Setup of Free Carotenoid Analysis Approach using State-of-the-Art Liquid Chromatography Coupled with High Definition Ion-Mobility qToF

Maxim Itkin, Life Science Core Facilities, Weizmann Institute of Science, Rehovot, Israel (maxim.itkin@weizmann.ac.il)
Sergey Malitsky, Life Science Core Facilities, Weizmann Institute of Science, Rehovot, Israel (sergey.malitsky@weizmann.ac.il)

Carotenoids belong to the largest (1200+ species) natural pigments group: yellow through orange to red. They are biosynthesized in all photosynthetic organisms and numerous non-photosynthetic bacteria, yeasts, and fungi. This nature’s widespread pigment group is essential in photosynthesis and photoprotection. Carotenoids are known to accumulate in animal species as xenochemicals. Biosynthesis of carotenoids conducts via Methylerythritol Pathway in Chloroplasts, resulting in C30-C50 molecules, which can be classified, based on their chemical composition, as either carotenes (hydrocarbon carotenoids) or xanthophylls (oxygen-containing carotenoids). Having an OH group, numerous xanthophylls can be subjected to mono- and diesterefication, glycosylation, epoxidation, and acetylation. The carotenoid cleavage oxygenases (CCDs) can catalyze the conversion of the carotenoids into apocaroteniods such as Vitamin A (11-cis retinol),  retinoic acid, and β-ionone (odor molecule).

In most biological carotenoid-related projects, the results were reported using Liquid chromatography coupled with a photo-diode array, Mass Spectrometer (LC-PDA, LC-MS), or both.

We used state-of-the-art Ion mobility High-Resolution Mass Spectrometer to detect and identify pigments and generate the pigment library utilizing different technologies such as a PDA detector, ion mobility, and Mass Spectrometer.

Using the current method, we tested different tissues, detecting 9 to 30 free carotenoids in mice feces, plants and alga. The suggested approach provides an excellent opportunity to analyze not just total carotenoids, which were done by previous analyses (hydrolysis), but to show the current state of the biosystem by analyzing the relative abundance of free carotenoids.

Using Ion Mobility measurement is an additional powerful dimension, complementary to MS and UV provides an enhanced understanding of biological systems.

---

Short Biography of Presenting Author

Upon receiving my BSc in Biology in BGU and MSc in Plant sciences in TAU under supervision of Prof. Isaac Barash , I joined at 2004 the lab of Prof. Asaph Aharoni at WIS and became his first PhD student. Using bioengineering tools, I worked on fruit ripening and on elucidation of steroidal glycoalkaloid pathway in Solanaceae. During postdoc at Asaph’s lab, I expanded the scope of my expertise to metabolomics, and was fascinated by this field of study. In a course of my postdoc and later my Researcher position at the lab of Dr. Ari Schaffer, Volcani, (2011-2017), I investigated fruit acidity and, elucidated the biosynthetic pathway of natural non-caloric sweetener in cucurbit fruit.  I also developed procedures to investigate cannabinoids in Cannabis sativa. During this period, in addition to the use of bioengineering, I broadened my knowledge in metabolomics and acquired an expertise in LC-MS equipment.

In 2017, I joined Metabolic Profiling unit (MPU) as senior intern, and participated in setting up the unit as part of the WIS LSCF. Since then i am on Staff Scientists track. MPU was established to provide comprehensive cutting-edge solutions for small molecule profiling to WIS scientists. Up to date more than fifty groups from WIS and more than 30 groups in Academia benefited from our service.