Lipidomics
Amity Lipidomics Research Facility houses a triple stage quadrupole/linear ion trap mass spectrometer, Q-Trap 4500 (SCIEX), equipped with ion sources (turbo V) Electrospray ionization (ESI), and Atmospheric pressure chemical ionization (APCI), autosampler, column oven with HPLC (Exion LC-AC) system. This instrument provides necessary resolution and sensitivity for a broad range of qualitative and quantitative lipid and metabolite analyses required for exhaustive characterization of lipid signaling molecules and metabolic pathways. We utilize ESI technique for generation of singly charged molecular ions with excellent concentration sensitivity and different scanning modes like precursor ion, neutral loss or MS/MS scan for detection of all species within a lipid/metabolite class. Separation and scanning of sample extracts in various scan modes give a complete and comprehensive profile of all molecular species in different lipid classes. Identification and quantification of unknown and known lipids/metabolites in biological samples such as cell extracts, tissue preparations, plasma, urine and saliva samples by using Data dependent (DDA) and data independent (DIA) analysis are done.
ALRF focuses on targeted and non-targeted analysis of lipids/metabolites. Depending on the lipid class and scientific question, analytical methods with (LC-MS/MS) or without (shotgun lipidomics) are used. LipidView and MultiQuant software are used for data processing.
Technology used for Lipid Profiling
We use a triple quad Q-TRAP 4500 (SCIEX) mass spectrometer with electrospray ionization source (ESI). Q-Trap 4500 functions like a standard triple quad and also doubles as a linear ion trap (LIT), a proprietary technology that can perform a multitude of additional workflows and is coupled to a HPLC system, Exion LC-AC (SCIEX). Direct infusions as well as LC-MS/MS methods are used for targeted and untargeted analysis of lipids/metabolites. ESI ionizes the lipid molecules, and forms protonated molecular ions ([M+H]+) and adduct molecular ions ([M+NH4]+, [M+Na]+, and [M+K]+) in ESI positive mode (ESI+). Deprotonated molecular anions ([M-H]-) and adduct anions ([M+Cl]- and [M+acetate]-) are formed ESI negative scan mode (ESI-). Fragmentation is done by collision activation and collision-induced dissociation (CID) by applying collision induced gas like (N2/Ar/He).
For targeted analysis we use Multiple Reactions Monitoring (MRM) scan mode or advanced scheduled MRM scan mode.
We use Information Dependent Acquisition (IDA) method for untargeted lipid molecules, where Precursor ion (Prec) and Neutral loss (NL) scans are performed that are specific to particular lipid classes; some classes are analyzed with Precursor scans, and others are analyzed with Neutral loss scans. The updated IDA method is capable for the profiling the lipids/metabolites in a single injection using additional enhanced product ion scan mode, which generate MS/MS spectrum that is used for library search or LipidView search for further confirmation.
Proteomics
ALRF provides service for targeted Proteomics analysis with focus on molecular weight confirmation, identification and their quantitation in complex biological samples. For Proteomics data analysis online Mascot and ProteinPilot 5.0 software are used for confirmation and structure identification of proteins molecules.
Application Support
- Sample preparation
- Mass spectrometric analysis
- Data processing