering short-chain FAs with 4 to 8 carbon atoms, medium-chain FAs with 10 to 15 carbon atoms and long-chain FAs with 15 to 22 carbon atoms, as can be seen in Figure 2, TAGs in sheep milk are characterized by having more sn-2 FAs with a medium chain length. Overall, both sheep and goat milk samples showed higher levels of saturated FAs at sn-2 (Figure 2). However, we can highlight that goat milk is characterized by more saturated fatty acids, while sheep milk contains higher levels of butyric acid (C4:0) and stearic acid (C18:0) at the sn-2 position (Figure 3A). At the same time, looking at FAs at the sn-1/3 positions (Figure 4A,B), butyric acid (C4:0) was more abundant in sheep milk, while stearic acid (C18:0) was higher in goat milk.  
Both milk samples were characterized by having predominant levels of oleic acid (C18:1) at the sn-2 position (Figure 3B). Sheep milk samples were characterized by higher levels of linoleic acid (C18:2), palmitoleic acid (C16:1) and gadoleic acid (C20:1) when compared to goat milk. Instead, higher levels of heptadecenoic acid (C17:1) and linoleic acid (C18:3) characterized goat milk.  
Figure 3: Triacylglycerlycerols’ relative abundance of saturated (**A**) and unsaturated (**B**) fatty acids at the sn-2 position.  
Figure 2: Average abundance values of the short, medium and long fatty acids at the sn-2 position.