– Proteomics Laboratory
The proteomics laboratory is mainly supported by The Malaysian Cohort Project (TMC). The main goal for establishing the proteomics lab was to facilitate research in cancer biomarker discovery research as well as other biomarker-based research for other diseases.
The main equipment in the lab is the Nano-Acquity UPLC-QTOF (Waters). This is a rather old system bought in 2007 and has been rather difficult to maintain. The equipment also has limited quantitative capabilities. Despite that there have been 3 publications on cancer biomarkers published using this equipment.
The lab is also equipped with the sample pre-fractionation device (Microrotofor Cell, BioRad), isoelectric focusing (IEF) units (Ettan™ IPGphor III, GE), second dimension gel separation units (Ettan™ DALTsix, GE), Ethan™ DIGE Imager (GE), 2D Imager (Ettan™ Image Scanner III, GE), and the robotic spot picker (Ettan™ Spot Picker). These state-of-the-art equipments allow us to conduct and provide various proteomics analysis such as separation, identification, characterization and quantification of proteins expressed in biological samples.
In addition, our laboratory is also supported by a very extensive data analysis software panel such as the ImageMaster Platinum (GE Healthcare), ImageQuant Tool (GE Healthcare), Progenesis SameSpots (NonLinear), Progenesis LC-MS (NonLinear) and ProteinLynx (Waters). All these top-end softwares allows us to apply rapid, objective and realiable statistical analyses in discovering compounds and proteins of interest for ‘omics’ research applications.
In UMBI, both top-down and bottom-up proteomics approaches are utilised for expression proteomics studies. Our group is the first user of the 2-Dimensional Differential Gel Electrophoresis (2D DIGE) in Malaysia since we first set it up in 2008. 2D-DIGE is an innovative 2D PAGE technology and has enhanced sensitivity and reproducibility than the conventional 2D-PAGE. It incorporates multiplex labelling protein technique using distinctive cyanine reactive dyes. Protein samples and internal standard with differential labelling can be separated on a single gel, and hence eliminating gel-to-gel variations. Internal standard on every gel allows reliable quantification and comparison.
The high throughput and accurate excision of protein spots separated on 2D gels are now made easier with the newly addition of Ettan™ Spot Picker in our proteomics laboratory. The instrument automates the time-consuming processes involved in pinpointing protein spots on 2D gels stained using a wide range of detection methods such as silver, Coomassie™ or fluorescence. Once protein spots are detected, the instrument then automatically picks and transfers them to the wells of a microplate for digestion and subsequent identification by mass spectrometry.
Protein identification and characterization are viable with the presence of nanoACQUITY UPLC-QTof (Waters) mass spectrometer (MS). MS/MS peptide fragmentation spectra are matched to comprehensive protein databases such as Swiss-Prot and UniProt to identify the peptides that subsequently are used to compile a protein hit list.
The Proteomics Laboratory is also equipped with various devices for downstream validation study (ELISA, Western blot and immunohistochemistry). The laboratory is also in the BIONEXUS Partners (BNP) Program of the Malaysian Biotechnology Corporation. Under this initiative, the laboratory is available for researchers from the industry for a very competitive charge.