Investigation into the feasibility of subcellular proteomics
Lead Research Organisation:
University of Surrey
Department Name: Chemistry
Abstract
The main aim of this research is to investigate the potential for subcellular proteomics (protein analysis) by using nanomanipulator-controlled extraction mass spectrometry methods.
Existing proteomic mass spectrometry techniques generally involve the pooling of cells/organelles. The advantage of analysis of single cells or single organelles is that, in particular, it allows observation of heterogeneity of different single cells of the same function, or of different single organelles of the same function. This has potential uses in disease diagnostics and monitoring.
We hope to achieve subcellular analysis through nanomanipulator-controlled extraction techniques such as Direct Analyte Probed Nanoextraction (DAPNe). DAPNe utilises a nanomanipulator unit mounted on a microscope slide. A capillary tip holder is attached to the nanomanipulator unit, and a pressure injector is attached to the capillary tip holder. The microscope is used to find a discrete region of interest, and the nanomanipulator is used to position the capillary tip in this discrete region of interest. The capillary tip has a 1 um internal diameter and is filled with approximately 10 uL of solvent. The pressure injector allows for the solvent to be injected either onto the surface, or inside a sample, and the resulting solvated sample is aspirated back into the capillary tip.
The capillary tip can then be transferred to a nano-electrospray ionisation source (NanoES, NSI) for direct introduction to the mass spectrometer, or the solvated sample can be dispensed into a well or a vial and subjected to liquid chromatograpy (LC) separation before being introduced to the mass spectrometer.
Further objectives of the research are to:
- Optimise the extraction conditions for proteins (successful extractions have been carried out using this technique for lipidomics (lipid analysis), and metabolomics (metabolite analysis)).
- Further reduce the dimensions of extraction without trading off with sensitivity.
- Evaluate the benefits of both NSI and LC and how both sample introduction methods are applicable.
- Compare the efficacy of techniques such as DAPNe against well-established proteomic techniques.
Existing proteomic mass spectrometry techniques generally involve the pooling of cells/organelles. The advantage of analysis of single cells or single organelles is that, in particular, it allows observation of heterogeneity of different single cells of the same function, or of different single organelles of the same function. This has potential uses in disease diagnostics and monitoring.
We hope to achieve subcellular analysis through nanomanipulator-controlled extraction techniques such as Direct Analyte Probed Nanoextraction (DAPNe). DAPNe utilises a nanomanipulator unit mounted on a microscope slide. A capillary tip holder is attached to the nanomanipulator unit, and a pressure injector is attached to the capillary tip holder. The microscope is used to find a discrete region of interest, and the nanomanipulator is used to position the capillary tip in this discrete region of interest. The capillary tip has a 1 um internal diameter and is filled with approximately 10 uL of solvent. The pressure injector allows for the solvent to be injected either onto the surface, or inside a sample, and the resulting solvated sample is aspirated back into the capillary tip.
The capillary tip can then be transferred to a nano-electrospray ionisation source (NanoES, NSI) for direct introduction to the mass spectrometer, or the solvated sample can be dispensed into a well or a vial and subjected to liquid chromatograpy (LC) separation before being introduced to the mass spectrometer.
Further objectives of the research are to:
- Optimise the extraction conditions for proteins (successful extractions have been carried out using this technique for lipidomics (lipid analysis), and metabolomics (metabolite analysis)).
- Further reduce the dimensions of extraction without trading off with sensitivity.
- Evaluate the benefits of both NSI and LC and how both sample introduction methods are applicable.
- Compare the efficacy of techniques such as DAPNe against well-established proteomic techniques.
Organisations
People |
ORCID iD |
| Melanie Bailey (Primary Supervisor) | |
| MASON MALLOY (Student) |