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Introduction to Proteomics

Proteomics involves the study of proteins. More formally, proteomics looks at ways proteins can benefit humanity through the diagnosis, elucidation and treatment of disease.  After the enormous impact genomics had on our understanding of biological systems through genome sequencing, it is now realised that proteomics promises even greater rewards. Clues to the solution of a myriad of biological problems in health and disease will be found through proteomics by revealing the identity of proteins to be found at any given time in a population of cells and, ultimately, in single cells. Proteomics is the characterization of complex protein mixtures by combining the skills and techniques of protein chemistry, mass spectrometry and bioinformatics.

Proteomics encompasses the identification and analysis of proteins, and particularly their interactions, their relationship to disease, genetics and environmental stresses, their structures and functions. The proteome is the entire complement of proteins and their interactions, including the modifications made to a particular set of proteins.

Our Proteomics Services Laboratory (PSL) will contribute to:

  • Protein Identification
  • Intact Protein Analysis
  • Glycan, Glycoprotein, Phosphoprotein and other post-translational modification analyses
  • Biomarker Discovery
  • Quantitative Proteomics

Modern proteomics has a cross-disciplinary and cross-platform nature as illustrated in the figure below.

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Proteomics and the Integration of Biotechnology Data Sources

Proteomics must be seen within the context of a broad range of complementary techniques, the integration of which leads to useful information.

The accepted methodology in describing processes and procedures is to understand the use of workflows. In proteomics, these may proceed via gel or Liquid Chromatography (LC)-based intermediary steps, as illustrated in figure below.

Workflows in Proteomics

  1. Gel-Based. Separation of complex protein mixtures are carried out by 2D gel-electrophoresis. 2D images are obtained by a range of imaging systems, one of which is being developed by deltaDOT.  Gel images are analysed by proprietary 2D image analysis software e.g. Nonlinear Dynamics or GE Healthcare etc. Spot picking is carried out either by a robotic spot picker or by hand and the spots are subjected to manual trypsin digestion. Picked proteins are identified by peptide mass fingerprint analysis on a Micromass MALDI micro MX using PLGS2 or MASCOT for database searches. Alternatively proteins are identified and quantitated by a high-end mass spectrometer such as the Waters SYNAPT™ HDMS.
  2. LC Based. A complex protein mixture is directly trypsin digested, tryptic peptides are separated by multi-dimensional liquid chromatography and as many peptides as possible are subjected to peptide sequencing in an LC-MS/MS instrument. Sequenced peptides are matched against the genome database of the analysed sample.

There are many excellent resources for a more detailed view on proteomics. Here are some suggested starting points:


http://www3.imperial.ac.uk/biomedeng/facilities/proteomics
http://www.uniprot.org
http://www.expasy.ch/
http://www.cancer.gov/cancertopics/factsheet/detection/proteomics
http://www.pnl.gov/biology/programs/msd/diabetes.stm

Mass spectroscopy (MS) is an analytical tool used for measuring the molecular mass of a sample. In the case of the PSL, we envisage MS systems to stand alongside the LFII tools from deltaDOT is providing the most powerful analyses of biomaterials. For large biomolecules such as proteins,  molecular masses can be measured to within an accuracy of 0.01% of the total molecular mass of the sample, ie. within a 4 Daltons (Da) or atomic mass units (amu) error for a sample of 40,000 Da. This is sufficient to allow minor mass changes to be detected. This has huge biomedical implications: one can measure the substitution of one amino acid for another, or a post-translational modification.

One of our tasks is to help elucidate cancer. From Ann Oncol. 2005 Jan;16(1):16-22: 

          Proteomics is an emerging field in medical science focused on the library of proteins specific to a given biosystem, the proteome, and understanding relationships therein. This field incorporates technologies that can be applied to serum and tissue in order to extract important biological information to aid clinicians and scientists in understanding the dynamic biology of their system of interest, such as a patient with cancer. These tools include laser capture microdissection, tissue lysate arrays and mass spectrometry approaches. These new technologies are more potent coupled with advanced bioinformatics analysis. They are used to characterize the content of, and changes in, the proteome induced by physiological changes, benign and pathologic. The application of these tools has assisted in the discovery of new biomarkers and may lead to new diagnostic tests and improvements in therapeutics. These tools additionally can provide a molecular characterization of cancers, which may allow for individualized molecular therapy. Understanding the basic concepts and tools used will illustrate how best to apply these technologies for patient benefit for the early detection of cancer and improved patient care.

For small organic molecules the molecular mass can be measured to within an accuracy of 5 ppm or less, which is often sufficient to confirm the molecular formula of a compound, and is also a standard requirement for publication in a chemical journal. This has implications for e.g. the analysis of oil and gas samples.

Structural information can be generated using certain types of mass spectrometers, usually those with multiple analysers which are known as tandem mass spectrometers. This is achieved by fragmenting the sample inside the instrument and analysing the products generated. This procedure is useful for the structural elucidation of organic compounds and for peptide or oligonucleotide sequencing.

Mass spectrometers are used in industry and academia for both routine and research purposes.

The following list is just a brief summary of the major CE + mass spectrometric applications:

biotechnology: the analysis of proteins, peptides, oligonucleotides

pharmaceutical: drug discovery, combinatorial chemistry, pharmacokinetics, drug metabolism

clinical: neonatal screening, haemoglobin analysis, drug testing

environmental: PAHs, PCBs, water quality, food contamination

geological: oil and gas composition