Radiant energy – Ionic separation or analysis – With sample supply means
Reexamination Certificate
2002-04-16
2004-10-12
Lee, John R. (Department: 2881)
Radiant energy
Ionic separation or analysis
With sample supply means
C250S281000, C250S282000
Reexamination Certificate
active
06803566
ABSTRACT:
FIELD OF THE INVENTION
This invention relates generally to methods and apparatus for transport, ionization and subsequent analysis of analytes, and more specifically to methods and apparatus for analyzing a plurality of sample spots on an array using electrospray mass spectrometry.
BACKGROUND OF THE INVENTION
The study of protein complements of cells, tissues or whole organisms is referred to as proteomics. Proteomics is of great interest and much progress has been made in recent years in large part because of new enabling analytical technologies. One theme in proteomics is to monitor the expression of proteins in a biological system as the system responds to a stimulus. Currently, two-dimensional gel electrophoresis (2-DE) is the most common and powerful platform for the measurement of such protein complements. This approach can support expression profiling of several thousand proteins in multiple samples.
However, 2-DE has several significant limitations. These limitations include, for example, difficulty in running membrane proteins, complicated gel image analysis and manual preparation and running of the gels. Moreover, 2-DE requires spot excising and clean-up to utilize the highly specific and sensitive mass spectrometric-based protein identification methods employed. Therefore, alternative measurement platforms for protein expression profiling within complex samples are being explored.
Protein “arrays” or “chips” are one potential alternative. In addition to protein expression profiling, this technology has potential uses in identifying protein-protein interactions, protein substrates or potential candidates in drug discovery processes. This approach to screening protein activity benefits from the same advantages as commercially available DNA microarrays for mRNA expression analysis, namely high-throughput parallel, quantitative microscale analysis. It also has advantages over DNA microarrays.
True expression analyses must be done at the protein level because the final active product of most genes is the protein and protein expression and mRNA expression are not necessarily quantitatively linked. Furthermore, proteins can be synthesized in both active and inactive forms. To understand the biological function of a gene, the amount of active gene product must generally be determined.
Analysis of nucleic acid chips is usually performed using a fluorescent probe reporter attached to the analyte. A number of problems are associated with using this approach for protein array read out. First, and foremost, the fluorescence approach requires that only the analytes bind to the capture molecule and that non-specific binding is minimal. This is usually not the case with proteins, especially when the binding conditions cannot be optimized for each specific interaction. A second problem is that labeling of the proteins with a fluorescent probe can change their binding characteristics and can destroy protein complexes that exist in solution. Finally, the fluorescence approach cannot distinguish among the different forms of a given protein. This includes situations wherein the active and inactive form of the protein are captured and give equivalent signals. Both of these problems plague the current parallel standard for protein detection and quantitation, enzyme linked immunosorbant assay (ELISA), which operates on a capture/detection format.
Mass spectrometry (MS) techniques offer advantages for both detecting and identifying proteins. At present there is no other technology that can rival the combination of speed of analysis, sensitivity, and high accuracy measurement of molecular mass afforded by mass spectrometry in protein analysis. High-resolution, accurate mass determination allows detection of post-translational modification of proteins, even in protein mixtures, which is difficult to assess by other available techniques. Peptide fragments of proteins generated enzymatically, and analyzed by mass spectrometry, are now routinely used to identify the whole molecule via on-line protein database searching (peptide mapping).
Alternatively, new methods allow protein identification from the fragments generated from intact proteins in the gas-phase using tandem mass spectrometry, eliminating the need for enzymatic digestions. Tandem mass spectrometry uses two stages of mass analysis, one to preselect an ion and the second to analyze fragments induced, for instance, by collision with an inert gas, such as argon or helium. This dual analysis can be tandem in time, or more commonly tandem in space. Tandem in space is implemented using two mass spectrometers in series.
Mass spectrometry is now regarded as having great potential as a method for protein microarray read out. There are currently two ionization methods commonly used to generate gas-phase ions from proteins for analysis by mass spectrometry. These methods are matrix-assisted laser desorption ionization (MALDI) and electrospray (ES) ionization. Of these two methods, the most common choice for protein array read out is MALDI-MS, which is a surface analysis technique.
MALDI-MS approaches to protein chip read out are currently being exploited by two different companies, Ciphergen Biosystems, Inc. (Fremont, Calif.) and Intrinsic Bioprobes, Inc. (Tucson, Ariz.). They each offer protein chips for MALDI-MS containing from four to eight interaction sites. These commercial products can be obtained with particular general affinities for protein capture built-in, e.g., hydrophobic or hydrophilic interaction, anion exchange, cation exchange, and immobilized metal affinity substrates for capturing metal binding proteins. Alternatively, special order chips can be obtained with immobilized receptor species of the investigator's choice. For example, the immobilized substrates can be a specific antibody. While the commercial products are not true arrays, there have been laboratory demonstrations of the preparation and MALDI-MS analysis of protein interaction arrays as large as ten by ten (100 spots).
The commercial availability of MALDI-MS protein chip products is an indication of their utility. Nonetheless, the use of MALDI-MS for chip read out presents significant analytical limitations. There is a low number density of analyte at any small point on a particular array spot where the laser beam interacts to generate ions. This negatively impacts detection levels. Detection levels in MALDI-MS precipitously decline above a molecular mass of about 15 kDa. This can severely limit the range of proteins that can be analyzed directly. Time consuming enzymatic digestion methods are also needed for generating low mass peptides that are more amenable to detection when larger proteins are analyzed. These digestions are also needed to generate peptides for protein identification by peptide mapping. Mass accuracies in MALDI-MS are usually no better than about 0.01% (e.g., +6 Da for bovine albumin, ca. 66,000 Da). Finally, analysis of the arrays requires removal of the analyte from the native liquid environment within which the interactions occur and the application of a chemical matrix to facilitate desorption and ionization, followed by a drying step.
Electrospray is an alternative to MALDI. Electrospray generally involves flowing a sample liquid into an electrospray ion source comprising a small tube or capillary which is maintained at a high voltage, in absolute value terms, with respect to a nearby surface. The nearby (e.g. 1 cm) surface is commonly referred to as the counter electrode. Conventional ES systems for mass spectrometry apply high voltage (relative to a ground reference) to the emitter electrode while holding the counter electrode at a lower, near ground reference voltage. For the positive ion mode of operation, the voltage on the emitter is high positive, while for negative ion mode the emitter voltage is high negative.
The liquid introduced into the tube or capillary is dispersed and emitted as fine electrically charged droplets (plume) by the applied electrical field generated between the tube or capillary which is held at high
Akerman & Senterfitt
Gurzo Paul M.
Lee John R.
UT-Battelle LLC
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