Abstract:
A method of scanning a sample plate surface mask in an area adjacent to a conductive area using mass spectrometry is disclosed. The method comprises the steps of providing a sample plate including a mask applied with a rough surface to the electrically conductive surface to produce a sample site comprising a central portion formed from the electrically conductive surface and a marginal portion of the mask, preparing an analyte comprising mixing a biomolecule with an organic solvent, an aqueous solution, and a matrix selected from the group of α-cyano-4-hydroxycinnamic acid and 3,5-dimethoxy-4-hydroxycinnamic acid; applying the analyte to the sample site; forming at least one crystal of the analyte in an area on the mask adjacent to the conductive area, and scanning the area on the mask adjacent to the conductive area with a laser beam.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application is a divisional of allowed U.S. patent application Ser. No. 10/426,226 filed Apr. 30, 2003, the content of which is incorporated herein by reference. 
     
    
     FIELD OF THE INVENTION 
       [0002]    The invention relates to a method of analyzing a sample plate in mass spectrometry using a sample plate having a rough hydrophobic surface. 
       BACKGROUND OF THE INVENTION 
       [0003]    Matrix-assisted laser desorption and ionization mass spectrometry (MALDI-MS) is an important analytical tool for the study and identification of biomolecules, particularly proteins, peptides, and nucleic acids such as DNA and RNA. 
         [0004]    MALDI-MS results in a mass spectrum that graphically identifies biomolecules according to peaks that correspond to the biomolecules&#39; concentration and mass. Using a library of known peaks, the biomolecules can be identified. 
         [0005]    Various methods exist for the preparation of samples for analysis by MALDI-MS, including the dried droplet method. In the dried droplet method an aqueous sample containing the subject biomolecule is mixed with an organic compound, the matrix, which is usually suspended in an easily evaporative aqueous-organic solvent. The resulting liquid mixture containing the biomolecule, the matrix, aqueous solution, and solvents is referred to herein as the specimen. 
         [0006]    The specimen is applied to the sample plate in a predetermined target area and allowed to dry. As the solvent begins to evaporate, and the biomolecule and matrix become more concentrated, the matrix molecules crystallize from solution while drying on the sample plate. The resulting crystals entrap the biomolecules on and/or within the crystals and in due course deposit on the sample plate. 
         [0007]    Other methods of applying the specimen to the sample plate are also known. In the electrospray deposition method, the specimen is applied to the sample plate as a fine mist of microdroplets that evaporates very quickly forming the specimen crystals. 
         [0008]    To analyze the biomolecules, the sample plate is inserted into the sampling compartment of a mass spectrometry instrument. A voltage is applied to the sample plate to permit the flow of electric current over the sample plate and prevent the possibility of an electrical charge buildup. To desorb the crystals, an ultra-violet (UV) laser scans the target area either by manual direction or in a predetermined automated fashion to irradiate the crystals. The laser beam radiation is absorbed by the matrix molecules, resulting in a vaporization of both the matrix molecules and the biomolecules. Once in the vapor phase, while still in close proximity to the target area, a charge transfer occurs as the matrix molecule loses a proton to the biomolecule. The ionized biomolecules are then drawn into the mass spectrometer where they are analyzed. Data processing yields a mass spectrum of a series of characteristic peaks corresponding to the biomolecules and matrix molecules. The signature of peaks is used to identify the biomolecules by reference to known peaks. 
         [0009]    Prior art of interest includes U.S. Pat. No. 6,287,872 (herein incorporated by reference) relating to sample support plates for the mass spectrometric analysis of large molecules, such as biomolecules, methods for the manufacture of such sample support plates and methods for loading the sample support plates with samples of biomolecules from solutions together with matrix substance for the ionization of the biomolecules using matrix-assisted laser desorption (MALDI). 
         [0010]    Also of interest is U.S. Pat. No. 5,958,345 (herein incorporated by reference) relating to a sample support for holding samples for use with an analysis instrument. The sample support is for use with analysis instruments, which rely on a beam of radiation or accelerated particles and a method for making the same. The holder includes a frame with one or more orifices covered by a support surface, typically in the form of a thin polymer film. The film is divided into hydrophobic and hydrophilic portions to isolate precise positions where samples can be placed to intersect a probe beam during analysis. 
         [0011]    MALDI-MS performance suffers chiefly from analysis insensitivity. The sample plates that are used in MALDI-MS are typically metallic plates due to the need to apply a voltage across the plate. Known trays have a smooth hydrophilic surface where the applied specimen drop spreads over a relatively large area before drying and forming crystals. Consequently, to effectively irradiate the crystals the UV laser has to scan this enlarged area requiring extra time. 
         [0012]    Another drawback of metallic plates is that they unfortunately often provide unsuitable results due to unintentional contamination from detergents. Since, metallic plates are also expensive, they are used repeatedly. Washing between each use may contaminate subsequent analysis. 
         [0013]    It is known, that specimens are non-homogeneously distributed on and/or within the lattice that located at the specimen periphery. It is further known that some of these matrix crystals bear more biomolecules than others. Thus, as the laser covers a likely search area at the specimen periphery, it scans “sweet spots” having a comparatively higher specimen concentration in the matrices. When irradiated and detected, the sweet spots provide an inaccurate concentration reading of the biomolecule. 
         [0014]    What is desired, therefore, is a sample plate for MALDI-MS analysis of a specimen wherein crystals are located in a sample site. 
         [0015]    What is also desired is a durable and cost effective sample plate which enables archiving of samples. 
         [0016]    What is also desired is a rough surface that is hydrophobic to enhance the formation of crystals in a sample site. What is further desired is a higher ratio of surface area to planar area of the hydrophobic mask. 
       SUMMARY OF THE INVENTION 
       [0017]    Accordingly it is an object of the invention to provide a method of analyzing a specimen. 
         [0018]    Another object of the invention is to provide a method of sample plate that overcomes known problems of analysis insensitivity. 
         [0019]    A further object of the invention is to provide a sample plate wherein crystals are located at predetermined positions. 
         [0020]    A still further object of the invention is to provide a durable and cost effective sample plate which enables archiving of samples. 
         [0021]    These and other objects of the invention are achieved by a method that crystallizes analyte in a reduced area. 
         [0022]    Therein, a method of scanning a sample plate surface mask in an area adjacent to a conductive area using mass spectrometry is disclosed. The method comprises the steps of providing a sample plate including a mask applied with a rough surface to the electrically conductive surface to produce a sample site comprising a central portion formed from the electrically conductive surface and a marginal portion of the mask, preparing an analyte comprising mixing a biomolecule with an organic solvent, an aqueous solution, and a matrix, applying the analyte to the sample site; forming at least one crystal of the analyte in an area on the mask adjacent to the conductive area, and scanning the area on the mask adjacent to the conductive area with a laser beam. 
         [0023]    In some embodiment the step of scanning the area on the mask adjacent to the conductive area with a laser beam further comprises scanning in a pattern. 
         [0024]    In some embodiments the method further comprises the step of determining a scanning pattern based on an algorithm having a confidence level. 
         [0025]    In some embodiments the step of preparing a specimen further comprises providing a matrix selected from the group consisting of α-cyano-4-hydroxycinnamic acid and 3,5-dimethoxy-4-hydroxycinnamic acid. 
         [0026]    In some embodiments the step of preparing a specimen further comprises providing an organic solvent. 
         [0027]    In some embodiments the step of preparing a specimen further comprises providing an aqueous solution. 
         [0028]    In some embodiments the step of preparing a specimen further comprises providing analyte wherein the analyte is at least one biomolecule. 
         [0029]    In some embodiments the step of preparing a specimen further comprises providing analyte wherein the analyte is selected from the group consisting of oligonucleotides, DNA, RNA, peptide, polypeptide, oligopeptide, protein, glycoprotein, lipoprotein, carbohydrate, monosaccharide, disaccharide, polysaccharide, and mixtures thereof. 
         [0030]    In some embodiments the method comprises applying a specimen to sample plate surface mask in an area adjacent to a conductive area comprising the steps of: providing a sample plate including a mask applied with a rough surface to an electrically conductive surface to produce a sample site comprising a central portion formed from the electrically conductive surface and a marginal portion of the mask; providing a liquid chromatography system for preparing a specimen comprising eluting a biomolecule with an organic solvent, an aqueous solution, and a matrix and applying the specimen; moving the sample plate cooperatively with the liquid chromatography system; and applying the specimen to the sample site. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0031]      FIG. 1   a  is an isometric view of a sample plate with a circular target area in accordance with one embodiment of the invention. 
           [0032]      FIG. 1   b  is an isometric view of a sample plate with a rectangular target area in accordance with one embodiment of the invention. 
           [0033]      FIG. 2   a  is an enlarged view taken at area A-A of  FIG. 1   a  of a section of a circle sample plate in accordance with one embodiment of the invention. 
           [0034]      FIG. 2   b  is an enlarged view taken at area A-A of  FIG. 1   b  of a section of a channel sample plate in accordance with one embodiment of the invention. 
           [0035]      FIG. 2   c  is a plan view of a circle sample site that includes a target area and a mask spot in accordance with one embodiment of the invention. 
           [0036]      FIG. 3   a  is a cross-section at section B-B of  FIG. 2   a  of a section of a circle sample plate in accordance with one embodiment of the invention. 
           [0037]      FIG. 3   b  is a cross-section at section B-B of  FIG. 2   b  of a section of a channel sample plate in accordance with one embodiment of the invention. 
           [0038]      FIG. 3   c  is an expanded elevation view of a circle sample site that includes a target area and a mask spot in accordance with one embodiment of the invention. 
           [0039]      FIG. 4   a  is a cross-section at section B-B of  FIG. 2   a  of a section of a circle sample plate with an electrically conductive coating applied to the substrate in accordance with one embodiment of the invention. 
           [0040]      FIG. 4   b  is a cross-section at section B-B of  FIG. 2   b  of a section of a channel sample plate with an electrically conductive coating applied to the substrate in accordance with one embodiment of the invention. 
           [0041]      FIG. 5   a  is an enlarged view taken at area A-A of  FIG. 1   a  of a circle sample plate in accordance with one embodiment of the invention wherein specimens have been applied on sample sites. 
           [0042]      FIG. 5   b  is an enlarged view taken at area A-A of  FIG. 1   b  of a channel sample plate in accordance with one embodiment of the invention wherein specimens have been applied on sample sites. 
           [0043]      FIG. 6   a  is a cross-section at section C-C of  FIG. 5   a  of a section of a circle sample plate in accordance with one embodiment of the invention wherein specimens have been applied on sample sites. 
           [0044]      FIG. 6   b  is a cross-section at section C-C of  FIG. 5   b  of a section of a channel sample plate in accordance with one embodiment of the invention wherein specimens have been applied on sample sites. 
           [0045]      FIG. 6   c  is a cross-section at section D-D of  FIG. 5   b  of a section of a channel sample plate in accordance with one embodiment of the invention wherein specimens have been applied on sample sites. 
           [0046]      FIG. 7   a  is an enlarged view taken at area A-A of  FIG. 1   a  of a circle sample plate in accordance with one embodiment of the invention wherein specimens of  FIG. 6   a  have begun to dry. 
           [0047]      FIG. 7   b  is an enlarged view taken at area A-A of  FIG. 1   b  of a channel sample plate in accordance with one embodiment of the invention wherein specimens of  FIG. 6   b  have begun to dry. 
           [0048]      FIG. 8   a  is a cross-section at section E-E of  FIG. 7   a  of a section of a circle sample plate in accordance with one embodiment of the invention wherein specimens of  FIG. 6   a  have begun to dry. 
           [0049]      FIG. 8   b  is a cross-section at section E-E of  FIG. 7   b  of a section of a channel sample plate in accordance with one embodiment of the invention wherein specimens of  FIG. 6   b  have begun to dry. 
           [0050]      FIG. 8   c  is a cross-section at section F-F of  FIG. 7   b  of a section of a channel sample plate in accordance with one embodiment of the invention wherein specimens of  FIG. 6   b  have begun to dry. 
           [0051]      FIG. 9   a  is an enlarged view taken at area A-A of  FIG. 1   a  of a circle sample plate in accordance with one embodiment of the invention wherein specimens of  FIG. 6   a  have dried. 
           [0052]      FIG. 9   b  is an enlarged view taken at area A-A of  FIG. 1   b  of a channel sample plate in accordance with one embodiment of the invention wherein specimens of  FIG. 6   b  have dried. 
           [0053]      FIG. 10   a  is a cross-section at section G-G of  FIG. 9   a  of a section of a circle sample plate in accordance with one embodiment of the invention wherein specimens of  FIG. 6   a  have dried. 
           [0054]      FIG. 10   b  is a cross-section at section G-G of  FIG. 9   b  of a section of a channel sample plate in accordance with one embodiment of the invention wherein specimens of  FIG. 6   b  have dried. 
           [0055]      FIG. 10   c  is a cross-section at section H-H of  FIG. 9   b  of a section of a circle sample plate in accordance with one embodiment of the invention wherein specimens of  FIG. 6   b  have dried. 
           [0056]      FIGS. 11 and 12  are isometric views of crystals that have crystallized in a halo effect around a sample site with different concentrations of matrix formulations in accordance with one embodiment of the invention. 
           [0057]      FIG. 13  is a cross-sections at section E-E of  FIG. 5   a  of a section of a sample plate in accordance with one embodiment of the invention wherein crystals are being scanned by an UV laser. 
           [0058]      FIGS. 14   a  and  14   b  are enlarged views of area A-A of  FIG. 1   a  of a circle sample plate wherein a path, in accordance with one embodiment of the invention, to irradiate crystals produced using CHCA or SA is illustrated. 
           [0059]      FIGS. 15   a  and  15   b  are enlarged views of one sample site of a channel sample plate wherein a path, in accordance with one embodiment of the invention, to irradiate crystals produced using CHCA or SA is illustrated. 
           [0060]      FIG. 16  is a photograph of a mask having a rough surface in accordance with one embodiment of the present invention. 
           [0061]      FIG. 17  is a photograph showing crystals that have crystallized around a sample site in accordance with one embodiment of the invention. 
           [0062]      FIG. 18  is a photograph showing crystals that have crystallized around a sample site in accordance with one embodiment of the invention using a specimen different than that shown in  FIG. 17 . 
           [0063]      FIG. 19  is a photograph showing DHB crystals in target area having large surface area. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0064]      FIGS. 1   a,    2   a,    3   a,  and  4   a  and  FIGS. 1   b,    2   b,    3   b,  and  4   b  are views of a sample plate with at least one circular target area and rectangular target area, respectively, in accordance with one or more embodiments of the invention. 
         [0065]    Therein,  FIGS. 1   a  and  1   b  are isometric views of sample plates with a circular target area and rectangular target area, respectively, in accordance with one or more embodiments of the invention. Sample plate  10  is characterized by any number of equally preferred embodiments of target area  24 . 
         [0066]    In one embodiment, referred to for simplicity as a circle sample plate  10 , and depicted in  FIG. 1   a,  sample plate  10  has a plurality of circular target areas  24 . In the second embodiment, referred to for simplicity as a channel sample plate  10 , and depicted in  FIG. 1   b,  sample plate  10  has a plurality of rectangular, linear, and/or curvilinear target areas  24 . Other embodiments, including combinations of geometries of target areas  24 , are also contemplated. 
         [0067]      FIGS. 2   a  and  2   b  are enlarged views taken at area A-A of  FIGS. 1   a  and  1   b,  respectively, of a section of a sample plate in accordance with one or more embodiments of the invention.  FIGS. 3   a  and  4   a  and  FIGS. 3   b  and  4   b  are cross-sections at section B-B of  FIGS. 2   a  and  2   b,  respectively, of a section of a sample plate in accordance with one or more embodiments of the invention. 
         [0068]    Sample plate  10  is a sample plate for applying a sample containing both matrix and biomolecules, referred to for convenience as specimen  40  (not shown for clarity in  FIGS. 1   a  and  1   b ), for subsequent analysis in a mass spectrometry instrument within a sample site  20  (not shown for clarity in  FIGS. 1   a  and  1   b ). Specimen  40  may be applied within sample site  20  by using the dried droplet method by spotting, i.e. in drop form, by streaking, i.e. in a continuous manner, by spraying, and/or any other form. Specimen  40  may also be applied within sample site  20  by the electrospray deposition method. 
         [0069]    Sample plate  10  includes substrate  12  having electrically conductive surface  12   a  and mask  14  which is selectively applied to surface  12   a  to form a mask that has a rough surface  14   a  where at least one target area  24  is located within sample site  20 , as will be explained further herein. 
         [0070]    Sample plate  10  is sized appropriately for usage for biological laboratory processing using automated and/or manual processing equipment. Thus, sample plate  10  may be appropriately sized as microtiter plate size comprising a rectangular plan size of 116.2 mm by 83 mm and/or any other convenient size. Sample plate  10  may be any suitable thickness for automated and/or manual processing. In accordance with one embodiment of the invention sample plate  10  is at minimum 0.5 mm thick with a maximum planar variance of 50 μm or less. For clarity, herein, sample plate  10  is described in relation to a rectangular plan and generally planar shape of the plate. However, sample plate  10  may have any plan shape and/or may have non-planar shapes as are and/or may become appropriate for usage. 
         [0071]    In accordance with one embodiment of the invention, sample plate  10  has an indicator, such as a notched corner, that aids in orienting sample plate  10 . Other indicators may instead or in addition be a central notch; one or more physical, chemical, optical, and/or electromagnetic markers; and/or any other type of indicator or indicating and/or orienting means. 
         [0072]    In accordance with one embodiment of the invention, sample plate  10  has a reference indicator for inventorying or archiving sample plate  10  before and/or after usage. Such an indicator may be a bar tag, alpha-numeric reference, chemical and/or luminescent reference, and/or any indexing and/or archival reference that is readable by a machine and/or a human, attached to and/or integral with sample plate  10  on one or more of its surface. 
         [0073]    In accordance with one embodiment of the invention, the reference indicator is sensitive to one or more wavelengths of the UV laser used in ionizing the crystals. Therein, the reference indicator is activated and/or marked by the UV laser leaving a permanent or semi-permanent reference readable by a machine and/or a human. 
         [0074]    Substrate  12  is preferably substantially planar and is made of any solid material and/or combination of material. Substrate  12  has a first surface  12   a  that is electrically conductive. Surface  12   a  has an electrical resistance of 100 meg. ohms-per-square or less. 
         [0075]    As illustrated in  FIGS. 3   a  and  3   b,  substrate  12  may be made of electrically conductive materials; as for example using metals, metal alloys, electro-conductive plastics, and/or combinations thereof. In accordance with one embodiment of the invention, surface  12   a  is made electrically conductive using an electrically conductive coating  16  that is applied to substrate  12 , as depicted in  FIGS. 4   a  and  4   b.  Coating  16  may be any type of applied mass that has an electrical resistance of 100 meg. ohms-per-square or less. Preferably, coating  16  maintains the substantially planar shape of substrate  12 . Coating  16  may be gold, copper, copper alloy, silver alloy, silver plating, conductive plastic, or a conductive polymer coating of any type. Preferably, the polymer coating includes Baytron P (3,4-polyethylenedioxythiophene-polystyrenesulfonate in water), CAS # 155090-83-8; polypyrrole, CAS # 30604-81, as a five percent (5%) water solution, or in a solvent-based solution; polyaniline as an emeraldine base, CAS # 5612-44-2; polyaniline as an emeraldine salt, CAS # 25233-30-1; and/or variants of polythiophenes, polyphenylenes, and/or polyvinylenes. 
         [0076]    Mask  14  is selectively applied to surface  12   a  to form a mask that has a rough surface  14   a  wherein target area  24  is centrally located within sample site  20 . Preferably, mask  14  has a thickness in the range of 1 to 100 μm and is made of a material that is relatively more hydrophobic than surface  12   a  and that maintains a suitable bond with substrate  12 . For example, mask  14  may be made of polytetrafluoroethylene, commonly known as Teflon® and manufactured, sold, and/or licensed by DuPont Fluoroproducts of Wilmington, Del., or any other suitable material. 
         [0077]    Rough surface  14   a  is a non-homogenous surface that is characterized by a coarse and/or an uneven surface quality and that is lacking uniform surface intensity, regardless whether surface  14   a  has a regular or repeating pattern or patterns of intensity, i.e. depth and/or graduations of the surface and/or material thickness. 
         [0078]    In accordance with one embodiment of the invention, mask  14  may be adulterated, i.e. doped, with one or more marking agents that upon mass spectrometric analysis is/are detected as one or more markers as a predetermined analytical result or is detected by another means such as visual reference by an operator who sees the color effect of a marking agent. Such marking agents may be used for instrument calibration; quality assurance of sample preparation, handling, laboratory procedures, and/or sample tracking; quality assurance during production of sample plate  10 ; and/or handling. Examples of marking agents may be carbon black, titanium oxide, ferrous oxide, aluminum trioxides, polymeric materials, coloring materials, and/or others. 
         [0079]    In accordance with one embodiment of the invention, mask  14  is applied to surface  12   a  with a predetermined rough surface  14   a.  For example, mask  14  is applied using a screening application process resulting in rough surface  14   a.  Preferably, mask  14  is applied utilizing Teflon® with a screen mesh sizes ranging from 30×30 μm to 500×500 μm such resulting rough surface being described by the mesh size. Other screen sizes may be employed equally well. Upon screening, sample plate  10  is allowed to air dry, and once dry is heated to at least 50 Celsius to bond mask  14  with substrate  12 . Referring to  FIG. 16 , a microscopic photograph of mask having rough surface is shown. The mask is made of black Teflon and is shown having a matted appearance. In this case, the matted appearance shows repeating substantially square shaped imperfections to the polymer surface substantially similar in size and shape as the mesh screen applied to the mask to form rough surface. 
         [0080]    In accordance with one embodiment of the invention, physical and/or chemical manipulation of the material of mask  14  is used to texture and create a rough surface  14   a.  For example etching, gouging, scraping, oxidation, photo-oxidation, lithographic printing, off-set printing, reverse image accessing, and/or any other means may be used. In manufacturing the invention, rough surface may be applied to mask while mask is being applied to the substrate, or after it has been fixed to the surface. 
         [0081]    Sample site  20  includes target area  24  and peripheral margin  22  of mask  14  that surrounds target area  24 . Target area  24  is an area of electrically conductive surface  12   a  and may have a number of equally preferred embodiments, including embodiments wherein target area  24  includes a mask spot or other structure. In accordance with one embodiment of the invention, target area  24  has a circular plan area as depicted in  FIG. 1   a  for a circle sample plate  10 . In accordance with one embodiment of the invention, target area  24  has a rectangular, linear, and/or curvilinear plan area as depicted in  FIG. 1   b  for a channel sample plate  10 . Target area  24  may also be embodied having other plan areas. 
         [0082]    As will be described further herein, target area  24  serves to substantially attract specimen  40  while it is in the liquid drop state. Specimen  40  is attracted to target area  24  because mask  14  is relatively more hydrophobic than target area  24 . 
         [0083]    In the circle sample plate  10  depicted in  FIG. 1   a,  sample site  20  includes target area  24  having a circular plan area of surface  12   a  and peripheral margin  22  coincident with the maximum diameter in plan view with specimen  40  upon spotting on target area  24 . Since the size of drops of specimen  40  may vary depending on investigative need, i.e. using a large drop to increase investigative sensitivity when biomolecules are in low concentration, target area  24  may be of different sizes to accommodate differently sized drops and sample plate  10  may be selected based upon a diameter of target area  24  that is sized appropriately for the drop size of specimen  40  that is to be investigated. 
         [0084]    As is easily understood, the volume of a drop of a liquid directly correlates to the diameter of any planar section of the drop. As is further understood, plan and radial dimensions of a drop of liquid may be predetermined by controlling the drop&#39;s volume and determining the relative hydrophilic and/or hydrophobic qualities of the surface to which it adheres. Thus, control of drop size may be achieved using pipetting or any other method to control the volume of specimen  40 . 
         [0085]    It is known that hydrophobic and/or hydrophilic qualities are relative to the contact angle between a drop and the surface to which it adheres. An angle of 0° indicates total hydrophilic wetting of the surface and an angle of 180° indicates total hydrophobicity of the surface. Teflon® typically has a contact angle of 140° to 160° for water. 
         [0086]    In channel sample plate  10  depicted in  FIG. 1   b,  sample site  20  includes target area  24  having a plan area of surface  12   a,  characterized by length exceeding width, and a peripheral margin  22  substantially parallel to target area  24  coincident with the maximum diameter in plan view of specimen  40  or the maximum diameter in plan view of a plurality of specimen  40 . Preferably, target area  24  has a width of 0.1 to 0.5 mm and sample plate  10  may be selected based upon a width of target area  24  that is sized appropriately for the volume of specimen  40  that is to be investigated. 
         [0087]    In accordance with one embodiment of the invention, sample site  20  includes target area  24  having a rectangular plan area. 
         [0088]    In accordance with one embodiment of the invention, sample site  20  includes target area  24  having a curvilinear plan area comprising a spiral, although other curvilinear plan areas such as a series of concentric plan areas are also contemplated. 
         [0089]    In accordance with one embodiment of the invention, mask  14  is additionally applied to a central region of target area  24  to form at least one mask spot  24   a.    FIG. 2   c  is a plan view of a sample site that includes a target area and a mask spot in accordance with one embodiment of the invention.  FIG. 3   c  is an expanded elevation view of a sample site that includes a target area and a mask spot in accordance with one embodiment of the invention. Mask spot  24   a  is further explained herein. 
         [0090]      FIGS. 5  through  FIGS. 10  depict the crystallization and crystals produced by the dried droplet method using specimen  40  on sample plate  10  in accordance with one embodiment of the invention.  FIG. 5   a  is an enlarged view taken at area A-A of  FIG. 1   a  of a circle sample plate in accordance with one embodiment of the invention wherein specimens have been applied on sample sites.  FIG. 5   b  is an enlarged view taken at area A-A of  FIG. 1   b  of a channel sample plate in accordance with one embodiment of the invention wherein specimens have been applied on sample sites. 
         [0091]      FIG. 6   a  is a cross-section at section C-C of  FIG. 5   a  of a section of a circle sample plate in accordance with one embodiment of the invention wherein specimens have been applied on sample sites.  FIG. 6   b  is a cross-section at section C-C of  FIG. 5   b  of a section of a channel sample plate in accordance with one embodiment of the invention wherein specimens have been applied on sample sites.  FIG. 6   b  is a cross-section at section D-D of  FIG. 5   b  of a section of a channel sample plate in accordance with one embodiment of the invention wherein specimens have been applied on sample sites. 
         [0092]      FIG. 7   a  is an enlarged view taken at area A-A of  FIG. 1   a  of a circle sample plate in accordance with one embodiment of the invention wherein specimens of  FIG. 6   a  have begun to dry.  FIG. 7   b  is an enlarged view taken at area A-A of  FIG. 1   b  of a channel sample plate in accordance with one embodiment of the invention wherein specimens of  FIG. 6   b  have begun to dry. 
         [0093]      FIG. 8   a  is a cross-section at section E-E of  FIG. 7   a  of a section of a circle sample plate in accordance with one embodiment of the invention wherein specimens of  FIG. 6   a  have begun to dry.  FIG. 8   b  is a cross-section at section E-E of  FIG. 7   b  of a section of a channel sample plate in accordance with one embodiment of the invention wherein specimens of  FIG. 6   b  have begun to dry.  FIG. 8   c  is a cross-section at section F-F of  FIG. 7   b  of a section of a channel sample plate in accordance with one embodiment of the invention wherein specimens of  FIG. 6   b  have begun to dry. 
         [0094]      FIG. 9   a  is an enlarged view taken at area A-A of  FIG. 1   a  of a circle sample plate in accordance with one embodiment of the invention wherein specimens of  FIG. 6   a  have dried.  FIG. 9   b  is an enlarged view taken at area A-A of  FIG. 1   b  of a channel sample plate in accordance with one embodiment of the invention wherein specimens of  FIG. 6   b  have dried. 
         [0095]      FIG. 10   a  is a cross-section at section G-G of  FIG. 9   a  of a section of a circle sample plate in accordance with one embodiment of the invention wherein specimens of  FIG. 6   a  have dried.  FIG. 10   b  is a cross-section at section G-G of  FIG. 9   b  of a section of a channel sample plate in accordance with one embodiment of the invention wherein specimens of  FIG. 6   b  have dried.  FIG. 10   c  is a cross-section at section H-H of  FIG. 9   b  of a section of a circle sample plate in accordance with one embodiment of the invention wherein specimens of  FIG. 6   b  have dried. 
         [0096]    Specimens  40 , each consisting of a drop in liquid form, are applied to sample plate  10  within sample site  20  and contact mask  14 . Therein, it is preferred that specimen  40  contact mask  14  at the sides over a distance of at least 0.1 mm. 
         [0097]    For circle sample plate  10 , specimen  40  contacts mask  14  in plan view at the perimeter of target area  24  by using a drop of specimen  40  where the drop&#39;s maximum diameter exceeds the diameter of target area  24 . Since, it is known that a drop with a volume of 0.5 μl has diameter of approximately 1.0 mm on the hydrophobic surface of Teflon®, it is preferred that each specimen  40  is between 0.1 to 4.0 μl in volume. 
         [0098]    For channel sample plate  10 , specimen  40  contacts mask  14  in plan view at peripheral margin  22  while the perimeter of specimen  40  also contacts target area  24 . In accordance with one embodiment of the invention, specimen  40  is applied within sample site  20  on channel sample plate  10  in a continuous manner, such as by spraying or streaking specimen  40 . Therein, the width or length of the application of specimen  40  exceeds the width or length of target area  24 , respectively, so that specimen  40  contacts mask  14 . 
         [0099]    In accordance with one embodiment of the invention, mask spot  24   a  is appropriately sized to form a drop of specimen  40  so that the drop contacts the side of mask  14  to enhance the deposition of crystals. 
         [0100]    Specimen  40  includes a biomolecule and a matrix mixed in a 1:1 ratio, by volume. Although other formulations including those with low solubility may also advantageously be used and the formulations presented herein are not intended to be limiting, the matrix may be made according to the following formulations: 
         [0101]    In a first formulation (CHCA formulation), α-cyano-4-hydroxycinnamic acid (C 10 H 7 NO 3 ) and an aqueous solution containing a solvent are mixed to produce a matrix. The solvent preferably is acetonitrile (C 2 H 3 N) and is mixed at a ratio of 30% to 50% acetonitrile and 70% to 50% water, respectively, with 0.1% trifluoroacetic acid (C 2 HF 3 O 2 ), pH 2.3, by volume, to produce a solvent. CHCA may be present at a concentration of 0.2 mg to 20 mg per 1 ml of solvent, although a concentration of 1 to 5 mg of CHCA per 1 ml of solvent is preferred. The first formulation may utilize other matrices such as other cinnaminic acids or other matrices of low solubility instead of CHCA. 
         [0102]    In a second formulation (SA formulation), 3,5-dimethoxy-4-hydroxycinnamic acid (C 11 H 12 O 5 ), commonly known as sinapinic acid, and an aqueous solution containing a solvent are mixed to produce a matrix. The solvent preferably is acetonitrile (C 2 H 3 N) and is mixed at a ratio of 30% to 50% acetonitrile and 70% to 50% water with 0.1% trifluoroacetic acid (C 2 HF 3 O 2 ), pH 2.3, by volume, to produce a solvent. Sinapinic acid may be present at a concentration of 0.2 mg to 20 mg per 1 ml of solvent, although a concentration of 1 to 5 mg of sinapinic acid per 1 ml of solvent is preferred. 
         [0103]    The CHCA formulation is preferred for analysis of biomolecules such as peptides and other biomolecules having molecular weights of less than 10,000 Daltons. The SA formulation is preferred for biomolecules such as proteins and other biomolecules having molecular weights of 10,000 Daltons and more. 
         [0104]      FIGS. 11 and 12  are isometric views of crystals that have formed in a halo effect in a sample site with different concentrations of matrix formulations in accordance with one embodiment of the invention. For simplicity, a sample site  20  of circle sample plate  10  is depicted. Therein, crystals  42  deposit on rough surface  14   a  in margin  22 , forming a halo effect around the perimeter of target area  24 . Further crystals  42  form as the sample solution dries and crystals  42  continue to form on surface  14   a  on margin  22  on sample site  20 . While crystals  42  are greatest in number in margin  22 , a significantly smaller amount deposit on target area  24 . 
         [0105]      FIG. 11  depicts crystals  42  on sample site  20  of circle sample plate  10  produced using CHCA matrix solution at a concentration of  1  mg of CHCA per 1 ml of solvent. Crystals  42  crowd margin  22  near the periphery of target area  24  approximately forming two concentric crystal rings around the periphery. A third ring is approximately present in some areas.  FIG. 17  is a photograph of a similar sample showing crystals on sample site of circle sample plate produced using CHCA matrix solution at a concentration of 1 mg of CHCA per 1 ml of solvent. Crystals crowd margin near the periphery of target area forming crystal rings around the periphery. 
         [0106]    Crystal rings are believed to result from the increase in matrix concentration during the concomitant decrease in solvent volume as the solvent evaporates. Crystalline lattices begin to form and are attracted to rough surface  14   a  known to induce crystalline formation. As the specimen drop dries, many matrix crystalline lattices precipitate from the solution at roughly the same time. Such precipitation occurs at regular intervals leading to deposition in ring. As the larger matrix crystals precipitate, smaller crystals form anew while the specimen drop continues drying. Eventually these smaller crystals  42  also are unsustainable in solution and precipitate from solution. In contrast, where a lower concentration of matrix is used, crystals  42  result in only one ring. Such crystals  42  are depicted in  FIG. 12  where crystal  42  on sample site  20  were produced using CHCA matrix solution at a concentration of 0.5 mg of CHCA per 1 ml of solvent.  FIG. 18  is a photograph of sample showing crystals on sample site of circle sample plate produced using 30% ACN matrix solution at a concentration of 1 mg of per 1 ml of solvent. Crystals crowd margin near the periphery of target area forming crystal ring around the periphery. 
         [0107]    To analyze the biomolecule, crystals  42  are irradiated using a UV laser (not shown for clarity) that scans crystals  42  directly using the energy of a laser beam. The UV laser generates a laser beam  48  typically at 337 nm wavelength, which may be any suitable ultra-violet laser beam such one having an effective beam diameter of 0.1 to 0.2 mm. As is easily understood, concentrating crystals  42  in margin  22  reduces the area required to be scanned by the laser in order to irradiate sufficient crystals  42  to obtain significant irradiation without compromising analysis sensitivity. Given the beam&#39;s relatively small effective diameter, reducing the requisite scanning area significantly enhances efficiency. 
         [0108]    The reduced area is advantageously illustrated in comparison to the area that must be irradiated when a traditional formulation is used to produce specimen  40 . To illustrate this example, 2,5-dihydroxybenzoic acid (C 7 H 6 O 4 ) known as DHB is used and is compared to the formulations of the present invention. In DHB formulations, crystals  42  occur in target area  24 . Thus, in order to irradiate crystals  42  of the DHB formulation, the entire target area  24  must be scanned. Thus, if target area  24  has a diameter of 1 mm, the area to be scanned is 0.25π mm 2 . In order to irradiate crystals  42  wherein the matrix is the CHCA or SA formulation and the sample site  20  has a diameter of 1.2 mm, the area to be scanned is the 0.11π mm 2 . This results in a required scanning area that is only 44% of the traditional scanning area.  FIG. 19  is a photograph of DHB crystals forming in the traditional target area, covering a large surface area. 
         [0109]      FIG. 13  is a cross-section at section E-E of  FIG. 5   a  of a section of a sample plate in accordance with one embodiment of the invention wherein crystals are being scanned by an UV laser.  FIG. 13  illustrates the scanning of crystals  42  that were produced using the CHCA and SA formulation. 
         [0110]    Laser beam  48  sweeps scanning pattern  50  (not shown for clarity) wherein it irradiates crystals  42  at a first position, marked by the letter A in  FIG. 13 . As pattern  50  continues, at a second position, marked by the letter B in  FIG. 13 , laser beam  48  irradiates another crystal  42  and proceeds further, where at a third position, marked by the letter C in  FIG. 13 , it irradiates another crystal  42 , and so forth. Therein, it is understood that scanning pattern  50  may be accomplished by maintaining the laser stationary and moving plate  10 , or by moving the laser and maintaining plate  10  stationary, and/or a combination of both. 
         [0111]      FIGS. 14   a  and  14   b  illustrate scanning pattern  50  in accordance with one embodiment of the invention. Pattern  50  may be any variety of patterns, circuit, or other traverse that irradiates crystal  42  efficiently by minimizing the length of the path while maximizing the number of crystals  42  that are irradiated. 
         [0112]      FIGS. 14   a  and  14   b  are enlarged views of area A-A of  FIG. 1  of circle sample plate  10  in accordance with one embodiment of the invention wherein a scanning path to irradiate crystals  42  produced using CHCA or sinapinic acid is illustrated. Laser beam  48  (not shown for clarity) utilizes pattern  50  that is confined by two predetermined boundaries; inner boundary  52   a  that is approximate with the perimeter of target area  24  and an outer boundary  52   b  that is within margin  22 . Boundaries  52   a  and  52   b  may be predetermined according to experience by an operator, statistical sampling, by an algorithm, or any other suitable means. 
         [0113]    One embodiment of scanning pattern  50  is illustrated in  FIG. 14   a . Pattern  50  is a cross-pattern that oscillates between boundary  52   a  on target area  24  and boundary  52   b  within sample site  20 . Another embodiment is illustrated in  FIG. 14   b.  Therein, pattern  50  is spiral pattern that starts at boundary  52   a  and in one or more circuits ends at boundary  52   b.  Other patterns or combinations of patterns may also be used for pattern  50 . 
         [0114]    For the cross pattern illustrated in  FIG. 14   a , an algorithm may include the number of oscillations, n, required to cover the area of margin  22  based on a certain confidence level, c, as expressed by a percentage or a ratio. A confidence level of 1 may mean certainty that all crystals  42  have been irradiated. Thus, 
         [0000]        n= (3× r   1   2 )/ r   2   2   ×c    Equation 1
 
         [0000]    where r 1  is the radius of target area  24  and r 2  is the effective radius of laser beam  48 . Therein, if target area  24  is 1 mm in diameter, laser beam  48  has an effective diameter of 0.1 mm, and a confidence level of 75% is desired, 56.25 oscillation are required if boundaries  52   a  and  52   b  are at perimeters of margin  22 . 
         [0115]    Similarly, pattern  50  may be advantageously employed to more to reduce the time and travel of UV laser and more efficiently irradiate crystals  42  on a channel sample plate.  FIGS. 15   a  and  15   b  are enlarged views of one sample site of a channel sample plate wherein a scanning pattern, in accordance with one embodiment of the invention, to irradiate crystals produced using CHCA or SA is illustrated. 
         [0116]    Therein, similarly, laser beam  48  utilizes pattern  50  that is confined by two predetermined boundaries; inner boundary  52   a  that is approximate with the perimeter of target area  24  and an outer boundary  52   b  that is within or coincident with sample site  20 . Boundaries  52   a  and  52   b  may be predetermined according to experience by an operator, statistical sampling, by an algorithm, and/or any other suitable means. Illustrated in  FIG. 15   a  is a scanning pattern  50  that alternates between boundaries  52   a  and  52   b,  and illustrated in  FIG. 15   b  is a path that is a spiral pattern  50 . Other patterns may also be used. 
         [0117]    In accordance with one embodiment of the invention, specimen  40  is applied on sample plate  10  using the electrospray deposition method. Critical to the electrospray deposition method is that specimen  40  is deposited in a smooth and constant application. Sample plate  10  moves on a platform while a liquid chromatography system elutes specimen  40  using a solvent and specimen  40  is applied by electrospray on sample plate  10  for mass spectrometry analysis. 
         [0118]    Sample plate  10  is a channel sample plate or a circle sample plate and cooperatively progresses from one location to another with a liquid chromatography system. In accordance with one embodiment of the invention, sample plate is located on a moving platform that operates at a predetermined speed. Preferably, the platform is operator controllable and adjustable, and further includes one or more check mechanisms to ensure a precise predetermined speed that cooperates with the electrospray deposition. 
         [0119]    A liquid chromatography system, such as micro-liquid chromatography system or nano-liquid chromatography system is provided to elute specimen  40  and apply it by electrospray on sample plate  10 . Typically, the liquid chromatography system includes a reversed-phase column. The liquid chromatography system is eluted with a matrix of the CHCA formulation at a concentration of 1 mg of CHCA per 1 ml of solvent or SA formulation at a concentration of 1 mg of SA per 1 ml of solvent; although other matrices may also be used. Therein, the percentage of acetonitrile varies from 0% to 70% over the course of eluting specimen  40  from the column during the time period of elution, typically 15 to 60 minutes. 
         [0120]    In accordance with one embodiment of the invention, specimen  40  is applied on channel sample plate  10  by direct application of specimen  40  in liquid form such as by streaking. Therein, sample plate  10  moves on a platform while a stationary liquid chromatography system applies specimen  40 . Specimen  40  is applied to channel sample plate  10  at a continuous rate over a predetermined length of target area  24 , preferably at a rate 1 μl per 1 mm of length of target area  24 , while specimen  40  is applied to circle sample plate  10  at a rate consistent with the size of target area  24 . Preferably, specimen  40  is applied within sample site  20  that is no more than 0.2 mm from the periphery of target area  24 . Specimen  40  then quickly forms crystals  42  that deposit on rough surface  14   a  from where they are irradiated using a UV laser. 
         [0121]    The present novel invention is also contemplated in additional embodiments. In accordance with one embodiment of the invention, sample plate  10  is produced includes sample site  20  wherein mask  14  is selectively applied with rough surface  14   a  to surface  12   a  so that mask  14  is surrounded by surface  12   a.  Specimen  40  may be applied to sample plate  10  using the dried droplet method by spotting, streaking, or spraying or by the electro-spray deposition method. Specimen  40  may also be applied by washing or submerging sample plate  10  with or in specimen  40 . Crystals  42  will then form on mask  14  in peripheral margin  22  and may be efficiently irradiated using laser beam  48 .