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Proteomics
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Proteomics

Overview of Proteomics »
Peptide Synthesis »
Ink-jet Chromatography »
Protein Analysis using the Chemical Ink-Jet Printer »
Protein Crystallization »


Overview of Proteomics

The term “proteome” was originally coined by an Australian scientist, Mark Wilkins (1995), to describe the "PROTEin complement of the genOME". The term "proteomics" is used to describe any and all of the collection of high throughput techniques that have emerged to enable scientists to analyze all the proteins expressed under a certain set of conditions within an individual cell or organism.

Proteomics involves the systematic separation, identification and characterization of the proteins present in a tissue or other biological sample. By comparing the proteins in samples from individuals affected by a particular disease with those present in samples from healthy individuals, it is possible to identify directly those proteins that are potentially related to that disease. These proteins may have commercial potential as targets for the development of drugs or as a basis for the development of diagnostics.

Although, the pattern of gene activity can be abnormal when a tissue is diseased, there is often a poor correlation between the level of activity of different genes and the relative abundance within the tissue of the corresponding proteins. Consequently, the information about a disease process that can be derived from analysis at the level of gene activity is limited. Moreover, since post-translational modifications are not directly encoded, knowing a gene sequence does not determine the complete structure of individual proteins encoded by that gene.

The analysis of proteins in tissues can be a laborious exercise in marked contrast with the sequence analysis of genes, which for several years has been a rapid automated process. For many years this meant that proteins were studied indirectly through their gene sequences. Proteomics has emerged as a field in which technology is being developed to enable proteins to be analyzed in a high throughput, automated way similar to how genes are being studied. This advance will have major implications for the understanding, diagnosis and treatment of disease and cancer including pharmaceutical research and development.

Ink-jet Chromatography

MicroFab Technologies has been awarded a NIH Phase II Small Business Innovative Research grant award for the development of a system to deliver microvolumes (0.1 to 100nL) of proteolysed peptides separated by liquid chromatography onto MALDI-TOF MS targets for mass spec analysis. Under this grant Microfab has developed an ink-jet microdispensing printhead having integrated liquid chromatographic capability. The piezoelectric liquid microdispenser having an integrated liquid chromatography (LC) column is for the purpose of separating and dispensing proteolysed peptides onto MALDI-TOF MS target plates for subsequent mass spec analysis. The figure below displays a magnified view of an ink-jet microdispensing with an integrated column producing drops of the eluted solution.



Left - An example of a glass capillary ink-jet device with an integrated liquid chromatography column (white area) dispensing elution buffer. Right (click on image to enlarge) - An overview of the ink-jet chromatography system (PiezoLC) showing the concept of using ink-jet devices to deliver of LC separated peptides to a MALDI-TOF target plate for MS analysis.



Left: Cross-section and porous structure of monolith. Right: FITC labeled angiotensin II captured on monolith in glass capillary.



Peptide mass fingerprinting (PMF) is a mass spectrometry (MS) protein identification technique based on peptide mass detection and is useful for differential expression analysis in the search for drug targets and for biomarker discovery. PMF by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) provides for protein identification in many instances. The efficiency of PMF experiments can be related to the number of proteolytic peptide fragments that are identified. When a larger number of peptides are detected the confidence level for protein identification derived from searching a primary sequence database is greater, and the sequence coverage for the protein increases. Maintaining the confidence level becomes more challenging when concentrations of protein in a sample are reduced, such as in the case with a limited number of cells or tissue. The problem of reduced peptide detection is further exacerbated by proton competition between the high and low abundance analyte ions. This can result in ion suppression and loss of detection of low abundance peptides or proteins, thus reducing the sequence coverage for the protein of interest. Small scale liquid chromatography (LC) separation can greatly enhance the detection of peptide and protein samples during MS analysis. The LC separation partitions the co-eluting low and high abundance analyte ions and reduces ion suppression effects. Ultimately this increases the sequence coverage for the protein of interest. A variety of small scale LC separation modes can be performed either separately or in combination and include affinity capture, ion-exchange, size exclusion chromatofocusing and reversed phase.

The quantity of protein and peptide materials available for PMF analysis is often limited and miniaturization of the fluid handling for the mapping process is desired. The PiezoLC inkjet device utilizes a polymeric monolithic column and requires a small volume of proteolysed material. Methacrylate-based polymeric separation media do not require the packing of beads or frits, which are difficult to incorporate in microfluidic devices and can interfere with fluid flow. The large pore size of the polymeric monolithic material results in low back-pressure, which is important for the efficient operation of the PiezoLC dispensing device. Polymerization by UV irradiation enables patterning and positioning of the monolithic column. Multiple chemistries/functions can be combined in one monolith with no dead volume between, i.e., strong cation exchange (SCX) and reversed phase (RP). Additionally, these columns are robust and have fast separation due to rapid convective mass transfer.

To perform the analysis, the peptide sample is loaded onto the integrated LC column (reversed phase). An elution buffer separates the peptides, as the buffer passes through the LC column. The eluted peptides exit the orifice of the piezoelectric device in the form of drops and are deposited onto a MALDI-TOF MS target plate. The peptide separation can be achieved through various mechanisms:

  1. Elution time: drops deposited later come from a solution that was in contact with the column for a longer period of time.
  2. Elution gradient: different solvent concentration can be used.
  3. Elution temperature: elution properties depend on the temperature and the microdispenser can be heated to the desired levels.

Bovine Serum Albumin (BSA) was used to obtain the standard peptide digest. The sequence coverage and the number of peptides identified for the isocratic-eluted BSA digest control was 30% and 17, respectively. The PiezoLC separation of the same BSA digest using a stepwise gradient elution resulted in sequence coverage of 56% with 38 peptides being identified This is an 87% increase in the sequence coverage and 124% increase in the number of peptides identified over the control percentage and number. The greatest number of BSA digest peptides identified was 19 during the 10% EBD1 gradient elution. No significant matches to BSA peptides were identified during the MASCOT search of the mass peak lists from the stepwise gradient elutions of 50%, 55%, 65% and 70%. These results (click here for spectra) were consistent with other PiezoLC BSA digest elution experiments.

Protein Analysis using the Chemical Ink-Jet Printer

The Chemical Ink-Jet Printer (ChIP) is the result of collaborative efforts between Proteome Systems, Ltd., (http://www.proteomesystems.com), MicroFab Technologies, Inc., and Shimadzu Biotech (http://www.shimadzu-biotech.net). These efforts have lead to the development of an instrument that delivers picoliter to nanoliter volumes of reagents or biochemicals to 2D-Page protein arrays that have been transferred to a PVDF membrane (see figure below). In essence this technology is a combination of a protein chip with 2 dimensional electrophoresis. The Chemical Ink-Jet printer utilizes piezoelectric drop-on-demand ink jet technology to deliver the small volumes of reagents and/or biofluids. It is possible to use this technology to perform in situ proteinase digests of selected membrane bound proteins with subsequent MALDI-TOF MS analysis directly from the membrane surface. Thus, the liquid handling steps associated with in gel digestion procedures are avoided. Additionally, it is possible to archive the sample retained on the protein blot for future interrogation.



Left - Close-up of the 4 ink jet device printhead positioned over a PVDF membrane during printing. Right - The Chemical Ink-jet Printer instrument [Images courtesy of Proteome Systems Ltd. and Shimadzu Biotech].

 

Additional Information (PDF)

Shimadzu Biotech brochure describing the Chemical Ink-Jet Printer.