WO2004055510A2

WO2004055510A2 - Method and system for maldi mass spectrometry

Info

Publication number: WO2004055510A2
Application number: PCT/EP2003/014318
Authority: WO
Inventors: Jan Axelsson
Original assignee: Amersham Biosciences Ab
Priority date: 2002-12-17
Filing date: 2003-12-16
Publication date: 2004-07-01
Also published as: GB0229337D0; WO2004055510A3; AU2003296657A1

Abstract

Method and system for preparing for MALDI mass spectroscopy analysis a sample containing an analyte wherein the method comprises the steps of:estimating the amount of analyte contained in said sample,calculating the amount of matrix material needed to obtain a satisfactory matrix-to-analyte ratio, isolating a known amount of said analyte, and,combining it with the amount of matrix needed to obtain a satisfactory matrix-to-analyte ratio. A system comprises means (17) for calculating the amount of analyte material containedin a sample spot (19) in a gel, means for calculating the amount of matrix material required to obtain a satisfactory matrix-to-analyte ratio (17), means for isolating a known amount of said analyte (14) and matrix dispensing means (15) for adding the amount of matrix needed to obtain a satisfactory matrix-to-analyte ratio to the isolated known amount of analyte.

Description

METHOD AND SYSTEM FOR MALDI MASS SPECTROMETRY Field of the Invention

The present invention relates to methods and systems for preparing samples for matrix- assisted laser desorption/ionisation (MALDI) mass spectrometry.

Prior Art

Matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry is a method in which a crystallised matrix made of light-absorbing small molecules is excited by a short laser pulse that creates vibrational movement of the matrix molecules. This movement releases some of the matrix molecules at the surface, and if there are other molecules, e.g. molecules of a sample being tested, embedded in the matrix, then some of these embedded sample molecules are also dragged out into the surrounding vacuum of the ion source. At some point during this process, a fraction of the sample and matrix molecules gets singly ionised, and this fraction of molecules is accelerated out of the ion source for mass-to-charge ratio (M/Z) analysis, often in a time-of-flight (TOF) system.

Before being examined by MALDI mass spectroscopy the sample being tested has to be prepared so that it is in a suitable form for MALDI mass spectroscopy. An example of a way of preparing a sample is as follows: a biological sample containing molecules of interest, e.g. protein molecules, is prepared and the protein molecules separated, for example by 2D gel electrophoresis. This results in a gel body containing a plurality of distinct spots. Each distinct spot only contains molecules having similar separation properties, e.g. molecular weight, isoelectric point, size or the like. These spots can be made visible by staining and the co- ordinates of any spots of interest recorded. The gel can then be destained and then any spots of interest can be picked, for example, by a spot-picking needle that is adapted to remove plugs of gel from the gel body. Each picked spot can be then treated to release the molecules of interest from the gel plug, leading to a solution (or possibly a suspension) of molecules of interest. In order to determine the composition of the molecules of interest in the solution, the solution is subjected to further analysis. Such further analysis could include MALDI mass spectroscopy. In that case, the sample may be prepared as follows:

A predetermined volume, e.g. 0.5 μl of solution containing the analyte being tested is placed on a MALDI target slide; a similar volume of user-prepared laser light absorbing matrix solution is added to drop of analyte solution on the MALDI target slide; and, the solvent is allowed to evaporate leaving crystals of sample/matrix on the target slide. The target slide is then placed inside a MALDI mass spectrometer and analysed.

A problem that may occur with such a method is that the amount of matrix added to the sample is not adapted to the amount of analyte being tested. Usually the user prepares only one concentration of matrix solution and the same volume of this solution is added to every drop of analyte solution on the MALDI slide. No attempt is made to match the volume of matrix solution to the amount of analyte in the drop of analyte solution. If there is too much matrix in relation to the amount of analyte present in the sample, i.e. the ratio of matrix to analyte in the crystals on the target slide is too high then when the sample/matrix crystals are excited by a laser pulse the resulting ions are mainly comprised of ionised matrix material. These ions dominate the spectrum recorded by the mass spectrometer, causing high background chemical noise and reducing the signal-to-noise ratio of the mass spectrometer. This leads to reduced sensitivity.

If, on the other hand, there is not enough matrix in relation to the amount of analyte present in the sample, i.e. the ratio of matrix to analyte in the crystals on the target slide is too low, then when the sample/matrix crystals are excited by a laser pulse then only a small number of sample molecules are ionised leading to a weak spectrum being recorded by the mass spectrometer. An optimal matrix-to-sample ratio is in the order of 10000 matrix molecules per sample molecule.

Summary of the Invention

According to the present invention, at least some of the problems with the prior art are solved by means of a method having the features present in claim 1 and a system having the features mentioned in claim 6.

Brief Description of the Figures

Figure 1 shows schematically a view of a system in accordance with a first embodiment of the present invention. Detailed Description of Embodiment Illustrating the Invention

Figure 1 shows a first embodiment of a system 1 in accordance with the present invention. System 1 comprises a gel plate holder 3 for holding a gel plate 5, an imaging device 7 such as a camera or scanner 7 adapted to take images of a gel plate 5 when it is in said gel plate holder 3, and a robotic spot picker 9 (e.g. EttanSpotPicker TM from Amersham Biosciences AB, Uppsala, Sweden) for picking spots of gel 19 from said plate and dispensing them to wells in a microplate. System 1 further comprises a robotic sample extracting device 11 for extracting a sample from a gel spot in a well in a microplate (e.g. EttanDigester TM from Amersham Biosciences AB, Uppsala, Sweden which can prepare gel spots for enzymatic digestion of proteins for the subsequent extraction of peptides), an extraction container 13, for example a test tube, for containing the released sample and a device for dispensing matrix solution 15. System 1 also comprises a control device 17, such as a computer and software, adapted to control imaging device 7, spot picker 9, sample extracting device 11 and matrix dispensing device for dispensing matrix solution 15.

System 1 is controlled by control device 17 to perform the following steps: a gel plate 5, containing a gel 19 in which a sample has been separated, is placed onto gel plate holder 3 either manually or by an optional laboratory robot (not shown) controlled by a control device, such as control device 17; gel 19 is treated, preferably before being placed on gel plate holder 3, for example by staining or by the addition of markers visible to the imaging device, so that spots 21 of material from the separated sample are visible to imaging device 7; imaging device 7 images gel 19 and produces a digital image of gel 19 which can be processed by software in control device 17; software in control device 17 identifies spots of material from the separated sample in the digital image and records the position of each spot with respect to reference marks on the gel; means for calculating the intensity of a spot , e.g. the software in control device 17, calculates the intensity of each spot of interest - the definition of a spot of interest can either be pre- programmed or can be manually selected by an operator; means for calculating the amount of analyte in a spot ,e.g. the software in control device 17, calculates how much analyte is contained in each spot of interest; the software calculates how much matrix material is required to achieve an optimum matrix- to-sample ratio for the amount of analyte contained in each spot of interest; each spot of interest is picked from the gel by spot picker 9, the analyte is extracted from each spot of interest by sample extracting device 11 and a known amount (e.g. a known volume) of analyte from each spot is isolated, for example by being injected by a means 14 for isolating a known amount of analyte, e.g. a pipette, into its own individual extraction container 13; and, the matrix dispensing device 15 is then controlled by control device 17 to dispense from one or more matrix solution reservoirs 18, 18' to each isolated known volume of analyte the calculated amount of matrix for that isolated known volume of analyte.

The mixture of extracted analyte and matrix is subsequently analysed in a MALDI mass spectrometer.

In the above example, a known volume of analyte is isolated by being placed into an extraction container and then the matrix solution added to the container. Other ways of isolating a known volume of analyte and combining it with the necessary amount of matrix are also conceivable , for example, applying a known volume of analyte to a MALDI slide and allowing it to dry, and then adding matrix solution to the dried spot of analyte. Alternatively, a known amount of matrix material in solution may be applied to a MALDI slide, allowed to dry and then the appropriate volume of analyte solution applied to the dried matrix material.

A first embodiment of a device for dispensing matrix solution comprises a single reservoir containing matrix solution at a known concentration (e.g. 0.1 mg/ml or 1 mg/ml or lOmg/ml, etc) and a dispensing means such as a pump, motorised syringe, automated pipette or the like. As the concentration of matrix in the matrix solution is known then after the software has calculated the amount of matrix material required to achieve an optimum matrix-to-analyte ratio for a spot, it calculates the necessary volume of matrix solution at the known concentration that is needed to provide the calculated amount of matrix material. The control device then commands the device for dispensing matrix solution to dispense the calculated necessary volume of matrix solution to the isolated known volume of analyte. For example if 0.95 mg of matrix material is needed for the amount of analyte isolated in an extraction container and the matrix solution has a concentration of 0.1 mg/ml then the dispensing means would dispense 9.5 ml of matrix solution into the container container.

A second embodiment of a matrix dispensing device in accordance with the present invention comprises a plurality of reservoirs, each reservoir containing matrix solution at known concentrations and a dispensing means such as a single pump or syringe or pipette or the like able to access all the reservoirs, or a plurality of dispensing means such as pumps and/or syringes and/or pipettes or the like, each able to access some or all of the reservoirs. In the case of a plurality of dispensing means, preferably each of the dispensing means has a different dispensing capacity i.e. each dispensing means is designed for a different maximum dispensing volume (e.g. 1 ml, 10 ml) or dispensing flow rate (e.g. 0.1 ml/s, 1 ml/s, 10 ml/s) in order to allow maximum flexibility in dispensing matrix solutions. Preferably each reservoir contains matrix solution at a concentration which is different to the matrix concentrations in the other reservoirs (i.e. a first matrix solution reservoir contains matrix solution at a first concentration and a second matrix solution reservoir contains matrix solution at a second concentration). Most preferably the concentrations in the reservoirs differ by a factor of 10 between reservoirs, e.g. if there are three reservoirs then the first reservoir could have a matrix solution concentration of 0.1 mg/ml, the second reservoir could have a concentration of 1 mg/ml and the third reservoir could have a concentration of 10 mg/ml. As described above, dispensing of matrix material is controllable by the control device which can calculate the amount of matrix material needed and command the device for dispensing matrix solution to dispense the necessary amount. For example, if a dried spot of analyte on a MALDI slide requires 0.0095 mg of matrix material then the device for dispensing matrix solution could command a first pump to dispense 0.009 ml of 1 mg/ml matrix solution and a pipette to dispense 0.005 ml of 0.1 mg/ml matrix onto the spot on the MALDI slide. Alternatively, the device for dispensing matrix may instead use a pipette to dispense 0.0095 ml of 1 mg/ml matrix solution.

A third embodiment of a matrix dispensing device in accordance with the present invention comprises two matrix solution reservoirs. A first reservoir contains matrix solution at a known high concentration (e.g. 10 mg/ml) and a second reservoir containing matrix solvent. The device also comprises dispensing means such as a single pump or syringe or pipette or the like able to access both the reservoirs, or a pair of dispensing means such as pumps and/or syringes and/or pipettes or the like, each able to access one of the reservoirs. This device can be controlled by the control device to first dispense the required quantity of matrix material into a container containing the extracted analyte and then to dispense solvent so that the total volume of liquid in the extraction container is sufficient to allow a pre-programmed number of drops of analyte and matrix mixture to be dispensed. For example, if the average volume of a drop that is dispensed onto a MALDI target is 2 μl and the analysis requires that 10 drops of each analyte are to be tested then the minimum volume of analyte and matrix mixture required is 20 μl. However it can be very difficult to fill a dispensing means accurately when there is only 20 μl of liquid available therefore it is preferable to add extra solvent so that the total volume of fluid in the extraction container is large enough to handle ,e.g. 200 μl, and then to only use a fraction of the total volume. The remaining analyte can then be stored for future use or used directly in other analytical systems.

h order to calibrate the software, i.e. to provide the software with means for determining the amount of sample material present in a spot, it is conceivable to add a known amount of a marker substance, e.g. a protein of known properties, which is not expected to occur in the sample being tested, to the sample being tested. The software can then identify the spot resulting from the marker sample after it has been separated in the gel, measure the intensity of the spot and correlate the intensity of the spot against the known amount of the marker substance. Optionally two or more marker substances can be used to assist in calibrating the software.

The above mentioned embodiments are merely intended to illustrate the present invention and are not intended to limit the scope of protection claimed by the following claims.

Claims

1. Method of preparing for MALDI mass spectroscopy analysis a sample containing an analyte comprising the steps of: estimating the amount of analyte contained in said sample, calculating the amount of matrix material needed to obtain a satisfactory matrix-to-analyte ratio, isolating a known amount of said analyte, and, combining it with the amount of matrix needed to obtain a satisfactory matrix-to-analyte ratio.

2. Method in accordance with claim 1 characterised by the step of preparing one or more solutions of matrix in a solvent at known concentrations and adding a volume of at least one of said solution or solutions of matrix at known concentrations to said isolated known amount of analyte until a satisfactory matrix-to-analyte ratio is achieved.

3. Method in accordance with any of the previous claims characterised in that said isolated known amount of analyte is isolated in a container and the amount of matrix needed to obtain a satisfactory matrix-to-analyte ratio to said isolated known amount of analyte is added to said container.

4. Method in accordance with any of claims 1-3 characterised in that said isolated known amount of analyte is isolated on a slide, allowed to dry and the amount of matrix needed to obtain a satisfactory matrix-to-analyte ratio to said isolated known amount of analyte is added to the dried analyte.

5. Method in accordance with any of claims 1-3 characterised in that said a known amount of matrix is isolated and the amount of analyte needed to obtain a satisfactory matrix-to-analyte ratio is combined with said known amount of matrix.

6. Method in accordance with claim 5 characterised in that said known amount of matrix is isolated on a slide, allowed to dry, and the amount of analyte needed to obtain a satisfactory matrix-to-analyte ratio is added to the dried matrix.

6. System for preparing a sample for MALDI mass spectroscopy characterised in that it comprises means (17) for calculating the amount of analyte material contained in a sample spot (19), means for calculating the amount of matrix material required to obtain a satisfactory matrix-to-analyte ratio (17), means for isolating a known amount of said analyte (14) and matrix dispensing means (15) for adding the amount of matrix needed to obtain a satisfactory matrix-to-analyte ratio to the isolated known amount of analyte.

7. System in accordance with claim 6 characterised in that it comprises a plurality of matrix solution reservoirs, wherein a first matrix solution reservoir contains matrix solution at a first concentration and a second matrix solution reservoir contains matrix solution at a second concentration.