Quantitative Macrolipidomics of Human Whole Blood for the Discovery of Novel Biomarkers of Omega-3 Polyunsaturated Fatty Acid Intake

The relationship between diet and blood levels of omega-3 PUFA has been studied extensively. Most of this research has been focused on overall fatty acid metabolism in total lipids or lipid fractions of plasma/serum and erythrocytes using gas chromatography-flame ionization detection (GC-FID). Fatty acid compositional data derived from GC-FID-based analyses rely on the hydrolysis and methylation or direct transesterification of fatty acyl chains on complex lipids to generate fatty acid methyl esters. Ultra-high performance liquid chromatography/tandem mass spectrometry (UHPLC-MS/MS)-based analyses have the potential to characterize complex lipids as they exist in their natural states.

Aristizabal-Henao, J. J., Meitei, N.S., Biltoft-Jensen, A.P., Stark, K.D.

67^th ASMS Conference on Mass Spectrometry and Allied Topics (2019),
Atlanta, GA

Lipid Profiling of Malaria Samples Using Orbitrap Velos Pro Mass Spectrometer with SimLipid Software

Rapid advancement in the technologies related to liquid chromatography (LC) and mass spectrometry (MS) leads to development of new lipidomics methods. We have developed LC-MS lipidomics method based on UHPLC coupled with high resolution Orbitrap Velos Pro MS instrument for lipid profiling of Plasmodium berghei samples. Lipid identification was achieved using MS/MS data from both positive and negative ion modes and quantitation of lipid species was performed using ion abundance corresponding to its monoisotopic peak of the isotope cluster and statistical analysis.

Meitei, N.S., Gupta, H., Rahlouni, F., Sullivan, D., Shulaev, V.

67^th ASMS Conference on Mass Spectrometry and Allied Topics (2019),
Atlanta, GA

FIA-HRMS-Based Lipidomics Method: Comparing Measured Lipid Concentration Calculated Using Parent Molecular Ion Abundance Versus Sum of Product Ions Abundances

Flow injection analysis (FIA) coupled with high-resolution mass spectrometry (HRMS) instrument based methods are increasingly becoming recognized as a suitable technique in Lipidomics studies. We present a lipidomic method using FIA coupled to an Orbitrap-based mass spectrometer. Lipid species were measured using data independent acquisition (DIA) based MS/MS data. The concentration of the lipid species present in the samples was measured using the parent ion abundance normalized to the internal standard belonging to the same class and compared to the product ion abundances of the same species.

Triebl, A., Torta, F., Gupta, H., Meitei, N.S., Goswami, R.

67^th ASMS Conference on Mass Spectrometry and Allied Topics (2019),
Atlanta, GA

Ultra-high sensitivity nanoflow lipidomics with Trapped Ion Mobility Spectrometry (TIMS) and Parallel Accumulation SErial Fragmentation (PASEF)

Despite consisting of few building blocks, lipids form a highly diverse group of biomolecules with important biological function. Established liquid chromatography – mass spectrometry (LC-MS) workflows sample the lipidome with high throughput, but limited selectivity and high starting amounts. We present a high-sensitivity workflow based on nanoflow separation and trapped ion mobility spectrometry (TIMS) [1]. By synchronizing TIMS separation with precursor selection (PASEF), we have recently demonstrated an over 10-fold increase in MS/MS acquisition rates without any loss in sensitivity.

Vasilopoulou, C.G., Sulek, K., Brunner, A., Meyer, S.W., Schweiger-Hufnagel, U., Meitei, N.S., Mann, M., Meier, F.

67^th ASMS Conference on Mass Spectrometry and Allied Topics (2019),
Atlanta, GA

The analysis of TAG in plant-based oils can be particularly challenging to measure due to their high structural diversity encompassing various fatty acyl chain lengths and isobaric/isomeric configurations. While TAG constitute most of the weight of vegetable oils, the measurement of other lipid species, such as diacylglycerols, phospholipids and sterols is relatively limited in the literature. Dual column chromatography was used for untargeted macrolipidomics of 10 commercially available plant-based oils.

Aristizabal-Henao, J., Meitei, N.S., Stark, K.D.

67^th ASMS Conference on Mass Spectrometry and Allied Topics (2019),
Atlanta, GA

Fast Supercritical Fluid Chromatography Separation and Shotgun Lipidomics with High Resolution Mass Spectrometry for the Study of Breast Cancer Metastasis

Breast cancer cells can undergo metabolic reprograming to adapt to different metastatic sites. 4T1 urine breast cancer cells are highly aggressive model of triple negative breast cancer that can spread to the lymph nodes, bone, lung, liver and brain. Their preferred metabolic program (glycolysis versus oxidative phosphorlyation) was found to change depending on the tissue site that the breasr cancer cells colonized. To further explore the metabolic adaptations that accompany organ-specific metastasis, parental 4T1 cells were subjected to an in vivo selection protocol to create subpopulations of cancer cells with increased selectivity for specific organs. These cells were shown to have distinct metabolic phenotypes (1,2). In order to explore the lipid adaptations that occur between the parental 4T1 cells and the liver-metastatic 2776 population, four replicate samples were generated from 8.87 million 4T1 cells per sample and 8.63 million 2776 cells per sample.

Mohsin, S., Meitei, N.S., Siegel, P., Avizonis D., Bridon, G.

67^th ASMS Conference on Mass Spectrometry and Allied Topics (2019),
Atlanta, GA

Automated Identification of Glycerolipid, Diacyl-, Monacyl- Glycerophospholipids with Detailed Analyses of the Fatty Acyl Composition using Tandem Mass Spectrometry

Tandem MS lipidomic data facilitates not only the identification of glycerophospho/ glycero-lipids based on their head groups but also the differentiation between saturated, moderately unsaturated (18:1 or 18:2) and highly unsaturated (20:4 or 22:6) acyl residues [1-2]. However, interpretation of MS/MS lipidomic data requires efficient software workflows. We present software workflows to facilitate identification of the fatty acyl composition in glycerolipid, diacyl-, monacyl- glycerophospholipids.

Meitei, N.S., Pujari, R., Gupta, H., Hufnagel, U.S., Goetz, S., Meyer, S.W., Barsch, A.

14^th Annual Conference of the Metabolomics Society (2018),
Seattle, Washington

Lipid Profiling of Grape Samples using Orbitrap Velos Pro Mass Spectrometer with SimLipid^® Software

Liquid chromatography—Mass spectrometry (LC-MS) provides one of the most popular platforms for lipidomics analysis. Recently, MS-based plant lipidomics research has been gaining popularity [1]. We developed LC-MS lipidomics method using Orbitrap Velos Pro Hybrid MS instrument to identify the lipid classes and lipid molecular species in grape samples. A challenge associated with this method was the analysis of large LC-MS data set. Besides the chemical complexity and large range of concentrations of thousands of lipid species that are present in the samples, there are co-eluting lipid species, especially from a class, in the LC domain and MS2 spectra contain product ions of multiple lipid species. SimLipid has been re-developed in order to streamline such large scale data analysis generated by the method.

Shulaev, V., Chitarrini, G., Gupta, H., Pujari, R., Vrhovsek, U., Mattivi, F., Meitei, N.S.

66^th ASMS Conference on Mass Spectrometry and Allied Topics (2018),
San Diego, CA

Application of SimLipid^® Software in Lipid Profiling of Secreted Lung Lipids by MSMS^ALL Shotgun Lipidomics

There has been rapid advancement in the field of analytical technology, particularly mass spectrometry that fuels the lipidomics research [1]. One of such MS based techniques was the sequential precursor ion fragmentation, MSMS^ALL, that delivers both qualitative and quantitative information for hundreds of lipid species in a short period of time without compromising data quality[2-3]. We present a comprehensive lipidomics workflow using automated large volume loop injection (FIA) coupled to a TripleTOF® 5600+ mass spectrometer (SCIEX, Concord, ON) with SimLipid software. The lipid species in the sample were identified using data from MSMS^ALL experiment and subsequently quantified using data generated by TOF-MS experiments. SimLipid software has been re-developed to streamline the data analysis from this workflow.

Meitei, N.S., Gupta, H., Pujari, R., Apte, A., Yin, H., Capka, V., Rowlands, D., Patel, S., Daya, S., Choy, K.

66^th ASMS Conference on Mass Spectrometry and Allied Topics (2018),
San Diego, CA

SimLipid^®: Informatics Support for Profiling Glycerolipid, Diacyl-, Monoacyl- Glycerophospholipids with Details of the Fatty Acyl Composition using Tandem Mass Spectrometry

The rapid advancement in mass spectrometry (MS) and chromatography technologies have led to the development of new lipidomics workflows[1]. MS technology facilitates the differentiation between saturated, moderately unsaturated (18:1 or 18:2) and highly unsaturated (20:4 or 22:6) acyl residues [2-3]. However, interpretation of MS/MS lipidomic data is complex because each lipid class has its own fragmentation patterns as well as ionization efficiency [4]. Hence, different platforms render significantly different lipid profiles [5]. A challenge faced by researchers is the lack of software tools to exploit the technology. We have developed new modules of SimLipid software for global lipid profiling using tandem mass spectrometry data, and also facilitating further identification of the fatty acyl composition in glycerolipid, diacyl-, monoacyl- glycerophospholipids.

Meitei, N.S., Pujari, R., Gupta, H., Apte, A., Hufnagel, U. S., Goetz, S., Meyer, S.W., Barsch, A.

66^th ASMS Conference on Mass Spectrometry and Allied Topics (2018),
San Diego, CA

Comparing Automatic Identifications in the Macrolipidomic Profiles of Human Whole Blood Across UHPLC-MS/MS Platforms and Acquisition Modes

Automated identifications of various lipid species are critical for untargeted or "macrolipidomic" profiling. Software can match analytical data to extensive libraries of spectra, but different instruments employing different technologies and different acquisitions modes can influence the ability to match spectra with library references. For this exercise, whole blood was examined due to its complex lipid profile and potential use of this matrix for biomarker discovery through dried blood spots.

Henao J.J.A., Meitei N.S., Chalil D., Smith R.W., & Stark K.D.

66^th ASMS Conference on Mass Spectrometry and Allied Topics (2018), San Diego, CA

Phosphatidylcholine Acyl-Lipid Species Identifications are Influenced by Sample Preparation

A major challenge in macrolipidomics is the ability to accurately identify the hundreds to thousands of individual lipids in biological samples. Automatic identification software has the ability to characterize various lipid species rapidly, but sample preparation, chromatographic separation and instruments settings can affect the number and quality of identifications.

Ralph L.T., Henao J.J.A., Chalil D., Meitei N.S., Smith R.W., & Stark K.D.

International Society For The Study Of Fatty Acids And Lipids (2018), Las Vegas, USA

Shotgun Lipidomics of Prostate Cancer Cells Using ESI-MS Shimadzu 8050 and Simplified Data Analysis by SimLipid Software

Lipidomics is a relatively recent omics field of research which includes complex lipidome analysis. It is an emerging field in biomedical research as lipids play an important role in cell, tissue and organ physiology and have potential as biomarkers of disease or treatment success. Shotgun lipidomics1 involves identification and quantification of lipids by direct infusion of complex lipid samples into the mass spectrometer without any chromatographic separation. In 2017 prostate cancer is the most commonly diagnosed cancer in Australia among males. It is estimated that the risk of a male being diagnosed with prostate cancer by his 85th birthday will be 1 in 7.

Gupta, R., Sadowski, M., Meitei, N.S., Blanksby, S.J.

13^th Annual Conference of the Metabolomic Society (2017), Brisbane, Australia

Utilisation of SimLipid for the Characterisation of Metabolic Syndrome Related Lipids Acquired Using a Novel Scanning Quadrupole DIA Acquisition Method

Lipidomics has become a rapidly increasing area of research over recent years with a focus on its use and application for disease processes including metabolic syndrome disorders, cancer and cardiovascular disease for example. Obesity, a metabolic disorder risk factor, is known to initiate inflammation, which in turn can lead to type 2 diabetes. The exact mechanism as to how this occurs is not understood.

In the method, a low-resolution quadrupole mass filter is scanned repetitively and both precursor and MS/MS data acquired at spectral rates approaching 2000 spectra/s. The method produces a high duty-cycle, highly specific and unbiased two-dimensional data that can be viewed and processed using readily available informatics.

Gethings, L.A., Meitei, N.S., Vissers, J.P.C., Heywood, D., Castro-Perez, J., Langridge, J.I.

13^th Annual Conference of the Metabolomic Society (2017), Brisbane, Australia & 65^th ASMS Conference on Mass Spectrometry and Allied Topics (2017), Indianapolis, IN

Development of an Integrated Workflow for Profiling and Semi-Quantitative Analysis of Lipids

The lipidome covers a range of lipids from non-polar to polar. Profiling experiments often need two or more chromatography separation schemes to cove the range of polarities: a Normal phase method and a reverse phase method. Quantitation of the annotated lipids requires additional investigation using a more targeted approach.

Mohsin, S.B., Williams, K., Renu, D., Meitei, N.S., Kalakoti, S., Madden, S., Kitagawa, N.

65^th ASMS Conference on Mass Spectrometry and Allied Topics (2017), Indianapolis, IN

Automated Lipid Profiling of a Plasma Sample Using Ultra-High Resolution Qq-Time-Of-Flight Impact II™ Mass Spectrometer with SimLipid Software

The field of lipidomics is fueled by analytical technology advances, particularly mass spectrometry and chromatography. The challenges with mass spectrometry based lipidomics lie in the complexity associated with identification of lipids that requires sophisticated software since automated interpretation of lipid MS/MS spectra is more challenging when compared to other biopolymers such as DNA, carbohydrates or peptides, since lipids show much less standardized fragment mass spectra. Each lipid class has its own fragmentation patterns as well as ionization efficiency.

Meitei, N.S., Gupta, H., Apte, A., Barsch, A., Alving, A., Henao, J.J.A., Stark, K.D.

65^th ASMS Conference on Mass Spectrometry and Allied Topics (2017), Indianapolis, IN

Automated Lipid Profiling of Malaria Samples Using Orbitrap Velos Pro Mass Spectrometer with Lipid Search and SimLipid Software

Liquid chromatography—Mass spectrometry (LC-MS) provides one of the most popular platforms for lipidomic analysis. Comparative studies of the complex lipid mixtures found in cells and tissues could potentially reveal lipid biomarkers. We have developed a lipidomics analysis method – LC coupled with high resolution Orbitrap Velos Pro MS instrument methods; lipid identification using database search of MS/MS data from both the positive, and negative modes; and multivariate statistical analysis – to identify the significant lipid species that are responsible for classifying multiple biological groups of malaria samples.

Meitei, N.S., Arun Apte, Rahlouni, F., Sullivan, D.J., Shulaev, V.

65^th ASMS Conference on Mass Spectrometry and Allied Topics (2017), Indianapolis, IN

SimLipid^®: Software platform for automating Shotgun, LC-MS and MALDI-MS based high-throughput lipidomics

A combination of different approaches namely ‘Shotgun lipidomics’, LC-MS, and MALDI-TOF is often necessary to detect the whole lipidome of an organism. Each lipid class has its own fragmentation patterns as well as ionization efficiency [1], thereby making the interpretation of MS/MS lipid spectra a challenging task.

Meitei, N.S., Gupta, H., Apte, A.

64^th ASMS Conference on Mass Spectrometry and Allied Topics (2016), San Antonio, Texas

Automatic lipid characterization using SimLipid® from normal phase and reverse phase liquid chromatography -MS, MS/MS data acquired in variable ion modes

LC-MS techniques using electrospray ionization (ESI) and reversed phase chromatography have successfully been used to profile complex lipid samples. Normal phase (NP) LC-MS separates phospholipids into their respective classes based on their respective polar head groups and with little influence from their sn-1 and sn-2 fatty acid substituents.

Meitei, N.S., Biswas, A., Apte, A., Madden, S., Sartain, M.

63^rd ASMS Conference on Mass Spectrometry & Allied Topics (2015), St. Louis

Automatic Characterization of Lipids Using Charge Remote Fragmentation Ions and Peaks Characteristic of Fatty Acid Fragmentation From MALDI MS/MS Data

MALDI mass spectrometry has been used for detecting phosphatidylcholines by direct tissue analysis (1-4). Recently, triacylglycerols and phosphatidylcholine in complex mixtures have been identified using MALDI MS/MS (5,6) wherein charge remote fragmentation (CRF) as well as other A-, B-, C-, G and J- ions are the determinants as described recently (7,8,9).

Meitei, N.S., Apte, A., Waidelich, D., Abdi, F., Glueckmann, M.

62^nd ASMS Conference on Mass Spectrometry and Allied Topics (2014), Baltimore

Automatic Lipid Characterization Based on Charge Remote and Fatty Acid Fragmentation in MALDI MSMS

MALDI mass spectrometry has been used for detecting phosphatidylcholines by direct tissue analysis (1-4). Recently, triacylglycerols and phosphatidylcholine in complex mixtures have been identified using MALDI MS/MS (5,6) wherein charge remote fragmentation (CRF) as well as other A-, B-, C-, G and J- ions are the determinants as described recently (7,8,9).

Meitei, N.S., Apte, A., Park, J., Waidelich, D., Abdi, F., Glueckmann, M.

Metabolomics 2014, 10^th Annual International Conference of the Metabolomics Society (2014), Tsuruoka

Lipid profiling of serum samples from calcific coronary artery disease patients using UPLC/TOF-HDMS^E and multivariate data analysis

The separation of glycans by chromatography prior to MS analysis can reduce sample complexity, minimize ion suppression, and increase dynamic range and separation of structural isomers.

Isaac, G., Vorkas, P.A., Want, E.J., McDonald, S., Naslund, U., Holmgren, A., Henein, M.Y., Millar, A., Holmes, E., Shockcor, J.P., Nicholson, J., Langridge, J.

60^th ASMS Conference on Mass Spectrometry and Allied Topics (2012), Vancouver

Untargeted and Targeted Lipidomics Using Ultra-High Resolution Qq-Time-Of-Flight Impact II™ Mass Spectrometer with SimLipid® Software

Improvements in analytical methods, especially liquid chromatography and mass spectrometry, have driven the recent advances in the development of lipid profiling methods. We have presented a robust workflow for the global lipid profiling of a plasma sample using an LC system coupled to an Impact II™ Ultra-High Resolution Qq-Time-Of-Flight (UHR-QqTOF) mass spectrometer (Bruker Daltonik, Bremen, Germany) with > 50,000 Full-Sensitivity Resolution and SimLipid® informatics software (PREMIER Biosoft, USA).

Meitei N.S., Gupta H., Apte, A., Barsch, A., Alving, A., PREMIER Biosoft & Bruker Daltonik (2016)

PREMIER Biosoft Technical Note No. TSL101

Phospholipid analysis using SimLipid software

Phospholipids (PLs) have a role of constituting a cellular membrane in a living cell and are also related to produce various fatty acids such as arachidonic acids, EPA and DHA which are precursors of bioactive lipids. Fluctuation of PLs concentration in a blood or a tissue is also known to be correlated with various disease.

T. Nakanishi (2017)

Shimadzu Document No. LAAN-A-LM-E120

Automated Lipid Identification Using UPLC/HDMS^E in Combination with SimLipid

Lipids constitute one of the largest classes of macromolecules, together with nucleic acids, proteins, and carbohydrates. Lipids play key biological roles in all organisms and therefore their analysis is of major interest in nutritional, pharmaceutical, and biological research.

Isaac, G., McDonald, S., Astarita, G. (2011)

Waters Application Note No. 720004169EN