Core Capabilities

Non-targeted Metabolite Profiling

A global (or non-targeted) approach to metabolite profiling involves monitoring as many metabolites in the entire metabolome as possible. The goal is to identify those few molecules that are either up or down regulated.  Depending on the general classes of molecules to study and sample complexity, we utilize HPLC/MS, GC/MS, and/or GCxGC/MS instrumentation.  Whereas HPLC/MS is able to monitor hundreds of peaks in a single run, the GCxGC/MS has a peak capacity in the thousands.  The bioinformatics analysis is performed using both commercial and in-house software applications.  An example of such an analysis includes plasma from healthy volunteers compared to those with a certain disease state. 

Lipid Profiling

HPLC/MS is utilized for non-targeted lipidomics. Complete lipid coverage is obtained by operating the mass spectrometer in positive and negative ionization modes. For lipidomics, samples are typically prepared using a Bligh-Dyer sample extraction. 

A method for general profiling of phospholipids using the LC/MS TOF or Thermo Orbitrap has been developed. The chromatography is designed to separate different phospholipid classes based on head group chemistry. The protocol was developed to analyze a typical lipid extract in negative ESI mode. Phosphatidylserine (POPS), phosphatidic acid (POPA), phosphatidylglycerol (POPG), phosphatidylethanolamine (POPE) and ceramides can be analyzed using this protocol. Phosphatidylcholines and sphingolipids can be analyzed using positive ESI mode. 

Small Molecule Quantitation

Many projects involve the quantitation of predetermined analytes in a complex biological matrix, such as in pharmacokinetic studies. This type of targeted analysis requires that analytical methods be developed and validated for specificity, linearity, range, precision, detection limit, quantitation limit, accuracy, robustness, and solution stability. For complex biological mixtures, internal standards are utilized to correct for the loss of analytes during sample preparation or introduction into the instrument. We have developed methods for most classes of small molecules, including hormones, sugars, amino acids, polyphenols, vitamins, pharmaceuticals, and lipids. 

Amino Acid Analysis

Amino acids have been successfully analyzed using GC/MS. The samples are derivatized to produce a TBDMS (tert-butyldimethylsilyl) derivative. This label will derivatize both the carboxyl and amino group of the amino acid, as well as any hydroxyl, carboxyl, thiol, or primary and secondary amine that may appear in the side chain. These derivatives are 10,000 times more stable than traditional TMS ethers. The separation can be conducted in 30 minutes. The detection limit is low picograms injected on-column. Useful internal standards are 2-aminobutyric acid and beta-alanine. Additionally, a HILIC HPLC method, coupled to our triple quadrupole mass spectrometer, can be employed for amino acid analysis. This newer approach has a lower detection limit and does not require sample derivatization. Our lab will perform free amino acid analysis. We require a minimum of 10 samples.

Sample Preparation

A variety of sample preparations can be performed. Liquid and liquid-liquid extractions are the most common. A variety of solid phase extraction (SPE) protocols are used in the lab. Sample homogenization can be done by sonication or with a bead homogenizer.  Large sample numbers can be prepared by robotic equipment through a collaborative effort with Bindley's Integrated Screening Technologies Facility.  

High-throughput Lipid Profiling

Many methods have been developed for fast (few minutes) and informative profiling of diverse phospholipid and glycerolipid classes by flow injection of lipid extracts into a triple-quadrupole mass spectrometer. Without chromatography, various scan modes (neutral loss and precursor ion scan) for profiling each lipid class are used. This analytical approach is ideal for exploratory lipidomics or high-throughput sample sets. Data analysis of lipid profiles by multivariate analysis can be used for biomarker identification and/or to drive further targeted analysis of specific lipid classes or compounds. 

Bioinformatics Consultation

The Computational Life Sciences and Informatics Core of the Bindley Bioscience Center provides a senior informatics scientist devoted to the analysis and modeling of complex data sets. The main focus of the core is on experimental design and data analysis related to proteomics and metabolomics data applied to human health, animal health, and agricultural investigations.

Individual Training

Studies can be conducted entirely by MPF staff members. For more extensive projects, students and post-docs may be trained to operate the instrumentation independently.

Contact

Amber Hopf Jannasch
Metabolomics Research Scientist
Phone: (765) 496-7028
Email: hopfas@purdue.edu

Purdue University, 610 Purdue Mall, West Lafayette, IN 47907, (765) 494-4600

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