# Ion Mobility-Mass Spectrometry Approach for the Comparison of Sheep and Goat Milk Lipidomes
## 4 Discussion  
In the negative ionization mode, PEs, SMs and PCs were the predominant lipid classes (Table 1). In particular, PE (18:1_18:1), PE (18:0_18:1), PE (16:0_18:1), SM d(34:1), SM d(41:1), SM d(42:1), PC (16:0_18:1) and PC (18:0_18:1) were the most abundant lipids in the two milk samples (Table 1). In this ionization mode, the percent levels of phospholipids were PE > SM > PS > PI. In particular, goat milk was characterized by higher percent levels of ether-phosphatidylethanolamine. The PC/PE ratio was 0.62 for goat milk, while for sheep milk, it was 0.42, thus reflecting the different sizes of fat globule membranes, which are smaller in goat milk (2.73 \(\pm\) 0.15 \(\upmu\)m and 3.63 \(\pm\) 0.27 \(\upmu\)m) [23]. Notably, sheep and goat milk were characterized by the unsaturated free fatty acid oleic acid (C18:1), while the saturated free fatty acid palmitic acid (C16:0) was found uniquely in sheep milk. On the other hand, in the positive ionization mode for both sheep and goat milk samples, TAG was the most abundant lipid class, but considering that the regiochemistry of FAs in the glycerol backbone influences TAGs' nutritional importance, we decided to separately study this lipid class. The positive ionization mode allowed us to annotate PC (16:0_18:1) and PC (18:0_18:1) as more abundant in sheep milk according to the literature ([PERSON] 2013). Furthermore, uniquely in sheep milk, we found several lysophosphatidylcholines, i.e., LPC (18:1), LPC (16:0) and LPC (18:0), which are hydrolysis products of PCs. Several studies have recently demonstrated the potential of dietary phosphatidylcholines as anti-inflammatory bioactive compounds in murine arthritis models [24]. Beneficial health effects are also provided by sphingomyelins (SMs) [25]. In our samples, SM levels were found to be higher in sheep milk (11.14 \(\pm\) 0.51%) when compared to goat milk (3.29 \(\pm\) 0.16%), and specifically, according to [PERSON] et al., SM d(34:1), SM d(36:1) and SM d(40:1) were the most abundant in sheep milk [26]. From the regiochemistry point of view, for SM, it is impossible to give the dominant lipid species due to the fact that isomers produce the same MS/MS spectra, particularly for SM d(34:1). A total of 18 potential lipids can be attributed to the same ion adducts and MS/MS fragments. Furthermore, it is known that dietary SMs may improve gut barrier function by preventing the translocation of inflammatory gut-bacteria-derived compounds and may protect against altered lipid metabolism, inflammation and gut dysbiosis [27]. Uniquely in sheep milk, we were able to annotate butyl carnitine C4:0 (ACar 4:0), and the percent levels found were 0.03 \(\pm\) 0.01%. Acylcarnitines are a group of ester substances formed by the combination of free carnitine and acyl-coenzyme A formed by fatty acids. [PERSON] et al. (2020) recently reported that liver levels of butyl carnitine were significantly increased when animals were under caloric restriction management, thus suggesting a favorable role in energy metabolism control while contributing to metabolic  
Figure 4: Triacylglycerlycerols’ relative abundance of saturated (**A**) and unsaturated (**B**) fatty acids at the sn-1/3 positions. flexibility under caloric restriction [28]. Furthermore, in the literature, carnitines were reported to be higher in sheep milk when compared with cow, horse, goat and human milk. Nevertheless, total carnitine did not show statistical differences between species, and the acid-soluble fraction of acylcarnitine was found to be higher in sheep milk [29]. Notably, in sheep and goat milk samples, we were able to annotate another bioactive lipid class corresponding to hexosyl non-hydroxy sphingosine ceramides, HexCer_NS d18:1/16:0 and HexCer_NS d18:1/23:0. The total percent levels of HexCer_NS found in sheep and goat milk were 0.15 \(\pm\) 0.01% and 0.11 \(\pm\) 0.01%, respectively. These ceramides serve as precursors of SMs, glycosphingolipid, sphingosine and FAs and are reported to reduce inflammation in a diet-induced obese mouse study [27].