# Ion Mobility-Mass Spectrometry Approach for the Comparison of Sheep and Goat Milk Lipidomes
## 3 Results  
### Milk Lipidome Profiles  
In this work, we investigated the lipid profiles of sheep and goat milk using an IM-QTOF-MS analytical platform. For the analysis, five different iterative injections of bulk milk samples were performed to evaluate the minimum number of runs with the maximumnumber of lipid species detected. Four chromatographic runs were enough to cover the maximum number of milk lipid species (Figure S1). The percent lipid class compositions for sheep and goat milk samples acquired in the ESI+/\(-\) mode are reported in Table 1.  
When compared to sheep milk, the percentage levels of TAGs (see Table 1) were higher in goat samples (84.30 \(\pm\) 4.21% vs. 65.25 \(\pm\) 3.26%). On the other hand, sheep milk samples showed higher levels of phosphatidylcholines (PCs) and sphingomyelins (SMs), when compared to goat milk. Notably, in the sheep milk samples, we found LysoPCs (0.30 \(\pm\) 0.01%), non-hydroxy fatty acid-sphingosine ceramides (Cer_NS) (0.06 \(\pm\) 0.01%) and acylcarnitines (ACar) (0.03 \(\pm\) 0.01%). In the ESI mode, goat milk samples showed higher levels of phosphatidylcholineethanolamines (PEs) (32.76 \(\pm\) 1.64% vs. 29.99 \(\pm\) 1.50%) (Table 1). While levels of SMs were comparable in the two groups, the milk levels of PCs, phosphatidylserines (PSs), ether-PEs and phosphatidylcholineits (PIs) were found to be higher in the goat milk samples. Importantly, non-esterified fatty acid levels were more abundant in the sheep milk samples (18.32 \(\pm\) 0.92%) when compared to goat milk samples (1.33 \(\pm\) 0.07%). Among fatty acids annotated in sheep samples, we found palmitic acid and stearic acid to be predominant. To enhance the confidence in the lipid annotations procedure, we decided to characterize the milk lipidomes by applying an ion mobility-mass spectrometry experiment to calculate CCS values, which, together with retention time and mass spectrometry characteristics, support the lipid annotation process. In Figure 1, we report an example of the usefulness of ion mobility spectrometry to discriminate TAG isomers. \begin{table}
\begin{tabular}{c c c} \hline
**Lipid Class** & \multicolumn{2}{c}{**\% Composition \(\pm\) SD**} \\ \hline
**Positive Ionization** & **Sheep** & **Goat** \\ \hline TAG & 65.25 \(\pm\) 3.26 & 84.30 \(\pm\) 4.21 \\ PC & 14.58 \(\pm\) 0.73 & 8.27 \(\pm\) 0.41 \\ SM & 11.14 \(\pm\) 0.51 & 3.29 \(\pm\) 0.16 \\ PE & 6.75 \(\pm\) 0.73 & 3.10 \(\pm\) 0.15 \\ PS & 1.71 \(\pm\) 0.08 & 0.89 \(\pm\) 0.04 \\ LPC & 0.30 \(\pm\) 0.01 & n.d. \\ HexCer\_NS & 0.15 \(\pm\) 0.01 & 0.11 \(\pm\) 0.01 \\ ACar & 0.03 \(\pm\) 0.01 & n.d. \\ DAG & 0.03 \(\pm\) 0.01 & 0.05 \(\pm\) 0.01 \\ Cer\_NS & 0.06 \(\pm\) 0.01 & n.d. \\ \hline
**Negative Ionization** & **Sheep** & **Goat** \\ \hline PE & 29.99 \(\pm\) 1.50 & 32.76 \(\pm\) 1.64 \\ SM & 19.28 \(\pm\) 0.96 & 19.84 \(\pm\) 0.99 \\ FA & 18.32 \(\pm\) 0.92 & 1.33 \(\pm\) 0.07 \\ PC & 12.86 \(\pm\) 0.64 & 20.61 \(\pm\) 1.03 \\ PS & 8.85 \(\pm\) 0.44 & 11.95 \(\pm\) 0.60 \\ PI & 4.68 \(\pm\) 0.23 & 6.41 \(\pm\) 0.32 \\ Cer\_NS & 2.72 \(\pm\) 0.14 & n.d. \\ Ether PE & 2.32 \(\pm\) 0.12 & 7.10 \(\pm\) 0.35 \\ LPE & 0.96 \(\pm\) 0.05 & n.d. \\ \hline n.d.: \(<\)0.01 mg/g fat. & & \\ \end{tabular}
\end{table} 
Table 1: Percent compositions based on relative abundance of lipid classes measured in sheep and goat milk samples. In Table S1, we report the mass spectrometry characteristics of each lipid, along with their sum composition and collision cross-section values (CCS, A2) measured in the ESI+ mode. With this approach, we were able to annotate a total of 26 PCs, 14 PEs, 8 PSs and 17 SMs. In this table, TAGs are not reported but are discussed successively. In the ESI mode, we annotated the following lipids: free FA, PCs, PEs, PSs, PIs and SMs (Table S2). Interestingly, plasmalogens, membrane glycerophospholipids containing a fatty alcohol with a vinyl-ether bond at the sn-1 position, were also found in the form of ether-linked phosphatidylethanolamine (ether-PE).