Radiant energy – Ionic separation or analysis – With sample supply means
Reexamination Certificate
2002-05-31
2004-01-20
Anderson, Bruce (Department: 2881)
Radiant energy
Ionic separation or analysis
With sample supply means
C250S281000, C250S282000, C436S173000
Reexamination Certificate
active
06680477
ABSTRACT:
BACKGROUND OF THE INVENTION
Matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) has become an increasingly common tool for protein analysis in biological research since its development in 1988 (Karas, et al 1988, Tanaka et al RCMS, Fenseleau). The simple sample preparation, short analysis time and sensitivity have made this a powerful technique for protein identification (Fenseleau, Anal. Chem. 1997). Furthermore, the ability to generate intact molecular ions for whole proteins directly from complex mixtures makes this a particularly attractive technique for biological samples. (Redeker et al Anal Chem 1998). Electrospray ionization has proven to be another powerful and widespread ionization technique for mass spectrometric analysis of proteins and peptides that provides a means to directly couple liquid separations and mass analysis (Washburn, M. P., D. Wolters, and J. R. Yates III Nat. Biotech. 2001—Veenstra, T. D., S. Martinovic, G. A. Anderson, L. Pasa-Tolic, and R. D. Smith JASMS 2000). However the necessity of a liquid phase for the samples prior to ionization is in contrast to MALDI-MS where the sample is generally allowed to dry on a surface in combination with matrix molecules. It is the ability to generate ions from a solid phase sample that has led to a unique application of this technique whereby the location of the analyte within a heterogenous sample can be determined along with its mass.
MALDI-MS has been used to obtain mass spectra for proteins and peptides from precise X-Y locations from within a complex biological sample such as single cells (Garden et al JMS 1996—Chaurand, Stoeckli, and Caprioli Anal Chem 1999.). An extension of this approach was later described where multiple mass spectra were obtained by rastoring across the sample in a grid like pattern across the sample to form the pixels of an image.—(Stoeckli, M., T. B. Farmer, and R. M. Caprioli JASMS 1999—Caprioli, Farmer, Gile Anal Chem 1997) Using this data, the mass spectra could then be reassembled to create an image detailing the two dimensional position for a particular m/z value and therefore the corresponding protein. This has been demonstrated with several types of tissues and most dramatically with tissue sections from a rat brain (Todd et al, 2001, Stockle et al 2001-Stoeckli, Caprioli Nat. Med. 2001).
Other types of desorption/ionization mass spectrometry have been used to generate an “ion image”, but have generally used relatively harsh ionization methods, such as secondary ion mass spectrometry and laser ablation, and were limited to examining low molecular weight species (Belu, A. M. et al Anal Chem. 2001, Todd et al, 2001,—Stockle et al 2001-Kossokovski et al 1998). Two of these reports achieved a very tightly focused laser beam with near field microscopy fibers (Stockle et al 2001-Kossokovski et al 1998). However, one limitation of the near field microscopy approach is the fluence achievable at the fiber optic tip. Fiber optic damage thresholds are too easily exceeded when the tip diameter is reduced to 150-200 nm. Precision control of laser intensity and beam profile is required to inhibit fiber optic tip heating and self-ablation. In addition, because of the necessity for the fiber optic tip to be in the proximity of the desorption surface; contamination of the tip surface is a constant concern. Coupled with the effect of laser heating, tip lifetime is compromised.
Several challenges to creating an image from mass spectral data have been identified in prior work. Among them, are the need to evenly distribute the matrix over the sample to generate a homogeneous surface, removal of contaminant peaks that may suppress the signal of other analytes, and visualization of the tremendous amount of data generated by this technique. These issues have been described in a recent review (Todd, P. J and R. M. Caprioli. JMS 2001). One limitation that exists is the resolution that can be achieved when creating the “protein image”. The picture resolution is limited by the pixel size achievable, which is in turn limited by the size of the laser spot used to perform the ionization. Typically the laser spot size used to obtain MALDI-MS spectra is on the order of 25 &mgr;m in diameter (Stockle, R., R. Zenobi Anal Chem) with limitations at the 1 &mgr;m level mentioned (Todd et al 2001). However, the laser spot size reliably used for MALDI imaging has remained at 5 to 100 &mgr;m. (ibid.). While this provides ample resolution to distinguish structures within tissue sections or single neurons from
A. californica
(cells on the order of 92 &mgr;m), smaller structures/cells cannot be resolved from one another using this pixel size (Rubakhin, S. S. et al J. Neurophys 1999). In particular, microbes are often on the order of 1-2 &mgr;m in length and great resolution is required (Auerbach, I. D. et al J. Bact. 2000). Reductions in laser spot size are needed in order to generate MALDI-MS images of cells and extracellular structures on the microbial scale.
An improvement in laser focus can also lead to additional benefits by improving the ability of MALDI-MS to ionize extremely small protein samples in the analysis of dense protein arrays. Currently, sample plates holding up to 384 sample wells (each ~2 mm in diameter), are used for high throughput protein analysis using MALDI-MS. Manufacturers have introduced sample plates such as the “Anchor chip™” with affinity or adsorptive surfaces to concentrate the sample on a small area (Bruker Daltonics Inc., Product information literature, 2001). These aid in concentrating the sample as well as potentially creating more tightly packed arrays of samples with smaller spots from 200 to 800 &mgr;m in diameter. Meanwhile there have been other notable developments in deposition of small sample spots for analytical arrays that may have application MALDI-MS protein analysis. Methods creating small protein spots by spray deposition have been described with spots ranging from 100 to 500 microns (Onnerfjord, P et al. Anal Chem 1998—Moerman, R., et al. Anal. Chem.2001). Furthermore, microstructured devices have been fabricated as microreactors with features on the 1 to 5 &mgr;m scale with reactor wells of 15 &mgr;m being produced (Grzybowski, B. A., R. Haag, N. Bowden, and G. M. Whitesides, Anal. Chem 1998). While these spots are still above the typical laser spot size used for MALDI-MS, a recent report of protein samples being deposited in 15 nm diameter spots has appeared (Perkel, C. The Scientist 2002,16[5], p.34,). Clearly, as technologies for depositing arrays of samples improve, methods for producing smaller laser spots for both ionization and imaging in association with MALDI-MS are needed.
The development and application of tightly focused MALDI in the present invention allows for generation of higher resolution images detailing intact protein location and the creation of “protein images” of a small sample area that can be compared to optical images to reveal their location within a 2-D sample.
SUMMARY OF THE INVENTION
One object of the present invention provides a system and process for focusing light to a spot size for matrix assisted laser desorption/ionization (MALDI). A coherent light source, such as a laser or infrared light source, may be directed through at least one confocal microscopic objective to create a desorption/ionization source at the surface of a MALDI sample plate adapted to receive a sample substrate within the focal working distance of the microscopic objective. The light is transported by at least one fiber optic cable to at least one confocal microscopic objective. At least one collimating fiber optic coupler is employed to collimate the light to an aperature of at least one fiber optic cable. A insulating microscopic objective holder holds the microscopic objective and insulates it from the electrical fields of the MALDI. At least one adapter secures the insulating microscopic objective holder. At least one X, Y, positioner moves the microscopic objective in the X, Y co-ordinates. At least one Z positioner moves the m
Beck Kenneth M.
Wunschel David S.
Anderson Bruce
Battelle (Memorial Institute)
El-Shammaa Mary
Harrington Todd J.
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