PROCESS AND APPARATUS FOR SEPARATING, ISOLATING AND ANALYSING BIO-MOLECULAR MATERIAL

Abstract: A process for isolating a bio-molecule from a sample of bio-molecules comprising the steps of: (a) separating the sample of bio-molecules so that the bio-molecules are displaced at separate locations in or on a support, (b) excising a selected first region of the support, (c) excising at least one further region of the support which is adjacent to but outside of the first region, (d) ascertaining the molecular profiles of the first and the at least one further region(s) of the support, and (e) analysing a bio-molecule as a function of the difference between the molecular profiles of the first and the at least one further region(s) of the support. The molecular profile of the first and the at least one further region(s) of the support is typically determined by mass spectrometry (including electrospray ionisation and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry), liquid chromatography or capillary electrophoresis or any combination of these means. Also claimed is an apparatus for carrying out the above process.

Description: PROCESS AND APPARATUS FOR SEPARATING, ISOLATING AND ANALYSING BIO- MOLECULAR MATERIAL FIELD OF THE INVENTION The invention relates to a process for isolating and analysing a bio-molecule and an apparatus for use in the process . BACKGROUND OF THE INVENTION The analysis of a cellular proteome requires a system which facilitates the rapid isolation of bio-molecules which are expressed in a cell. The system should have the capacity to isolate all species of bio-molecule, regardless of the relative abundance of each species of bio-molecule in the cell. A bio-molecule present in a sample of heterogenous bio- molecules may be isolated by separating the bio-molecules in the sample according to their physical characteristics and excising selected areas of a support that carry the bio-molecule . Separation of the sample of heterogenous bio-molecules is typically achieved by effecting two-dimensional gel electrophoresis of the sample in or on a support comprising, for example, polyacrylamide, agarose, nitrocellulose, polyvinyldiflouridine (PVDF) or teflon. Electrophoresis of the sample in a first dimension separates the bio-molecules in the sample so that each bio- molecule is displaced at a location in or on the support from other bio-molecules in the sample according to molecular weight. Electrophoresis in a second dimension separates the bio-molecules in the sample so that each bio- molecule is displaced at a location in or on the support from other bio-molecules in the sample according to molecular weight and molecular charge characteristics. The isolation of a bio-molecule from the sample is completed by excising selected areas of the support which contain the bio-molecule. The excising function is typically effected by a coring means and may be automated, for example by the use of a computer driven coring device. The employment of two-dimensional electrophoresis combined with an automated excision process allows rapid isolation of bio-molecules which have a high relative abundance in a sample. However, it is difficult to apply the procedure to the isolation of bio-molecules which have a low relative abundance in a sample. Two-dimensional electrophoresis of a complex mixture of heterogenous bio-molecules results in the displacement of bio-molecules which have similar molecular weight and molecular charge characteristics to closely adjacent locations on the support. However, this separation can be imperfect and bio-molecules may be associated with contaminating agents. The problem is exacerbated when the bio-molecule to be isolated is of low relative abundance. Further, two-dimensional gel electrophoresis produces an atypical gradient of electrophoresis buffer concentrations and an atypical concentration of reagents that are used prior to the separation of the bio-molecules in the support. Thus an excised portion of the support is rarely, if ever, perfectly pure with respect to the bio-molecule contained in the excision. Thus, when the bio-molecule is present at low abundance in a cell or is at low concentration in the excised portion, it is difficult to determine isolation of the bio-molecule. This problem is exacerbated further by contaminants introduced during chemical processing and sample handling prior to subsequent analysis, by contaminating agents present in water, buffers and chemicals employed for endoproteinase digestion, for example . As a consequence, a serious limitation applies to the usefulness of the combination of two-dimensional electrophoresis and excision in the analysis of a cellular proteome. This is particularly so because usually about 10% of proteins present in any biological sample represent about 50% of the protein content of cells. There is a need for a system which is suitable for the analysis of a cellular proteome. SUMMARY OF THE INVENTION The present invention seeks to minimise the above- mentioned limitation by providing a process for isolating a bio-molecule from a sample of bio-molecules and which comprises the steps of : (a) separating the sample of bio-molecules so that the bio-molecules are displaced at separate locations in or on a support , (b) excising a selected first region of the support, (c) excising at least one further region of the support which is adjacent to but outside of the first region, (d) ascertaining the molecular profiles of the first and the at least one further region (s) of the support, and (e) isolating a bio-molecule as a function of the difference between the molecular profiles of the first and the at least one further region (s) of the support. As indicated previously, the region of the support on which a bio-molecule is located will typically contain the bio- molecule the subject of the isolation and, also, contaminating agents. The region may include contaminants introduced during preparative steps prior to ascertaining the molecular profile of each region. By comparing and differentiating the molecular profiles of the first and the at least one further region (s), the process of the invention enables the isolation of a bio-molecule regardless of the presence of contaminating agents that influence the molecular profiles of the first and the at least one further region (s). This is particularly useful for the isolation of low abundance proteins which are otherwise difficult to detect against the contaminating agents. An advantage of the process is that it provides for increased sensitivity of detection of bio-molecules in a high throughput assay format . In one embodiment, the first region is selected as one at which a bio-molecule is perceived to located. In another embodiment, one only further region of the support is excised. The process which is used to separate the sample of bio- molecules is dependent on the type of bio-molecules comprised in the sample. In one embodiment, the bio- molecules are separated by two dimensional gel electrophoresis. As noted above, two dimensional gel electrophoresis is capable of separating a bio-molecule from other bio-molecules according to the physical characteristic of the bio-molecule. When two-dimensional gel electrophoresis is used to separate the bio-molecule from the bio-molecules in the sample, the bio-molecules are separated according to molecular weight and molecular charge characteristics. It is to be understood however, that the bio-molecules may be separated according to molecular weight or molecular charge characteristics only, or any other characteristic which is capable of being exploited so as to separate the bio-molecule from the bio- molecules in the sample. Further, the type of support which is used in the separation process will depend on the type of bio-molecules comprised in the sample to be separated. The support may comprise a nitrocellulose, PVDF, teflon or other solid or particulate membrane or material. In one embodiment, the support comprises polyacrylamide or agarose . The bio- molecules may be separated on the support or in the support . Any bio-molecule, for example, a peptide or polypeptide, nucleic acid, lipid or sugar molecule, or combinations thereof can be isolated in accordance with the process of the invention. In one embodiment the bio-molecule is a polypeptide. The sample of heterogenous bio-molecules can be derived from any source. In one embodiment, the sample is derived from a cell, tissue or whole organism. The first and the at least one further region (s) of the support can be excised by any cutting means. In one embodiment, the support is excised by a coring device. In another embodiment the support is excised by a laser. Preferably the laser is a C0 2 impact laser. The use of a laser for the excision of the support comprising the bio-molecule to be isolated is advantageous because the bio-molecules are typically displaced in or on the support in an asymmetrical shape at the completion of the separation process. The laser can more easily excise an asymmetrical shape than a coring device. It is recognised however, that a coring device may be used in the process of the invention by multiple applications of the coring device so as to emulate more or less well the particular asymmetrical shape. A further advantage of the laser is that the circumference of the laser excision avoids unwanted dilution or loss of bio-molecules at the completion of the isolation process and also allows the excision of bio-molecules which have a low relative abundance in the sample . The molecular profile of the first and the at least one further region (s) of the support is typically determined by mass spectrometry, including electrospray ionisation and matrix-assisted laser desorption/ionisation time-of flight mass spectrometry, liquid chromatography and capillary electrophoresis and/or any combination of these means. The most suitable means are those which are able to detect all bio-molecules regardless of relative abundance, and all contaminating reagents introduced in the separation and/or preparative processes (for example, contaminating agents present in water, solvents, buffers, chemicals, enzymes), so that the signal to noise ratio can be enhanced. In a second aspect, the invention provides an apparatus for isolating a bio-molecule from a sample of bio-molecules comprising means for excising a selected first region of the support and for excising at least one further region of the support which is adjacent to but outside of the first region, and means for ascertaining the molecular profiles of the first and the at least one further regio (s) of the support and for isolating a bio-molecule as a function of the difference between the molecular profiles of the first and the at least one further region (s) of the support. Any means capable of excising the first and the at least one further region (s) of the support may be used in the apparatus of the invention. In one embodiment the means is a laser. Preferably, the laser is a C0 2 impact laser. The means for ascertaining the molecular profiles of the first and the at least one further region (s) of the support and for isolating a bio-molecule as a function of the difference between the molecular profiles of the first and the at least one further region (s) of the support is typically a mass spectrometer, alone or in combination with other analytical platforms, as described above. BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a representation of known methods of isolation of a species of bio-molecule after separation on a support. The empty circle represents a region excised from the support. The filled circle represents a species of bio- molecule at a region on the support. (A) shows excision of all bio-molecules of the species together with a surrounding region. (B) shows excision of some of the bio- molecules of the species together with adjacent regions. (C) and (E) show excision of some of the bio-molecules of the species. (D) shows excision of all bio-molecules of the species together with adjacent regions. In (A) to (E) , the excision process does not allow for optimal isolation of the species of bio-molecule, or for optimal signal to noise ratio. Figure 2 is a representation of the method of isolation of the invention. (A) The filled circles represent individual species of bio-molecules located at or on a first region of a support after separation. The empty circles represent regions excised from the support. (B) The filled circle shows a species of bio-molecule (protein) in an acrylamide gel after separation. (C) shows the optical intensity of the bio-molecule. Figure 3 is a representation of the method of isolation of the invention using (A) laser excision or (B) repeated coring of fixed size, of a first region carrying the bio- molecule and a second region adjacent the first region. (C1,C2 and C3) are representation of the mass spectrographs of the second and subsequent regions adjacent the first region. (C4) is a representation of a mass spectrograph of the first region. The molecular species marked with an asterisk are also present in Cl, C2 and C3 and are therefore contaminating molecules. Figure 4 shows a computer image of a first region carrying the product of E. coli K-12 gene Yid Q with an observed pi/ Mr (molecular mass) of respectively 6.2 / 15,000 Da (as determined by 2 dimensional electrophoresis) , and second an subsequent regions located adjacent to but outside the first region. Figure 5 (A, B, C and D) shows a mass spectrograph of second and subsequent regions adjacent but outside the first region shown in Figure 4 carrying the product of E. coli K-12 gene Yid Q. (E) shows a mass spectrograph of the first region carrying the product of E. coli K-12 gene Yid Q. DETAILED DESCRIPTION OF THE INVENTION A sample comprising the E. coli K-12 Yid Q gene product was electrophoresed by two dimensional gel electrophoresis and transferred to a support by standard techniques. Detection of stained gene product was conducted using imaging software (Phoretix Software, Newcastle, U. K. ) specifically designed for applications in two dimensional electrophoresis. Software controlling XYZ robotic space for membrane and liquid handling was supplied by Cyberlab (Connecticut) . The imaging software interacts with the software controlling robotic space by an interface. The region of the support carrying the gene product , and the regions adjacent but outside the region carrying the gene product were excised from the support using a Lumonics C02 impact laser (Ottawa, Canada) and were analysed on a Finnigan Mat LCQ Ion trap mass spectrometer using standard techniques .

Claims: WE CLAIM : 1. A process for isolating a bio-molecule from a sample of bio-molecules and which comprises the steps of: (a) separating the sample of bio-molecules so that the bio-molecules are displaced at separate locations in or on a support , (b) excising a selected first region of the support, (c) excising at least one further region of the support which is adjacent to but outside of the first region, (d) ascertaining the molecular profiles of the first and the at least one further region (s) of the support, and (e) isolating a bio-molecule as a function of the difference between the molecular profiles of the first and the at least one further region (s) of the support. 2. A process according to claim 1 wherein the first region is selected as one at which a bio-molecule is perceived to be located. 3. A process according to claim 1 or 2 wherein one only further region of the support is excised. 4. A process according to claim 1 wherein the bio- molecule in the sample is separated according to the molecular weight and/or molecular charge of the bio- molecule. 5. A process according to claim 4 wherein the bio- molecule is separated by 2 dimensional electrophoresis. 6. A process according to claim 1 wherein the bio- molecule is separated in or on the support. 7. A process according to claim 6 wherein the support is one selected from the group consisting of nitrocellulose, PVDF, teflon, agarose and polyacrylamide . 8. A process according to claim 1 wherein the bio- molecule is one selected from the group consisting of a peptide, polypeptide, nucleic acid, lipid, or sugar. 9. A process according to claim 8 wherein the bio- molecule is a polypeptide. 10. A process according to claim 1 wherein the sample is derived from a cell, tissue or whole organism. 11. A process according to claim 1 wherein the means for excising the first and/or the at least one further region (s) of the support is a coring device or a laser. 12. A process according to claim 11 wherein the laser is a C0 2 impact laser. 13. A process according to claim 1 wherein the molecular profile of the first and/or the at least one further region (s) of the support is ascertained by mass- spectrometry, chromatography, capillary electrophoresis, or any combination thereof. 14. An apparatus for isolating a bio-molecule from a sample of bio-molecules comprising means for excising a selected first region of the support and for excising at least one further region of the support which is adjacent to but outside of the first region, and means for ascertaining the molecular profiles of the first and the at least one further region (s) of the support and for isolating a bio-molecule as a function of the difference between the molecular profiles of the first and the at least one further region (s) of the support. 15. An apparatus according to claim 14 wherein the means for excising the first and/or the at least one further regio (s) of the support is a laser. 16. An apparatus according to claim 15 wherein the laser is a C0 2 impact laser. 17. An apparatus according to claim 14 wherein the means for ascertaining the molecular profiles of the first and/or the at least one further region (s) of the support is a mass spectrometer, means for chromatography, capillary electrophoresis, or any combination thereof.

Inventors: HUMPHERY-SMITH IAN, HUMPHERY-SMITH, IAN
Applicants: HUMPHERY SMITH IAN, UNIV SYDNEY, HUMPHERY-SMITH, IAN, THE UNIVERSITY OF SYDNEY
Application year: 2000
Main classification: G01N33
IPC codes: G01N27, G01N30, H01J49