The present invention relates to a means of delivering ions to a mass spectrometer. Mass spectrometry is an important tool in the analysis of a wide range of chemical compounds. Specifically, mass spectrometers can be used to determine the molecular weight of sample compounds. The analysis of samples by mass spectrometry consists of three main steps—formation of ions from sample material, mass analysis of the ions to separate the ions from one another according to ion mass, and detection of the ions. A variety of means exist in the field of mass spectrometry to perform each of these three functions. The particular combination of means used in a given spectrometer determine the characteristics of that spectrometer.
To mass analyze ions, for example, one might use a magnetic (B) or electrostatic (E) analyzer. Ions passing through a magnetic or electrostatic field will follow a curved path. In a magnetic field the curvature of the path will be indicative of the momentum-to-charge ratio of the ion. In an electrostatic field, the curvature of the path will be indicative of the energy-to-charge ratio of the ion. If magnetic and electrostatic analyzers are used consecutively, then both the momentum-to-charge and energy-to-charge ratios of the ions will be known and the mass of the ion will thereby be determined. Other mass analyzers are the quadrupole (Q), the ion cyclotron resonance (ICR), the time-of-flight (TOF), and the quadrupole ion trap analyzers.
Before mass analysis can begin, however, gas phase ions must be formed from sample material. If the sample material is sufficiently volatile, ions may be formed by electron ionization (EI) or chemical ionization (CI) of the gas phase sample molecules. For solid samples (e.g. semiconductors, or crystallized materials), ions can be formed by desorption and ionization of sample molecules by bombardment with high energy particles. Secondary ion mass spectrometry (SIMS), for example, uses keV ions to desorb and ionize sample material. In the SIMS process a large amount of energy is deposited in the analyte molecules. As a result, fragile molecules will be fragmented. This fragmentation is undesirable in that information regarding the original composition of the sample—e.g., the molecular weight of sample molecules—will be impossible to determine.
For more labile, fragile molecules, other ionization methods now exist. The plasma desorption (PD) technique was introduced by Macfarlane et al. in 1974 (Macfarlane, R. D.; Skowronski, R. P.; Torgerson, D. F., Biochem. Biophys. Res Commoun. 60 (1974) 616). Macfarlane et al. discovered that the impact of high energy (MeV) ions on a surface, like SIMS would cause desorption and ionization of small analyte molecules, however, unlike SIMS, the PD process also results in the desorption of larger, more labile species—e.g., insulin and other protein molecules.
Lasers have been used in a similar manner to induce desorption of biological or other labile molecules. See, for example, VanBreeman, R. B.: Snow, M.: Cotter, R. J., Int. J. Mass Spectrom. Ion Phys. 49 (1983) 35; Tabet, J. C.; Cotter, R. J., Anal. Chem. 56 (1984) 1662; or Olthoff, J. K.; Lys, I.: Demirev, P.: Cotter, R. J., Anal. Instrument. 16 (1987) 93. Cotter et al. modified a CVC 2000 TOF mass spectrometer for infrared laser desorption of involatile biomolecules, using a Tachisto (Needham, Mass.) model 215G pulsed carbon dioxide laser. The plasma or laser desorption and ionization of labile molecules relies on the deposition of little or no energy in the analyte molecules of interest. The use of lasers to desorb and ionize labile molecules intact was enhanced by the introduction of matrix assisted laser desorption ionization (MALDI) (Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y.; Yoshica, T., Rapid Commun. Mass Spectrom. 2 (1988) 151 and Karas, M.; Hillenkamp, F., Anal. Chem. 60 (1988) 2299). In the MALDI process, an analyte is dissolved in a solid, organic matrix. Laser light having a wavelength that is absorbed by the solid matrix but not by the analyte is used to excite the sample. Thus, the matrix is excited directly by the laser, and the excited matrix sublimes into the gas phase carrying with it the analyte molecules. The analyte molecules are then ionized by proton, electron, or cation transfer from the matrix molecules to the analyte molecules. This process, MALDI, is typically used in conjunction with time-of-flight mass spectrometry (TOFMS) and can be used to measure the molecular weights of proteins in excess of 100,000 daltons.
Recently, MALDI has been especially gaining acceptance as a way to ionize large molecules such as proteins. MALDI requires that samples applied to the surface of a sample support must be introduced into the vacuum system of the mass spectrometer. According to the prior art, a relatively large number of sample are introduced together on a support, and the sample support is moved within the vacuum system in such a way that the required sample is situated specifically in the focus of the laser's lens system. The analyte samples are placed on a sample support in the form of small drops of a solution, which dry very quickly and leave a sample spot suitable for MALDI. Normally a matrix substance is added to the solution for the MALDI process and the sample substances are encased in the crystals when the matrix substance crystallizes while drying. There are other methods known in the prior art, such as the application of sample substances to an already applied and dried matrix layer.
Current methods use visual control of the sample spots via microscopic observation. Thus, these are not truly automated. True automation opens up the possibility of processing large numbers of samples. It is well established within the art that microtiter plates are used for parallel processing of many samples. The body size of these plates is 80 by 125 millimeters, with a usable surface of 72 by 108 millimeters. There are commercially available sample processing systems which work with microtiter plates of this size. These originally contained 96 small exchangeable reaction vials in a 9 mm grid on a usable surface of 72 by 108 millimeters. Today, plates of the same size with 384 reaction wells imbedded solidly in plastic in a 4.5 mm grid have become standard.
The use of Atmospheric pressure ionization (API) is also well known in the prior art. Typically, analyte ions are produced from liquid solution at atmospheric pressure. One of the more widely used methods, known as electrospray ionization (ESI), was first suggested by Dole et al. (M. Dole, L. L. Mack, R. L. Hines, R. C. Mobley, L. D. Ferguson, M. B. Alice, J. Chem. Phys. 49, 2240, 1968). In the electrospray technique, analyte is dissolved in a liquid solution and sprayed from a needle. The spray is induced by the application of a potential difference between the needle (where the liquid emerges) and a counter electrode. By subjecting the sample liquid to a strong electric field, it becomes charged, and as a result, it “breaks up” into smaller particles if the charge imposed on the liquid's surface is strong enough to overcome the surface tension of the liquid (i.e., as the particles attempt to disperse the charge and return to a lower energy state). This results in the formation of finely charged droplets of solution containing analyte molecules. These droplets further evaporate leaving behind bare charged analyte ions.
Electrospray mass spectrometry (ESMS) was introduced by Yamashita and Fein (M. Yamashita and M. B. Fein, J. Phys. Chem. 88, 4671, 1984). To establish this combination of ESI and MS, ions had to be formed at atmospheric pressure, then introduced into the vacuum system of a mass analyzer via a differentially pumped interface. The combination of ESI and MS affords scientists the opportunity to mass analyze a wide range of samples, and ESMS is now widely used primarily in the analysis of biomolecules (e.g. proteins) and complex organic molecules.
In the intervening years a number of means and methods useful to ESMS and API-MS have been developed. Specifically, a great deal of work has focused on sprayers and ionization chambers. In addition to the original electrospray technique, pneumatic assisted electrospray, dual electrospray, and nano electrospray are now also widely available. Pneumatic assisted electrospray (A. P. Bruins, T. R. Covey, and J. D. Henion, Anal. Chem. 59, 2642, 1987) uses nebulizing gas flowing past the tip of the spray needle to assist in the formation of droplets. The nebulization gas assists in the formation of the spray and thereby makes the operation of ESI easier. Nano electrospray (M. S. Wilm, M. Mann, Int. J. Mass Spectrom. Ion Processes 136, 167, 1994) employs a much smaller diameter needle than the original electrospray. As a result the flow rate of sample to the tip is lower and the droplets in the spray are finer. However, the ion signal provided by nano electrospray in conjunction with MS is essentially the same as with the original electrospray. Nano electrospray is therefore much more sensitive with respect to the amount of material necessary to perform a given analysis.
Sample preparation robots (e.g. Gilson) have been used in the prior art for the automated injection of sample aliquots into an ESI source. In such a case, solution is pumped continuously from a resevoir to the sprayer of an ESI source. Sample aliquots are injected into this solution stream and are thereby carried through a transfer line to the sprayer.
Many other ion production methods might be used at atmospheric or elevated pressure. For example, MALDI has recently been adapted by Victor Laiko and Alma Burlingame to work at atmospheric pressure (Atmospheric Pressure Matrix Assisted Laser Desorption Ionization, poster #1121, 4th International Symposium on Mass Spectrometry in the Health and Life Sciences, San Francisco, Aug. 25–29, 1998) and by Standing et al. at elevated pressures (Time of Flight Mass Spectrometry of Biomolecules with Orthogonal Injection+Collisional Cooling, poster #1272, 4th International Symposium on Mass Spectrometry in the Health and Life Sciences, San Francisco, Aug. 25–29, 1998; and Orthogonal Injection TOFMS Anal. Chem. 71(13), 452A (1999)). The benefit of adapting ion sources in this manner is that the ion optics and mass spectral results are largely independent of the ion production method used.
An elevated pressure ion source always has an ion production region (where ions are produced) and an ion transfer region (where ions are transferred through differential pumping stages and into the mass analyzer). The ion production region is at an elevated pressure—most often atmospheric pressure—with respect to the analyzer.
In much of the prior art the ion production region will often include an ionization “chamber”. In an ESI source, for example, liquid samples are “sprayed” into the “chamber” to form ions. The design of the ionization chamber used in conjunction with API-MS has had a significant impact on the availability and use of these ionization methods with MS. Prior art ionization chambers are inflexible in that a given ionization chamber can be used readily with only a single ionization method and a fixed configuration of sprayers. For example, in order to change from a simple electrospray method to a nano electrospray method of ionization, one had to remove the electrospray ionization chamber from the source and replace it with a nano electrospray chamber (see also, Gourley et al. U.S. Pat. No. 5,753,910, entitled Angled Chamber Seal for Atmospheric Pressure Ionization Mass Spectrometry). In a co-pending application entitled Ionization Chamber For Atmospheric Pressure Ionization, this problem is addressed by disclosing an API ionization chamber providing multiple ports for employing multiple devices in a variety of combinations (e.g., any type of sprayer, lamp, microscope, camera or other such device in various combinations). Further, any given sprayer may produce ions in a manner that is synchronous or asynchronous with the spray from any or all of the other sprayers. By spraying in an asynchronous manner, analyte from a multitude of inlets may be sampled in a multiplexed manner.
Analyte ions produced via an API method need to be transported from the ionization region through regions of differing pressures and ultimately to a mass analyzer for subsequent analysis (e.g., via TOFMS, Fourier transform mass spectrometry (FTMS), etc.). In prior art sources, this was accomplished through use of a small orifice or capillary tube between the ionization region and the vacuum region. An example of such a prior art capillary tube is shown in FIG. 1. As depicted, capillary 7 comprises a generally cylindrical glass tube 2 having an internal bore 4. The ends of capillary 7 include a metal coating (e.g., platinum, copper, etc.) to form conductors 5 which encompass the outer surface of capillary 7 at its ends, leaving a central aperture 6 such that the entrance and exit to internal bore 3 are left uncovered. Conductors 5 may be connected to electrical contacts (not shown) in order to maintain a desired space potential at each end of capillary 7. In operation, a first electrode (one of conductors 5) of capillary 7 may be maintained at an extreme negative potential (e.g., −4,500V), while the other electrode (the other of conductors 5), which may form the first stage of a multi-stage lensing system for the final direction of the ions to the spectrometer, may be maintained at a positive potential (e.g., 160 volts).
It is often observed that the capillaries used in MS analysis acquire deposits over time. Therefore, through normal operation the capillaries need to be regularly cleaned or even replaced. To do so, the MS system must be turned off before the capillary can be removed—requiring the pumps to be shut down and the vacuum system to be broken—thereby rendering the system unavailable for hours and even days at a time.
More recently, Lee et al. U.S. Pat. No. 5,965,883 attempted to solve this problem in the manner shown by FIG. 2. Shown in FIG. 2 is capillary 8 which comprises an outer capillary sleeve 9 surrounding an inner capillary tube 10. Sleeve 9 has substantially cylindrical inner surface 11 and outer surface 14. Similarly, tube 10 has substantially cylindrical inner surface 12 and outer surface 13. The innermost channel, or bore, of capillary 8 is substantially formed by inner surface 12 of tube 10. Capillary 8 is substantially radially symmetrical about its central longitudinal axis 15 extending from an upstream end 16 to a downstream end 17. At each end, capillary 8 has conductive end caps 18 comprising the unitary combination of a tubular body having cylindrical inner surface 20 and outer surface 21 and an end plate 22 having inner surface 23 and outer surface 24 with a central aperture. The tubular body of end cap 18 encompasses and is in circumferential engagement with a reduced diameter portion 25 of sleeve 9 adjacent to the respective ends of capillary 8, such that the external diameter of end cap 18 is substantially the same as the external diameter of sleeve outer surface 14.
In order to remove tube 10, end cap 18 at the upstream end of capillary 8 is first removed. A removal tool (not shown) is inserted into the tube as to engage the tube's inner surface 12. It is further suggested by the prior art that in order to remove tube 10 it may be necessary to apply a slight torque orthogonal to axis 15, or other appropriate means such as bonding a removal tool to the tube using an adhesive. Once the tube is withdrawn, a replacement tube may be inserted into sleeve 9. However, this too is difficult and cumbersome, requiring tools to remove and replace the inner capillary tube.
Such prior art designs for the transfer capillary have inherent limitations relating to geometry, orientation, and ease of use. The capillary according to these prior art designs is substantially fixed in the source. Only if the instrument—or at least the source—is vented to atmospheric pressure can the capillary be removed. The geometric relation of the capillary is therefore fixed with respect to the source and all its components. This implies that the ion production means—e.g. an electrospray needle, atmospheric pressure chemical ionization sprayer, or MALDI probe—must be positioned with respect to the capillary entrance. In order to change from one ion production means to another—e.g. from an electrospray needle to a nano electrospray needle—the first means must be removed from the vicinity of the capillary entrance and the second must then be properly positioned with respect to the capillary entrance. For any production means, there will be an optimum geometry between the means and the capillary entrance at which the ion current passing into the analyzer is maximized. To achieve this optimum, a positioning means must be provided for positioning the ion production means with respect to the capillary entrance. This might take the form of precision machined components, a translation stage on which the ion production means is mounted, or some other device. If the ion production means is required or desired to be remote from the source, a long, fixed length capillary would have to be produced and installed (in a fixed position) in the source.
Another limitation of prior art capillaries relates to the orientation of the capillary bore with respect to the ion production means. Such orientation can be important for the operation of the source. One major consideration in the operation of an electrospray source is the formation of large droplets from the analyte solution at the spray needle. Such droplets do not readily evaporate. If these droplets enter the capillary, they may cause the capillary to become contaminated with a residue of analyte molecules and salts. In view of this, Apfel et al. in U.S. Pat. Nos. 5,495,108 and 5,750,988 describe apparatuses for API sources wherein the axis of the bore of the capillary 110 is at an angle of 90° with respect the axis of the bore of the spray needle 111, as depicted in FIG. 3. According to Apfel et al., certain experimental conditions lead to the production of large droplets by the spray needle. These large droplets will move away from the spray needle along the axis of the sprayer. However, an electric field between the spray needle and the capillary will cause ions formed from the spray to move towards the capillary. In this way, the ions are separated from the spray droplets and the droplets do not enter the capillary. However, this orientation is fixed in the prior art source of Apfel. To change this orientation, one would have to move the spray needle.
Prior art capillaries are further limited in the geometry of the capillary bore. That is, prior art capillaries as depicted in FIGS. 1–3, are substantially straight (i.e., cylindrically symmetric) and fixed (i.e., the geometry of the capillary and its bore is fixed at the time of manufacture). However, as described in the co-pending application METHOD AND APPARATUS FOR A MULTIPLE PART CAPILLARY DEVICE FOR USE IN MASS SPECTROMETRY Ser. No. 09/507,423 a capillary which can be cleaned or replaced without the need to shut down the entire mass spectrometer in which it resides now exists. The use of this capillary within the system described herein allows ionization to occur within the MALDI tray as opposed to occurring within the vacuum.
Others have disclosed atmospheric pressure matrix-assisted laser desoprtion/ionization (AP-MALDI). Laiko et al. disclose an AP-MALDI apparatus for the transfer of ions from an atmospheric pressure ionization region to a high vacuum region, which is pneumatically assisted (PA) by a stream of nitrogen gas. (Victor V. Laiko, Michael A. Baldwin and Alma L. Burlingame, “Atmospheric Pressure Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry”, Analytical Chemistry, Vol. 72, No. 4, Feb. 4, 2000) The invention of matrix-assisted laser desoprtion/ionization (MALDI) and electrospray ionization (ESI) are considered the most powerful tools for detection, identification, and characterization of biopolymers such as peptides, proteins, and DNA. MALDI and ESI enable the production of intact heavy molecular ions from a condensed phase, where MALDI is for solids and ESI is for liquids. Although, MALDI's target material density drops rapidly after laser desorption, from a high value characteristic of the initial solid phase to a very low value. Hence, a new ionization source combines atmospheric pressure and MALDI, which was called atmospheric pressure (AP) MALDI. AP-MALDI produces a uniform ion cloud under atmospheric pressure conditions. The apparatus disclosed in Laiko, i.e., for PA-AP-MALDI, is readily interchangeable with electrospray ionization on an orthoganal acceleration TOF mass spectrometer. According to Laiko, PA-AP-MALDI can detect low femtomole amounts of peptides in mixtures with good signal-to-noise ratio and with less discrimination for the detection of individual peptides in a protein digest. Thus, total sample consumption is higher for PA-AP-MALDI than vacuum MALDI, as the transfer of ions into the vacuum system is relatively inefficient.
Yet another high throughput MALDI elevated pressure mass spectrometry technique and apparatus is disclosed by Schevchenko et al. (“MALDI Quadrupole Time-of-Flight Mass Spectrometry: A Powerful Tool for Proteomic Research”, Analytical Chemistry, Vol. 72, No. 9, May 1, 2000). More particularly, Shevchenko et al. disclose use of a MALDI QqTOF mass spectrometer to achieve high mass resolution and accuracy in the identification of proteins. The apparatus disclosed by Schevchenko includes interfacing an orthogonal injection TOF MS to a hybrid quadrupole TOF MS (QqTOF) to form a MALDI QqTOF instrument, whereby a collisional damping interface cools the ions before they enter the analytical quadrupole Q. According to Schevchenko, once the ions are cooled, they can be transported through the quadrupoles more efficiently for measurement of the whole mass spectrum. A precursor ion can be selected in the quadrupole Q and fragmented in the collision cell q. Measurement of the product ions in the TOF section then provides a MS/MS spectrum of the selected precursor, thus carrying out both peptide mass mapping and MS/MS measurement on the same target in the same experiment. This process provides a high mass selection of precursor ions, precise tuning of the collision energy, and a much simplified calibration procedure. Also, Schevchenko et al. suggest that such an analytical approach lends itself to automation in obtaining MALDI spectra. However, Schevchenko et al. are silent as to how this might be achieved.
Also, Franzen et al. U.S. Pat. No. 5,663,561 (Franzen) teaches a device and method for the desorption and ionization of labile substance molecules at atmospheric pressure by MALD followed by chemical ionization (APCI). The method of Franzen consists of desorbing the analyte substances, which are mixed with decomposable substances (matrix substances) in solid form on a solid support, by laser irradiation at atmospheric pressure into a gas stream, and to add sufficient ions for proton transfer reactions to the gas stream. The objective of the method and apparatus of Franzen et al. is to transfer large molecules on solid sample support from solid state to a state of ionized gas phase molecules to be subjected to mass spectrometric analysis in an efficient manner.
The system disclosed in Franzen et al. generates ions from macromolecular substances in an area outside the vacuum, instead of within the vacuum, and separates the ionization process from the desorption process. Since new development of ion transfer from atmospheric pressure have become possible, external ionization has become effective and relatively economical. Thus, Franzen et al. recognized the problem of evaporating the non-volatile analyte substances into the surrounding gas. Therefore, the method and apparatus of Franzen et al. support the desorption process by photolytic and thermolytic processes triggered by laser photons. Consequently, the matrix material would decompose explosion-like into small gas molecules which can blast the analyte molecules into the surrounding gas. Then, the matrix molecules in the photolytic and thermolytic processes are broken down into smaller molecules. According to Franzen et al., if a matrix substance is selected in such a way that the product of its decomposition is gaseous in its normal state, the large, embedded analyte molecules would be catapulted into the gas phase. Of course, the matrix material then has to be selected such that the transfer of heat to the analyte molecules is minimal.
Moreover, in each of these systems, the samples are positioned outside of the vacuum system of the mass spectrometer for ionization (e.g., a MALDI target, sample plate, etc.). The present invention recognizes this and provides a simple and efficient method and apparatus for ionizing samples and introducing the sample ions into a mass spectrometer with the sample positioned outside of the vacuum system of the mass spectrometer.
Also, it has been recognized that a need exists for a simple, fast, efficient and reliable means of integrating a robot with various ionization sources for automating the preparation and introduction of samples into a mass spectrometer, and more particularly into an atmospheric pressure MALDI mass spectrometer. The present invention provides a novel solution to this problem.