Mass Spectrometry (MS) is an extremely powerful analytical technique that underpins all modern day biomolecular science. It measures molecules to determine their weight, thus facilitating the identification and characterization of key components in biological processes (proteins, peptides, carbohydrates, DNA, drugs). MS is the best approach to the analysis of the signalling events (e.g. protein phosphorylation) that drive these processes within cells.
Proteomics is the ability to study proteins in our cells and tissues on a large scale as well as one at a time. The successful study of important cellular proteins depends largely on the experimental design and requires a great deal of careful planning.
Biomedical Proteomics at CMRI combines applied proteomics with the development of Liquid chromatography–mass spectrometry (LC–MS) - based qualitative and quantitative methods for protein identification and characterisation.
The facility supports CMRI and the Westmead Research Hub, as well as collaborations with other groups and individuals by offering access to equipment and methodology, as well as expertise in the assessment of feasibility and strategy development for projects and programs involving MS-based proteomics analysis.
LC–MS is an extremely powerful analytical strategy for the identification, quantitation, and characterization of biomolecules (e.g. proteins, peptides, carbohydrates, DNA, drugs) in cells, tissues, biological fluids, and organisms. Modern LC-MS workflows can identify and measure thousands of proteins/protein groups. This enables the study of protein–protein interaction, post-translational modifications, and protein expression and provides insights into the physiological changes occurring between two or more states of interest (e.g. disease vs. control; treated vs. nontreated).
Currently the Biomedical Proteomics facility at CMRI houses ten LC-MS systems:
SCIEX Triple TOF 5600 Plus
The Triple TOF 5600 Plus is an accurate-mass instrument with the speed and sensitivity to deliver comprehensive qualitative exploration, rapid profiling, and high-resolution quantitation in complex matrices.
SCIEX QTrap 6500 Plus
This system is a fast and sensitive QTRAP enabling the detection and quantitation of a wide range of chemical compounds in challenging matrices. Improved polarity switching and MRM3 speeds allow faster chromatography and better throughput. The built in QTRAP functionality enables quantitative MRMs and qualitative QTRAP scans in the same injection to maximize throughput. The system is ideal for demanding applications requiring ultimate sensitivity for ultra-low-level quantitation of both small and large molecules.
SCIEX QTrap 5500
The 5500 QTRAP is specifically designed for label-free quantitative analysis by SRM/MRM. High selectivity results from monitoring one pair of precursor/fragment ions characteristic of a single peptide. Many such “transition pairs” can be specified in a LC-MS/MS run, allowing many proteins to be quantified in parallel.
SCIEX MALDI TOF/TOF 5800
The instrument is ideal for quality control of sample preparation and for identification of proteins in relatively simple mixtures (e.g. bands/spots cut from gels).
Thermo Orbitrap Elite
The Thermo Orbitrap LTQ is an ion trap configuration mass spectrometer. Its high sensitivity in combination with a very high resolution make it suitable for discovery and targeted proteomics research.
Thermo Q Exactive Plus
The Thermo Q Exactive Plus system enables rapid and confident identification and quantitation of proteins, peptides, and posttranslational modifications. It significantly improves the depth of proteome analysis, due to its compatibility with the Tandem Mass Tag (TMT) multiplexing technology.
Shimadzu LCMS 2020
The Shimadzu LCMS 2020 is a single quadrupole mass spectrometer offering faster measurements and higher detection sensitivity for quicker and more accurate detection of trace impurities in a variety of applications.
Example proteomics project: Dynamin
The question: Is it possible to control nerve communication to ultimately treat various brain and nerve disorders?
Answer: Many years ago, the Cell Signalling Unit discovered that the protein dynamin plays a central role in a process called endocytosis, which is crucial to nerve communication (or nerve cell signalling).
The group initially discovered dynamin as a phosphoprotein (a protein with a phosphate molecule attached to it) that is rapidly de-phosphorylated upon stimulation of nerve terminals. Using various techniques, including proteomics, the group deciphered the molecular mechanisms of dynamin’s phosphorylation cycle, and they have now identified all of the specific sites of phosphorylation in dynamin.
Their work revealed that each site plays an essential role in endocytosis, and that they act cooperatively for maximal effect. They were also able to discover dynamin’s protein partner for endocytosis in neurons. These findings have led directly to the development of a new class of drugs to potentially treat epilepsy and to greater understanding of nerve communication that may one day lead to treatments for other nerve disorders.
If you believe an MS-based proteomics approach is suitable for your research, please discuss your needs with the Facility Manager.
George Craft, Biomedical Proteomics
Facility Located at: Children's Medical Research Institute, 214 Hawkesbury Road, Westmead, NSW