Types of Analysis

Proteomic approaches have enabled the identification of thousands of differentially expressed proteins in complex mixtures and also ushered the capability of discriminating disease subtypes/aggresiveness that are not recognised by traditional criteria.  Thus, it offers a platform for the identification and quantification of proteins and may uncover new therapeutic targets.  Translation of protein biomarkers in FDA approved diagnostic can be carried out at BMSF.

Some examples of what we can do include:

• Quantitative analysis (iTRAQ, comparative proteomics, MRM)
• Protein modification determination
• Separations (pre-fractionation, SDS-PAGE, 2D-gels)
• Analysis of tryptic digests, gel-fractions and peptides in biological extracts
• Determine pancreatic polypeptide levels in human plasma
• Mass determinations of intact proteins/glysosylations level estimation
• Top/Middle Down protein analysis by ECD, ETD, and IRMPD
• Development of phosphorylation assays used to investigate cell-signalling
• Phosphopeptide analysis by TiO2, IMAC and anti-phosphotyrosine enrichment
• Low mass peptide/protein (<25kDa) enrichment from complex proteomes
• nanoLC-MS/MS protein digest analysis (LysC, AspN, Chyotrypsin etc)
• De novo sequencing of peptides and small proteins
• Mascot, SEQUEST, MaxQuant or PEAKS searches for protein identification
• iTRAQ analysis and quantitation
• SILAC analysis and quantitation
• High resolution UPLC label free protein quantification 
• Static nanospray of peptides and proteins including antibodies
• MALDI-ToF tissue imaging
• Interpretation of results
• SWATH (data independent acquisition)

Alternations in cellular metabolism are hallmark features of metabolic dysfunction, cancer, infection, inflammation, and neurobehavioural disorders.  The BMSF Small Molecule team trains students and researchers on the use of MS-based methods to conduct targeted measurements of small metabolites (<1kDa) using stable isotopes and in global metabolite profiling.

These metabolites provide valuable understanding of major metabolic pathways and their quantification helps elucidate mechanisms underlying cell energy/homeostasis and disease pathology.  Biological samples include cultured cells, bodily fluids, and tissue extracts.

Some examples of what we can do include:

• Acylcarnitines
• Aminoacids
• Organic acids
• Isotope dilution GC/MS or LC/MS assays
• Analysis of catecholamines
• Kynurenine Pathway metabolite identification
• Nitrates and Nitrites analysis
• Glucose quantification
• Hormone identification and quantification
• Acetylcholine and other quaternary ammonium biological amines
• Quantification of Vitamin D and metabolites
• Assay for the quantification of dityrosine in urine and protein hydrolysates
• Detection of lactones in microbial extracts
• Analysis of coal tar samples for molecular weight distribution
• Analysis of borate complex structure in plant extracts
• Metabolite profiling by LC-MS or GC-MS
• Untargeted metabolomics
• Orbitrap LC-MS for accurate mass small molecule analysis
• Triple quadrupole LC-MS for small molecule analysis
• Ion trap LC-UV-MS for small molecule analysis
• Small molecule quantitation by mass spectrometry
• Drug quantitation in biological fluids by mass spectrometry
• Targeted quantitation of specific molecules in biological fluids/tissues by mass spectrometry
• Folate quantitation

Our small molecule analysis work includes accurate mass analysis with mass-to-charge measurement accuracy of 2ppm using an LTQ-Orbitrap XL MS with static nanospray.  This is used to confirm synthetic target structures and to narrow the range of possible elemental formulae for unknowns.  Isotopic fine structure data for unknowns can also be determined.

Volatile organic compounds (VOC's) in the headspace above aqueous sample solutions can also be determined by both direct headspace sampling and by solid phase microextraction (SPME).  Typical applications include odor analysis, novel cancer screening technologies, quantifying methanogenesis in the headspace above cultured bacteria and analysis of permanent organic and inorganic gases.

Lipids have low water solubility and comprise a broad range of structurally diverse molecules, including fatty acids, waxes, eicosanoids, monoglycerides, diglycerides, triglycerides, phospholipids, sphingolipids, sterols and steroids, terpenes, prenols and fat-soluble vitamins.  Lipids are commercially important as food, in the production of biodiesel and other manufacturing processes, and they perform a wide variety of biological functions.

Key roles include energy storage, cell membrane structure and signalling molecules.  Considerable research demonstrates that changes in lipid species and lipid damage significantly influence the development of disease by a range of mechanistic pathways.

We have experience in:

• Isotope dilution GC/MS or LC/MS quantification
• Lipid analysis or profiling by high resolution LC-MS and MS/MS
• Quantification of lipids by nanospray
• Lipid extraction
• "Non-targeted" qualitative and quantitative analysis of
  • Glycerolipids
  • Phospholipids
  • Sphingolipids
• "Targeted" quadrupole MS analysis of
  • Sterols and steroids
  • Total fatty acid profiles

The MALDI Biotyper (MBT) platform identifies microorganisms using MALDI-TOF (Matrix Assisted Laser Desorption Ionization - Time of Flight) mass spectrometry by measuring primarily highly abundant ribosomal proteins that represent a unique molecular fingerprint of a microorganism (https://www.bruker.com/products/mass-spectrometry-and-separations/maldi…). 

This characteristic protein fingerprint is used to accurately identify a particular microorganism by matching thousands of reference spectra from microorganism strains contained within a proprietary database. The MALDI- Biotyper (MBT) platform at the BMSF contains over 8000 fingerprints derived from wide range of Gram-negative, Gram-positive bacteria, yeasts and molds.  Custom libraries also may be created to enhance analysis of novel species. 

The MBT platform provides a rapid and cost-effective way of accurately identifying microorganisms and can be employed in several disciplines but not limited to clinical microbiology, environmental and water analysis, pharmaceutical microbiology and taxonomical research.   

Procedures for Microbial Identification:

• Protein extraction: There are two preparatory methods for MALDI-TOF based bacterial identification. 1) Direct transfer method works best for non-spore-forming microorganisms (i.e. bacteria). 2). Tube extraction method (ethanol - formic acid extraction) is more time intensive but results in more consistent extraction and good quality spectra.
• Data acquisition and analysis: BMSF utilizes a Bruker UltraflexExtreme TOF/TOF instrument, that offers rapid and high-throughput data acquisition in an automated mode. Biotyper software allows us to identify microorganisms at species/subspecies level with high sensitivity and accuracy.
• Training: Our team offers training in sample preparation, data acquisition as well as use of the MALDI Biotyper system. 

Note: To achieve good quality spectra, standard protocols should be followed along with using freshly cultured bacterial colonies is highly recommended.

The BMSF has capabilities for the stable isotope analysis of carbon and oxygen isotopes for small inorganic carbonate samples, such as used in the analysis of stalagmites, coral, and bone apatite. Applications include environmental sciences, geosciences, forensic science, and archaeological sciences, measured using a MAT253 and Kiel IV carbonate device.

We also have compound specific and bulk organic isotope capabilities for carbon, nitrogen, oxygen, and hydrogen isotopes.

Our EA/GC-IRMS has a wide range of environmental and forensic applications, including tree cellulose records of environmental change, environmental lipids and food web studies, and the analysis of archaeological collagen.

We offer services with:

• Kiel/IRMS analysis of carbonate
• Elemental Analysis/IRMS

The DESI technology is a simple, sensitive, gentle, and versatile ionization method that allows for the direct sampling of surfaces without any sample preparation and under ambient temperature and pressure conditions. This patented technology (patent #7335897) was first disclosed in Science, Vol. 306, #5695, pp. 471-473, October 2004.

• Imaging Mass Spectrometry (IMS)
• Analysis of synthetic polymers and large synthetic organometallics
• HPLC separations
• UPLC separations
• nano-DSC
• nano-iTC
• Data analysis and bioinformatics software
• Synthetic Biology.  Many of the analytical capabilities of the BMSF may be utilized in research area.