Patent Application
20080078931
The foregoing and other features and aspects of the present invention will be best understood with reference to the following detailed description of specific embodiments of the invention, when read in conjunction with the accompanying drawings, wherein:
The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular description of preferred embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention.
A description of preferred embodiments of the invention follows.
In the disclosure that follows, in the interest of clarity, not all features of actual implementations are described. It will of course be appreciated that in the development of any such actual implementation, as in any such project, numerous engineering and technical decisions must be made to achieve the developers' specific goals and subgoals (e.g., compliance with system and technical constraints), which will vary from one implementation to another. Moreover, attention will necessarily be paid to proper engineering practices for the environment in question. It will be appreciated that such a development effort might be complex and time-consuming, but would nevertheless be a routine undertaking for those of ordinary skill in the relevant fields.
The present invention exploits and expands the use of the collimated hole structure (CHS) sample plate disclosed in U.S. Ser. No. 11/138,127, filed May 26, 2005. Accordingly, in one embodiment the collimated hole structure comprises a regular array of holes 0.025 mm in diameter arranged in hexagonal close-packed configuration with 0.035 mm spacing between holes. The collimated hole structure is 102×108×1.5 mm and is mounted in a 400 series magnetic stainless steel frame 124×127×3 mm. The total number of 0.025 mm diameter holes in the plate is approximately 10,384,000.
In another embodiment the hole diameters and spacings are as above, but the dimensions of the collimated hole structure is 30×30×1.5 mm and it is mounted in a stainless steel frame 50×50×1.5 mm. These are for illustration only since any size hole and plate can be used in the invention.
In yet another embodiment holes are formed in a 400 series magnetic stainless steel plate. In one such embodiment the outside dimensions of the plate are 124×127×3 mm, and the holes are 3 mm in diameter in a square array with 4.5 mm between holes within the central 102×108 mm region for a total of 528 holes in an array of 24×22 holes. In another such embodiment the holes are 1.5 mm in diameter in a square array with 2.25 mm between holes for a total of 2112 holes in an array of 48×44 holes.
In a preferred embodiment the holes in CHS plates are filled with a porous monolithic structure designed to preferentially adsorb samples of interest. One such embodiment employs formation of reversed phase mode styrene-divinylbenzene based monoliths. The reversed phase separation mode was chosen due to its near ubiquitous ability to capture peptides; a characteristic directly in-line with the initial applications of the plates. The initial formulations used in monolith construction herein were taken from published procedures for capillary HPLC monolithic column formation. C. W. Huck, G. K. Bonn, “Poly(Styrene-Divinylbenzene) Based Media for Liquid Chromatography, Chem. Eng. Technol. 28:457-1472 (2005). S. Xie, R. W. Allington, F. Svec, J. M. J. Frechet “Rapid Reversed-Phase Separation of Proteins and Peptides using Optimized ‘Moulded’ Monolithic Poly (Styrene-co-Divinylbenzene) columns, J. Chrom. A. 865:169-174 (1999).
For all polymer formulations, monolithic plate construction was paralleled with the creation of fused-silica capillary columns so that the separation behavior and loading capacity of the resulting media could be characterized by HPLC.
The collimated hole structure plates of the present invention have a high capture capacity. As used herein a “high capture capacity” means that the collimated hole structure plate captures samples of interest even when the volume of liquid effluent containing said samples of interest delivered to any predetermined location on the collimated hole structure plate is larger than the internal volume of the collimated hole structure at that location.
In one embodiment of the interface system of the invention, the liquid effluent delivered to one surface of the collimated hole structure sample plate at a predetermined location is efficiently vaporized at the opposite surface of the plate so that nonvolatile samples of interest in the liquid effluent are efficiently captured either within that portion of the collimated hole structure plate or on the opposite surface even though the volume of liquid delivered to that location may be larger than the internal volume of that portion of the collimated hole structure.
Referring now to
In operation the CHS sample plate is loaded either manually or using a mechanical transfer device 107 into the sample deposition apparatus 104 where liquid effluent 108 is transferred from the liquid separations device 101 onto predetermined locations on the CHS sample plate 103.
In one embodiment employing a CHS plate with 3 mm holes in a 24×22 array, the liquid effluent is initially directed to a hole at one corner of the array. After a predetermined time the effluent is directed to an adjacent hole in the array, and this procedure is repeated until effluent has been directed to all of the desired holes in the array, or until the separation is completed. After the plate is loaded with samples of interest, the sample plate 103 is transferred either manually or mechanically using a mechanical transfer device 109 to a matrix deposition apparatus 105 for adding MALDI matrix. In the matrix deposition apparatus 105 a solution of MALDI matrix in an appropriate solvent is added to one surface of the plate and caused to flow through the holes in the plate. The solvent composition is chosen so that samples of interest are eluted from the holes to the opposite surface of the plate. Solution reaching the opposite surface is evaporated to form matrix crystals incorporating samples of interest. The sample plate 103 is then transferred either manually or mechanically using a mechanical transfer device 110 to a MALDI mass spectrometer 102 where the surface of the plate containing matrix crystals with incorporated samples of interest are exposed to a laser beam, and the samples of interest are ionized and analyzed by mass spectrometry.
Referring now to
Referring now to
In one embodiment the end of the connection tube is in close proximity to the surface of the CHS plate, but does not necessarily make contact with the plate. In another embodiment the end of the tube is enclosed in a sleeve that presses against the plate so that the flow is directed through the plate by the liquid pressure within the sleeve The CHS plate is rigidly mounted in a frame 3 and the frame is mounted onto a plate holder 4 and the chamber 5 is effectively isolated from the ambient air surrounding the connection tube 1. The chamber 5 is adjacent to a second surface of the CHS plate 2B of the plate and communicates with a vacuum generator (not shown) via a vacuum coupling 6 and an outlet flow controller 7. The vacuum generator need not be directly mounted to or on the interface apparatus. It need only be operably linked to the apparatus, preferably by a vacuum coupling. As used herein a vacuum coupling may be any tube, pipe, or system which affords fluid communication between the vacuum generator and the interfacing system.
Operationally, liquid effluent from the LC entering the inlet 15 is supplied to the first surface of the CHS plate 2A of the collimated hole structure plate 2 through connection tube 1, and the liquid flows through the plate driven by a pressure differential. The pressure differential can be generated across the plate, in which case the differential is that between the atmospheric pressure on opposite sides of the plate (i.e., determined by the gas flow). The flow across the plate may also be driven by a pressure differential generated between the pressure of the liquid effluent and the atmospheric pressure on opposite side of the plate from the effluent side. Optimum performance of the interface requires that samples of interest are captured on monolithic structures within the holes or on the second surface 2B. If samples of interest are only weakly bound to the monolithic polymer, then it is desirable that the liquid be completely vaporized as it emerges from the monolithic polymer structure at the opposite side, the second surface 2B of the plate. This assures that all of the nonvolatile solutes are retained on the plate even in cases in which binding to the polymeric structure is weak. On the other hand if samples are strongly retained within the holes, or if the volume of liquid supplied to each hole is less than the internal volume, then vaporization is not required.
If vaporization is required then the temperature of the plate and the partial pressure of the liquid vapor in the gas at the opposite surface of the plate must be controlled to assure that vaporization occurs at the surface.
To this end, a source of make-up air 8 is supplied and coupled to chamber 5 through an inlet flow controller 9 and inlet coupling 10. In one embodiment the make-up air is heated by a heater 11 sufficient to supply the heat of vaporization of the liquid. Inlet flow controller 9 and outlet flow controller 7 are used to set the desired pressure and total flow in the chamber 5 measured by pressure gauge 12 which is operably connected to the system via a pressure gauge coupling 13.
In supplying the sample-containing effluent to the surface of the plate the connection tube can move in both the x and y directions to any predetermined or directed spot on the CHS plate.
Referring now to
The apparatus illustrated in
Before elution of samples in matrix solution, it is desirable in many cases to wash the plates to remove any nonvolatile salts that may be co-deposited, or other water soluble impurities that are not retained by the reversed-phase media employed in the monolithic structures. The apparatus illustrated in
Those skilled in the art will recognize that the same apparatus could be used for both matrix deposition and sample washing merely by changing the solvent employed and other operating conditions. Similarly, these functions could be incorporated into the apparatus employed for depositing samples from the separation device onto the CHS plate. Thus, the invention is not limited to the case described above in which these operations are carried out in separate apparatus, but rather encompasses any combination that accomplishes the required functions.
After the samples are dried in crystals on surface 2A the plate 2 is moved to the MALDI mass spectrometer and installed on the x-y table 20 within the vacuum system 21 as illustrated in
In one example the flow rate is 1 mL/min and the nominal spot size is 3 mm. The distance between the first and second position is 4.5 mm, as is the distance between the second and third, etc. Thus, 24 spots may be placed in a row along the 108 mm dimension of the plate, and 22 rows of such spots along the 102 mm dimension for a total of 528 spots. This application may employ a version of the CHS plate that has a separate hole filled with a reversed-phase monolithic structure at each location. One of the advantages of this system over interfaces employing solid sample plates is that the time that effluent is placed on a particular spot is not limited by the size of the spot. Thus, the time resolution and volume of liquid applied to a spot can be determined by the requirements of the chromatography, rather than limitations of the interface.
Any sampling time between 0.1 and 10 seconds can easily be accommodated with this embodiment operating at a flow of 1 mL/min. On the other hand, a solid sample plate operating at 1 mL/min would limit the sampling time for a 3 mm diameter spot to less than 0.2 sec. With typical peak widths on the order of 5-6 seconds, the maximum sample concentration in a spot is reduced by a factor of about 25, and the plate is filled with less than 2 minutes of chromatography.
According to the present invention, a mass spectrometry-LC interface has been developed that allows nonvolatile samples in LC effluents at any flow rate from 1 uL/min to 1 mL/min to be deposited on a collimated hole structure (CHS) with no loss in sensitivity. The area of sample spots on the MALDI plate is proportional to the flow rate, and range in diameter from 3 mm at a flow of 1 mL/min down to about 0.1 mm at a flow of 1 uL/min. Thus, the sample concentration on the surface (mole/mm2) depends only on the concentration of the sample in the LC effluent and the deposition time, but is independent of flow rate.
Unlike other LC-MS systems, here chromatography can be designed according to the total amount of sample available rather than the limits of the mass spectrometer. In applications such as the present one where the total amount of sample available may be relatively large (at least 1 mL) the concentration of trace components is limiting sensitivity rather than the absolute amount of sample. In this case, high capacity separation systems are required using large columns and high flow rates. The interface of the present invention directly interfaces these to the MALDI plate.
The detection limit in MALDI is primarily limited by chemical noise and is typically about 1 fmole/mm2 for proteins down to about 1 attomole/mm2 for some peptides. The area of the spot for 1 mL/min flow is about 7 mm2; thus with about 6 second deposition the detection limit for proteins corresponds to an average concentration of about 70 fmoles/mL in a 0.1 mL fraction. The detection limits expressed in concentration units are independent of flow rate, but the larger total amount of sample per spot and larger sample spot produced at higher flow rates allows larger numbers of laser shots to be employed when necessary, for example in MS-MS. The deposition time can be chosen to match the chromatography. For example, if the deposition time is approximately equal to the peak width (FWHM), then in the worst case at least one-half of the total sample will be in one spot. Since the samples can be washed to remove salts before the MALDI matrix is added, any type of chromatography can be employed without sacrificing performance of the MS.
From the foregoing detailed description of specific embodiments of the invention, it should be apparent that methods and apparatuses for MALDI-TOF mass spectrometric LC interfacing using a collimated hole structure sample plate have been disclosed. Although specific embodiments of the invention have been disclosed herein in detail, this has been done solely to describe various features and aspects of the invention, and is not intended to be limiting with respect to the scope of the invention. It is contemplated that various substitutions, alterations, and modifications may be made to the embodiments disclosed herein, including but not limited to those implementation variations and alternatives that have been specifically discussed herein, without departing from the spirit and scope of the invention as defined in the appended claims, which follow.
While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.
