Untargeted LC-MS Based Shotgun Lipidomics

TM in Lab

Lipidomics approaches can provide valuable new insights into the role of lipid molecular species in human health and disease and may identify potential lipidomic biomarkers that can be developed for diagnostic/prognostic and therapeutic use. Our strategy is to use a ABSCIEX 5600 triple TOF MS that combines high-sensitivity detection, high resolution with the fast acquisition speeds, and stable mass accuracy over days of acquisition accompanied by RPUPLC methodology. Identification of lipids is accomplished by data-dependent MS/MS product ion information of human plasma lipid species in both positive and negative ionization modes. During the electrospray ionization, molecular ion adducts such as [M+H]+, [M+Na]+ and [M+NH4]+ or [M−H]−, and [M+CH3COO]− are formed in both positive and negative modes. Data-dependent MS/MS acquisition provides information on the nature of the head group and/or neutral loss of the head group from the molecular ion adducts. The information on fatty acids composition of the lipids is obtained in the negative mode.

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Please contact the Managing Director, Maureen Kachman, if you have questions not answered here, or to discuss details.

More background information about the Shotgun Lipidomics platform. Lipids are extracted from biological samples using a modified Bligh-Dyer method [1] using liquid-liquid extraction at room temperature after spiking with internal standards. Analysis of lipids is performed on reversed phase HPLC, followed by MS analysis that alternates between MS and data-dependent MS2 scans using dynamic exclusion in both positive and negative polarity and yields excellent separation of all classes of lipids. Quality Controls (QC) are prepared by pooling equal volumes of each sample, in addition to a well characterized plasma pools, and are injected at the beginning and end of each analysis and after every 10 sample injections, to provide a measurement of the system's stability and performance as well as reproducibility of the sample preparation method [2]. Lipids are identified using the LipidBlast [3] library (computer-generated tandem mass spectral library of 212,516 spectra covering 119,200 compounds from 26 lipid compound classes), by matching the product ions MS/MS data. The method allows us to measure >70% of lipids with an intensity RSD value below 20% belonging to eight different lipid classes which includes phospholipids like lysophosphotidylchioline (lysoPC), lysophosphotidylchioline (PC), phosphotidylethanolamine (PE), phoaphotidylserine (PS), phosphotidylglycerol (PG), phosphatidylinositol (PI), glycerolipids like triglyceride (TG), diglyceride (DG) and monoglyceride (MG) and sphingholipods like sphingomyleon (SM) and ceramides (Ce) in a single RP UPLC-QTOF MS/MS acquisition. The lipids are quantified using Multiquant and normalized by internal standards. This method displays excellent reproducibility, mass accuracy and no significant carryover. The method is successfully utilized for the comprehensive lipidomic profiling of complex biological samples like tissues, plasma, serum, urine, saliva, cells etc.

REFERENCES
1. Bligh, E. G.; Dyer, W. J. A Rapid Method of Total Lipid Extraction and Purification. Can. J. Biochem. Psysiol 1959, 37, 911–917.
2. Gika, H. G.; Macpherson, E.; Theodoridis, G. A.; Wilson, I. D. Evaluation of the repeatability of ultra-performance liquid chromatography−TOF-MS for global metabolic profiling of human urine samples. J. Chromatogr., B: Anal. Technol. Biomed. Life Sci. 2008, 871 (2), 299−305<br>
3. Kind, T.; Liu, K.-H.; Lee, D. Y.; DeFelice, B.; Meissen, J. K.; Fiehn, O. LipidBlast in silico tandem mass spectrometry database for lipid identification, Nat. Meth 2013, 10, 755−758.</p>

Services & Fees

Service Charge per Sample (*Internal Rate) Assay Description (PDF file)
Shotgun Lipidomic - Biofluids $305 / per sample
Shotgun Lipidomic - Tissue $331 / per sample