An International Symposium of the Association of Biomolecular Resource Facilities

(SW2) Current Status and Challenges in Lipidomics Based Biomarker Development

Workshop Sponsors


 

 
 
Organizers:
Amrita Cheema, Ph.D., Georgetown University
Maryam Goudarzi, Ph.D., Georgetown University
 
Instructors:
Gary Suizdak, Scripps Center for Metabolomics and Mass Spectrometry
Amrita Cheema, Ph.D., Georgetown University
Baljit Ubhi, SCIEX
Roy Martin, Waters Corporation
John Asara, Beth Israel Deaconess Medical Center
 
Sponsor:
Waters Corporation
 
The workshop is scheduled as an afternoon session, from 1:00 pm to 5:00 pm. Lunch is included from 12 noon to 1:00 pm.
 
Topics Covered:
1. Introduction to mass spectrometry based lipidomics technologies - Gary Suizdak
2. Sample preparation and solid phase extraction - Amrita Cheema
3. The Lipidyzer™ Platform: A Revolutionary Tool for Understanding the Role of Lipids in Disease - Baljit Ubhi
4. Post processing of lipidomics data - Roy Martin
5. Importance of lipidomics in the systems biology context -John Asara
6. Challenges - Panel Discussion chaired by Gary Suizdak
 
Introduction:
Lipids are important signaling molecules mediating a large number of structural and functional activities for the maintenance of homeostasis in the cells. The structural importance of lipids is due to maintaining the integrity of cell membrane while their functional importance stems from their involvement in energy metabolism and cell communication and signaling. Perturbations in lipid metabolism have been implicated in pathogenesis of several disease such as atherosclerosis, insulin-resistant diabetes, schizophrenia, and Alzheimer. Based on the functional and structural dynamic of lipids more studies have recently focused on the qualitative and quantitative analysis of lipids. Lipidomics was first introduced by Han and Gross in 2003 as a branch of metabolomics. Lipidomics focuses on the comprehensive identification and quantitation of all lipids in various samples such as serum, plasma, tissue, or cells. Their characterization encompasses their interactions with other lipids and proteins as well as protein expression associated with lipid metabolism and function. Lipidomics has gained popularity in a wide array of applications including biomedical research. As a result in 2004 National Institute of General Medicine supported the LIPID MAPS project as a database for lipid classicification and structural identification. However, due to their heterogeneity and chemical structure diversity, lipids are divided into 8 categories: fatty acyls, glycerolipids, glycerophospholipids (GP), sphingolipids, sterol lipids, prenol lipids, saccharolipids and polyketides. This workshop will focus on the applicability and advances in LC-MS-based lipidomics, sample preparation, high-throughput lipidomic approaches, data analysis as well as the challenges facing the field.
 
Approaches and
Analytical Advances:
Lipids are considered important disease biomarkers and drug targets in the recent years. The analytical tools commonly used for lipidomics are NMR and MS. The combination of MS with pre-separation LC provided higher resolution for structure identity in a high-throughput manner. More recently, ion mobility and imaging MS have not only improved the resolution but also made in vivo real-time analysis possible. We will discuss targeted and non-targeted approached for lipidomics studies.
 
Sample Preparation:
Lipidomics sample collection and preparation is of particular importance in order to maintain the integrity of the sample. The samples are flash-frozen at -80C to reduce the rate of oxidation, peroxidation and enzymatic activities. The solid samples need to be homogenized while preparation of biofluids is easier. All samples proteins are eliminated as they interact with lipids. In some cases solid phase extraction is used to eliminate impurities. The number of freeze-thaw cycles need to be minimized to prevent hydrolysis or oxidation of lipids in the sample. Major extraction methods include single organic solvent extraction, liquid-liquid extraction, solid phase extraction, supercritical fluid extraction, and ultrasonic assisted extraction. We will focus on various strategies in place/in development for lipid enrichment and analysis. In this context exosomal lipidomics will also be discussed.
 
Shotgun lipidomics:
Used for screening various classes of lipids, this method provides low cost high throughput yet low resolution profiling data. Advances in ESI-LC-MS now allows for determination of the entire lipidome with MS/MS providing reliable structural. ESI source can provide a more detailed structure information, such as the indication position of double bonds and the fatty acyl group position in the glycerol backbone. Thus, these new advances in MS allowed the clear identification of isobaric components of lipids. Shotgun lipidomics is routinely performed on a quadrupole time-of-flight MS (QTOF/MS) or orbitrap MS which provid higher mass resolution and accuracy than the traditional triple quadrupole MS. Several MS/MS-based techniques are used for shotgun lipidomics on QTOF/MS including multiple precursor ions scanning and data-dependent MS/MS experiments. MSE technique where each MS scan (low energy scan) is followed by a high-collision scan provides fragmentation information in one injection which provides structural information in addition to profiling data. It is a challenge to analyze broad classes of lipids in a single analytical run which is especially useful when the sample is limiting. We will discuss advances in this direction that would help researchers to pursue these broad based studies leveraging the available technologies for this purpose.
 
Data Analysis:
Data analysis of lipids includes statistical analysis such as univariate significance test and ANOVA related methods. Principle component analysis, and heat-map generation are two of the most common visualization methods of the data. In addition LIPDMAPS is used to acquire biological relevance and pathway information. Deconvolution of lipid spectra for identification and quantification remains a challenge; we will discuss the current status and advances that are needed. This session will also feature some case studies where we will discuss an experimental data set and try to identify lipids associated with the spectra.
 
Challenges:
The field lipidomics is still in its infancy compared with proteomics and metabolomics. The current focus primarily on biomarker searching and structure identification, which are limited in scope. Also complexity of lipid metabolism presents a series of technical and bioinformatics challenges. High resolution and sensitive analytical techniques are of urgent need. We will open the floor for an open panel discussion and get a feedback from participants about their views about how ABRF can help via organizing hands on workshops or user sessions.
 

Platinum Sponsors of the ABRF