Kolla, Varaprasad. Identification of screening biomarkers for chromosomal anomalies and pregnancy-related disorder using quantitative plasma proteomics. 2011, PhD Thesis, University of Basel, Faculty of Science.
Official URL: http://edoc.unibas.ch/diss/DissB_9399
One of the routes explored for NIPD was via the enrichment of fetal cells, specifically erythroblasts, from maternal blood. After the enrichment, the putative fetal cells were examined by FISH analysis for the presence of a chromosomal anomaly. In the large a multi-centre NIFTY study it was concluded that although promising, the sensitivity and specificity was below the required clinical application.
Proteomics is defined as the analysis of whole protein component of a tissue (e.g. brain), cell (e.g. yeast) or a body fluid (blood or urine). More precisely it involves the determination of identity of the protein present in the mixture and its relative and absolute quantity. Recently it is also used to identify protein modifications (e.g. phosphorylation, glycosylation). Applications of proteomics are very wide, with the major application in clinical research being applied for the better understanding of biological processes and disease state. Examples are, to find and validate new biomarkers (diagnostic and prognostic) or to understand the pharmacodynamics and pharmacokinetics of a drug compound.
A specific proteome is very dynamic and can provide lot of information on the expression pattern on normal vs. disease or control vs. treated. To study the proteome in its complexity, advanced tool are required, and in this context mass spectroscopy has emerged as a powerful technique for proteomic analysis.
A typical mass spectroscopy based proteomics workflow involves the digestion of protein in-gel or in liquid. Online or off-line fractionation of the peptides is performed by liquid chromatography (LC) and followed by ionization. The peptides are converted into ions and mass analysis is done on these ions, following which the mass to charge ratio is recorded. The resulting fragment masses are used to search of large protein databases search, resulting in the identification of the peptide and protein.
Plasma is an attractive entity for the proteomics studies. It contains different proteins from the various organs in high or low abundance. Many pathology or disease associated proteins are often present in plasma. Due to there low abundance, a number of different strategies have been developed to detect these in the plasma proteome, of which a few are discussed below.
In traditional 2D gel electrophoresis the most preferred staining method is by Coomassie Brilliant Blue (CBB) or sliver stains. After staining intensity of the protein spot is used for relative quantitation when compare with the gel which is run in parallel. But CBB has poor detection sensitivity, where as sliver stain is not compatible with the down stream mass spectroscopy analysis. To over come this problem the proteins were label with fluorescent cyanine dye (cy2, cy3 and cy5) before the 2D separation. This method in know as 2-D differential gel electrophoresis (2D DIGE) to avoid the gel to gel variation. In same gel one can run control and experimental sample as well the internal standard. Internal standard is made by mixing equal amount of control and experimental sample and is used for the relative quantitation. After the run the gel is scanned with the special Typhoon variable mode imager and DeCyder software is used for the differential analysis.
Over a decade MS has evolved as a powerful technique. This has lead to the development of shotgun proteomics, which is a useful tool as it bank ready quantification using special reagent and technique. Lately there are different techniques available for the labeling which enables the quantification of the protein like stable isotope labeling of amino acid in cell cultures (SILAC), isotopic-coded affinity tags (ICAT) and isobaric tags for relative and absolute quantitation (iTRAQ).
iTRAQ is an isobaric chemical labeling approach currently the only technique capable of multiplexing up to eight different samples for relative quantification. 8-plex chemically identical iTRAQ reagents are available, named 114, 115, 116, 117, 118, 119, and 121 which have the same overall mass. Each label is composed of a peptide reactive group (NHS ester) and an isobaric tag of 145 Da that consists of a balancer group (carbonyl) and a reporter group (based on N-methyl piperazine), between the balancer and the reporter group is a fragmentation site. The peptide reactive group attaches specifically to free primary amino groups – N-termini and ε-amino groups of lysine residues.
Each sample to be analyzed is tryptic digested and labeled with the single iTRAQ label after which sample are pooled for tandem mass analysis. The peptide product ion spectra is then used for the identification of the proteins and relative quantitation is derived from the peak intensities of the 8-plex iTRAQ reporter ions detected in the 114-121 m/z region of the fragmented ion spectra. Data acquired is always compared to a reference sample, and the quantity of each peptide is expressed as a ratio relative to the reference sample. As the field of shotgun proteomics is evolving rapidly, it is likely to play a role in the detection of biomarkers. iTRAQ has been shown to permit very reliable quantitation of proteins in complex mixture such as plasma, serum or urine. As such it has been suggested to be a useful tool for the detection of biomarkers. Hence, it is likely to play a key role in this field.
Selected Reaction Monitoring (SRM) is very rapidly developing tool for the MS based quantitation. It exploits the unique future of the triple quadrupole. The first and third quadrupole act as a filter to select the ion of defined m/z and the second quadrupole is a collision cell. The research carried out in this thesis does however demonstrate that it is possible to mine the maternal plasma proteome for new biomarkers. Their verification will however need to be carried out in much larger studies, and using more convenient techniques.
|Committee Members:||Jenal, Urs and Jenö, Paul|
|Faculties and Departments:||03 Faculty of Medicine > Departement Biomedizin > Department of Biomedicine, University Hospital Basel > Prenatal Medicine (Hahn)|
|Bibsysno:||Link to catalogue|
|Number of Pages:||132 S.|
|Last Modified:||30 Jun 2016 10:41|
|Deposited On:||25 Mar 2011 10:09|
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