Method and apparatus for maldi analysis

• US 20020005478A1
• Filed: 01/14/2000
• Published: 01/17/2002
• Est. Priority Date: 09/19/1996
• Status: Active Grant

• Current Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Original Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Inventors: Hillenkamp, Franz, Reuter, Dirk, Lough, David M., Higgins, G. Scott
• Granted Patent: US 6,423,966 B2
• Time in Patent Office: Days
• US Class Current: 250/288
• CPC Class Codes: B01J 2219/00387 Applications using probes
• CPC Class Codes: C40B 60/14 for creating libraries
• CPC Class Codes: H01J 49/0418 for laser desorption, e.g. matrix-assisted laser desorption/ionisation [MALDI] plates or surface enhanced laser desorption/ionisation [SELDI] plates
• CPC Class Codes: H01J 49/164 Laser desorption/ionisation, e.g. matrix-assisted laser desorption/ionisation [MALDI] sample holders H01J49/0418

Abstract

Matrix assisted laser desorption/ionization is performed in a manner to thermalize large analyte ions in a plume of desorbed material for spectroscopic analysis. The thermalized ions have a low or zero mean velocity and are presented at a well-defined instant in time, reducing artifacts and sharpening the spectral peaks. In one embodiment the light is delivered to a matrix or sample holder having a cover, baffle or compartment. The baffle or compartment impedes or contains a plume of desorbed material and the analyte undergoes collisions to lower its mean velocity and directionality. Thus “thermalized” the analyte ions are passed to a mass analysis instrument. In a preferred embodiment an optical fiber butts up against a thin transparent plate on which the specimen resides, with the matrix side in a vacuum acceleration chamber. A mechanical stage moves the specimen in both the x- and y- directions to select a point on the specimen which is to receive the radiation. The use of a fiber optic illuminator allows the entire stage assembly to be subsumed essentially within the dimensions of a conventional stage. In other embodiments, a thermalizing compartment is provided in a capillary tube about the end of the illumination fiber and the sample matrix is deposited along the inner cylindrical wall of the tube, so the capillary forms a migration path to the outlet for thermalization of the desorbed analyte. In other embodiments microstructures having the shape of a small lean-to, overhang or perforated cover plate, or providing a high aspect surface texture, provide the necessary containment to promote thermalization of the released analyte. A thin layer or cover of fibrous or permeable material may also be used to thermalize the analyte before mass analysis, and in other embodiment this material may also act as the substrate. An automated instrument may include a fixed array of illumination fibers which are illuminated at different times to eject samples from a corresponding array of points on the specimen.

Claims

1. An improved method for matrix assisted laser desorption/ionization (MALDI) of an analyte material, such method comprising the steps of

preparing a specimen comprised of a major portion of matrix material and a minor portion of analyte material, wherein said matrix and analyte are deposited as a sample on a support

illuminating said matrix such that material including the analyte is desorbed from a surface of the sample and ionized,

and initially confining said desorbed material such that desorption momentum is at least partially directionally randomized by collisional interaction

thereby conditioning the desorbed analyte to control its initial conditions for analysis.

2. The improved method of claim 1, further comprising the step of providing a collision buffer gas while confining said desorbed material, said collision buffer gas being selected from among gases having a property to promote at least one of

i) collisional dissociation,

ii) chemical ionization, and

iii) molecule-molecule or ion-molecule reaction with the analyte.

3. The method of claim 1, wherein the step of illuminating includes the step of illuminating with a beam output from an optical fiber.

4. The method of claim 3, wherein the step of illuminating is effected by imaging the fiber output end on the sample.

5. The method of claim 3, wherein the step of illuminating is effected directly by placing the fiber close to an optically transparent substrate carrying the sample and illuminating through the substrate.

6. The method of claim 1, wherein the step of confining is effected by confining with a baffle.

7. The method of claim 1, wherein the step of confining is effected by confining in a partially closed compartment.

8. The method of claim 1, wherein the step of confining includes permeating said desorbed material through a porous layer of material.

9. The method of claim 8, wherein said porous layer of material is the substrate.

10. The method of claim 1, wherein the step of confining is performed by depositing said specimen as a permeable bed on the support and illuminating an underside of the bed so that desorbed material is confined by the support.

11. The method of claim 1, wherein the step of confining includes confining with at least one electric potential defining structure, said electric potential defining structure having a voltage selected to provide a residence time effective for said ions to initially thermalize by collisional interaction.

12. The method of claim 1, wherein the step of confining is effected by applying an extraction voltage only after a delay following desorption of said material thereby only extracting or sampling a portion of desorbed material which has undergone collision.

13. A method of sampling a matrix assisted laser desorbed ionized material comprising the steps of

illuminating a matrix to desorb material, and

passing said material through a permeable barrier effective to at least partially thermalize the desorbed material thereby controlling initial conditions for analysis of said material.

14. Apparatus for providing an ionized sample of material to an analysis instrument such as a mass spectrometer or the like, the apparatus comprising

a stage for holding a specimen from which said sample is to be provided

means directed at the stage for illuminating the specimen to cause desorption of material from the sample and ionization thereof, whereby the desorbed material has a directional momentum, and

means for confining said desorbed material sufficiently to at least partially thermalize said directional momentum so that large molecules in said material are reduced in velocity,said means for confining having an outlet communicating with the analysis instrument to provide the large molecules of reduced velocity thereto.

15. Apparatus according to claim 14, wherein the means directed at the stage includes an optical fiber spaced from said specimen to illuminate a defined region thereon.

16. Apparatus according to claim 15, wherein said stage and said fiber are mounted for mutual relative motion to selectively scan the defined region of said specimen past an illumination position for sampling portions thereof.

17. Apparatus according to claim 14, wherein the means for confining includes porous material across a path to said outlet.

18. Apparatus according to claim 14, wherein the means for confining includes electrostatic means for maintaining desorbed material in a limited region to enhance collisional interaction before said outlet.

19. Apparatus according to claim 14, wherein the stage contains a conductive region defining a flat field adjacent said specimen.

20. Apparatus for matrix assisted laser desorption of a large molecule analyte, such apparatus comprising a support stage including means for holding a sample on a substrate in a defined region of the stage an optical fiber having a first end adapted to couple to a light source for receiving a light input, and a second end held by said stage in a position to illuminate a spot region on the sample, the stage positioning the second end so that its output beam passes through the substrate to directly illuminate said spot region on the sample with a spot of defined size at a fluence effective to desorb the analyte from the sample, whereby the desorbed material has a directional momentum and the apparatus further comprises a means for confining said desorbed material sufficiently to at least partially thermalize said directional momentum.

21. Apparatus according to claim 20, comprising a plurality of optical fibers in an array arranged to illuminate an array of separate spot regions of the sample, each fiber being separately actuable to desorb material from a respective spot region of the sample at a distinct time whereby plural samples of desorbed analyte may be provided at successive instants to a common mass analyzer.

22. Apparatus according to claim 20, wherein the stage is operative to move the substrate with the sample in at least two lateral dimensions relative to the second end of the optical fiber for aiming at arbitrary locations on the sample to desorb material.