# Ion Mobility-Mass Spectrometry Approach for the Comparison of Sheep and Goat Milk Lipidomes
## 1 Introduction  
Sheep milk is gaining worldwide interest for its nutritional and functional characteristics. The Italian sheep dairy industry accounts for 6.31 million head of ewes, mainly located in Sardinia (21.8%) and characterized by the breeding of razza SARa animals [1]. Sheep and goats are managed in semi-extensive systems, in which extensive use consists of artificial or natural pastures representing the first source of nutrients and constituting roughly 80% of the dry matter ingested annually by the animals. The latter factor influences the acid profile of the milk fat, which plays a fundamental role in defining the technological and nutritional characteristics of milk. In particular, sheep milk is characterized by a higher lipid content (6-7%) when compared to goat milk (4-5%) [2]. The lipid profile of a small ruminant's milk consists of triglycerides (TAGs), together with diglycerides (DAGs), phospholipids, ceramides, cholesterol, squalene, lanosterol and free fatty acids [3]. Milk fatty acids, representing 95% of the triglyceride structure, originate from the diet, both from rumen microbial activity, after which they are transferred to secretory cells via the blood and lymph, and from de novo synthesis in the endoplasmic reticulum of secretory cells. The milk lipid fraction is organized into fat globules with cores of neutral lipids (TAGs and cholesterol) and membranes with three or more layers. The milk fat globule membrane (MFGM) produced by secretory cells in the mammary gland consists of lipids, proteins and cholesterol. In addition to the major glycerophospholipids, i.e., phosphatidylcholines (PCs) and phosphatidylethanolamine (PEs), MFGM contains sphingomyelins (SMs), glucosyl-(HexCer) lactoseylceramides and gangliosides [4; 5]. The complexity of the milk composition also makes its in-depth study extremely difficult. Furthermore, milk fat contains over 400 individual fatty acids and their isomers,whose composition is influenced by various factors, such as the diet [6; 7], lactation stage and animal conditions [8]. These reasons make the open profile study of the milk lipidome very difficult. Similar to other -omics sciences, lipidomics is commonly used in profiling lipids present in cells, tissues and body fluids. Nowadays, this holistic technique represents a relatively new, stimulating and complex research area in the field of human nutrition, encompassing metabolic, nutritional, physiological, epidemiological and clinical aspects [9]. Lipidomics was recently proposed as a method for the profiling of goat milk [10; 11], the digestibility evaluation of sheep and goat milk [12; 13] and the identification of the geographical origin and lactation stage of goat milk [14]. Recently, [PERSON] et al. reported a phospholipidomics approach for measuring phospholipids in sheep milk [15]. Nevertheless, despite the use of high-throughput mass spectrometers, it is difficult to elucidate important structural features of lipids, such as constitutional isomers' identification, carbon-carbon double-bond positions and the presence of acyl-chain branching. However, ion mobility coupled with time-of-flight mass spectrometry may be used to separate ions in the gas phase based on their dimensions and shapes. Mixtures of ions can be separated based on changes in mobilities through a gas phase and then analyzed by a mass spectrometer measuring mass-to-charge (m/z) ratios. The great advantage offered by this analytical system is the acquisition of a further layer of separation compared to LC alone, as well as the use of an extra physiochemical property, i.e., collision cross-section (CCS, A), which, in combination with the retention time and m/z value, can help in the lipid identification process [16]. Over the years, ion mobility spectrometry (IMS) has been increasingly central to the field of chemistry for the analysis of food composition, including nutrient and bioactive components, as well as for ensuring food safety, preventing food fraud and optimizing technological processes [17].