Radiant energy – Ionic separation or analysis – With sample supply means
Reexamination Certificate
2002-01-23
2003-06-10
Nguyen, Kiet T. (Department: 2881)
Radiant energy
Ionic separation or analysis
With sample supply means
Reexamination Certificate
active
06576896
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a device and method for directing analytes to an analyzer and, more particularly, to a fluidic device and method that directs analytes to a mass spectrometer through electroosmotic pumping.
BACKGROUND OF THE INVENTION
A protein can be conclusively identified, without the need for de novo sequence determination, by correlating the information contained in its amino acid sequence with the corresponding object in a sequence database. The rapidly growing size of genomic, cDNA, and expressed sequence tag sequence databases has made this approach increasingly valuable. Several methods differing in the type of information extracted from the protein sample and the degree of automation have been described. The most conclusive and reliable methods are based on correlating collision-induced dissociation (CID) mass spectra of peptides with sequence databases. CID spectra of peptides are most frequently generated by electrospray ionization tandem mass spectrometry (ESI-MS/MS) on triple quadrupole or ion trap mass spectrometers.
For the generation of CID spectra in ESI systems, peptide samples have been introduced into the mass spectrometer by continuous infusion (flow injection) or on-line from liquid chromatography (LC) or capillary electrophoresis (CE) systems. The development of micro- and nano-electrospray ion sources has significantly improved the limit of detection achievable in ESI-MS to the low femtomole to attomole level. For the purpose of protein identification using peptide CID spectra, micro- and nano-electrospray ionization techniques have been successfully used with techniques to separate the peptides contained in protein digests and also in continuous flow analysis. In the continuous flow mode, the sample is sprayed into the MS at a flow rate of a few nl/min without peptide separation. CID spectra are sequentially generated from selected constituent peptides and used to search sequence databases or for de novo sequence determination. The most sensitive implementation of the continuous flow nanospray method is slow, tedious and difficult to automate and therefore does not realize the potential of the flow injection approach for high sample throughput and automated protein analysis. The methods involving on-line peptide separation have demonstrated the benefits of sample purification from contaminants, resolution of analytes and sample concentration. However, these methods are technically more involved and the most sensitive implementations have been difficult to automate.
The shift from the analysis of single genes and isolated proteins to the comprehensive analysis of biological systems and pathways has been one of the most dramatic recent developments in biological research. The shift is a direct consequence of the development of automated, high throughput genomic technologies which are now used for whole genome sequencing (e.g.,
Haemophilus influenzae
Rd and
Saccharomyces cerevisiae
) and for the establishment of comprehensive mRNA expression maps. However, there is currently no equivalent technology available for the analysis of biological systems on the protein level. Because proteins are the most significant class of biological control and effector molecules, a complete model of a biological process cannot be established without knowledge of the identity, function and state of activity of the proteins involved. Therefore, there is a demonstrated need for the development of a technology for the rapid and conclusive identification of proteins.
The current technologies for protein analysis described above have reached a level of sensitivity which permits the identification of essentially any protein which is detectable by conventional protein staining. However, protein sample throughput is orders of magnitude lower than the throughput of DNA-based technologies and some of the most sensitive protein analysis techniques developed to date are difficult to automate.
The implementation of steps required for protein identification by MS/MS (e.g., protein isolation. enzymatic or chemical fragmentation, peptide separation, and delivery of peptides to a mass analyzer) on microfabricated devices is a promising approach to enhance sample throughput and the sensitivity of protein analysis.
Microfabricated devices have been used previously for capillary electrophoresis and capillary gel electrophoresis where the analytes are detected on the device, typically by laser-induced fluorescence providing little structural information. Accordingly, there exists a need for a device and method for analyzing small quantities of proteins and providing structural information for the protein. Preferably, such a device and method is rapid, efficient, and readily automated. The present invention seeks to fulfill this need and provides further related advantages.
SUMMARY OF THE INVENTION
The present invention provides a device and method for delivering analytes to an analyzer to obtain structural information for the analyte. The device and method are useful in identifying proteins by mass spectrometry. The present invention is adaptable to many analytical problems beyond protein analysis that require rapid, unambiguous, automated, and sensitive analysis of complex mixtures of analytes.
In one aspect of the present invention, a device for delivering analytes to an analyzer is provided. The device includes one or more reservoirs connected by channels that direct sample flow to the analyzer. The device's reservoirs can include sample and buffer reservoirs, each of which includes an electrode that is in contact with the liquid sample contained within the reservoir. The electrode is connected to a power supply and, on applying a voltage to a reservoir, mobile analytes contained within the reservoir are directed to an analyzer, for example, a mass analyzer by means of a counterelectrode maintained at a stable electrode. The potential difference between the activated reservoir and the counterelectrode induces electroosmotic flow on the device and from the device. Preferably, the device is coupled to mass spectrometer through an interface, such as a microelectrospray interface, to introduce an analyte into a mass spectrometer for analysis.
In one embodiment, the invention provides a device and method for automating the continuous injection approach to protein identification by providing for the sequential infusion of different peptide samples into an electrospray ionization mass spectrometer without the need for sample manipulation. As noted above, the device includes sample and buffer reservoirs that are etched into the device and connected by channels, which are also etched into the device, and direct the reservoir contents to a microelectrospray ion source. Peptide samples, such as unseparated tryptic digests of proteins, can be applied to different reservoirs. A flow of liquid originating from a specific reservoir can be generated and selectively directed toward the microsprayer and the mass spectrometer by electroosmotic pumping. The analyte proteins can be identified by searching sequence databases either with the peptide masses generated by chemical or enzymatic protein fragmentation or with collision-induced dissociation (CID) spectra of selected peptides. The system achieves a limit of detection in the low fmol/&mgr;l range for peptide standards and can conclusively identify proteins at the low fmol/&mgr;l level. Furthermore, samples deposited in different reservoirs can be sequentially mobilized and analyzed without cross-contamination.
In another aspect, the present invention provides a system for the automated analysis of multiple analytes such as protein samples. The system includes a fluidics device, preferably a fluidics device such as described above, having multiple sample reservoirs for containing and directing samples to an analyzer; a computer-controlled array of high voltage relays for sequentially mobilizing analytes contained in the reservoirs and electroosmotically pumping the analyte to the analyzer; and an analyzer such
Aebersold Ruedi
Figeys Daniel
Christenson O'Connor Johnson Kindness PLLC
Nguyen Kiet T.
University of Washington
LandOfFree
Electroosmotic fluidic device and related methods does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Electroosmotic fluidic device and related methods, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Electroosmotic fluidic device and related methods will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3146291