Method and apparatus for performing peptide digestion on a microfluidic device
First Claim
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1. A method of processing a protein sample in a microfluidic device, the method comprising the steps of:
- providing a microfluidic device comprising a first fluid network that includes a sample reservoir, a waste reservoir, a plurality of parallel channels connecting the sample reservoir to the waste reservoir, the plurality of parallel channels defining a first region proximate the sample reservoir, a second region adjacent the first region, and a third region proximate the waste reservoir, and a plurality of reagent reservoirs that introduce reagents at various points along each of the plurality of parallel channels, wherein each of the reagent reservoirs is in fluid communication with at least two of the plurality of parallel channels;
maintaining the first region at 50°
C. and the third region at a temperature below 50°
C. but above room temperature;
placing immobilized trypsin in a portion of each of the plurality of channels in the third region;
placing the protein sample into the sample reservoir;
placing the reagents required to carry out reduction, alkylation, and quenching into the reagent reservoirs;
applying a single negative pressure driving force to the waste reservoir that generates a continuous flow of fluid from the sample reservoir, through the plurality of channels, to the waste reservoir, wherein the flow sequentially exposes the protein sample to the reduction reagent in the first region, and the alkylation and quenching reagents in the second region before the protein sample flows through the immobilized trypsin in the third region; and
collecting the fluid including the protein sample in the waste reservoir.
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Abstract
The invention provides methods and apparatuses that allow a protein sample to undergo reduction, alkylation, and digestion in a continuous flow process carried out within a microfluidic device. Methods and apparatuses in accordance with the invention can be employed as part of an automated proteomics analysis carried out in an integrated proteomics system.
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Citations
16 Claims
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1. A method of processing a protein sample in a microfluidic device, the method comprising the steps of:
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providing a microfluidic device comprising a first fluid network that includes a sample reservoir, a waste reservoir, a plurality of parallel channels connecting the sample reservoir to the waste reservoir, the plurality of parallel channels defining a first region proximate the sample reservoir, a second region adjacent the first region, and a third region proximate the waste reservoir, and a plurality of reagent reservoirs that introduce reagents at various points along each of the plurality of parallel channels, wherein each of the reagent reservoirs is in fluid communication with at least two of the plurality of parallel channels; maintaining the first region at 50°
C. and the third region at a temperature below 50°
C. but above room temperature;placing immobilized trypsin in a portion of each of the plurality of channels in the third region; placing the protein sample into the sample reservoir; placing the reagents required to carry out reduction, alkylation, and quenching into the reagent reservoirs; applying a single negative pressure driving force to the waste reservoir that generates a continuous flow of fluid from the sample reservoir, through the plurality of channels, to the waste reservoir, wherein the flow sequentially exposes the protein sample to the reduction reagent in the first region, and the alkylation and quenching reagents in the second region before the protein sample flows through the immobilized trypsin in the third region; and collecting the fluid including the protein sample in the waste reservoir. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)
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16. A method of processing a protein sample in a microfluidic device, the method comprising the steps of:
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providing a microfluidic device comprising a fluid network that includes a sample reservoir, a waste reservoir, a plurality of parallel channels connecting the sample reservoir to the waste reservoir, the plurality of parallel channels defining a first region proximate the sample reservoir, a second region adjacent the first region, and a third region proximate the waste reservoir, and a plurality of reagent reservoirs that introduce reagents at various points along each of the plurality of parallel channels, wherein each of the reagent reservoirs is in fluid communication with at least two of the plurality of parallel channels; maintaining the third region at 37°
C. and the first region at a temperature above 37°
C.;placing immobilized trypsin in a portion of each of the plurality of channels in the third region; placing the protein sample into the sample reservoir; placing the reagents required to carry out reduction, alkylation, and quenching into the reagent reservoirs; applying a single negative pressure driving force to the waste reservoir that generates a continuous flow of fluid from the sample reservoir, through the plurality of channels, to the waste reservoir, wherein the flow sequentially exposes the protein sample to the reduction reagent in the first region, and the alkylation and quenching reagents in the second region before the protein sample flows through the immobilized trypsin in the third region; and collecting the fluid including the protein sample in the waste reservoir.
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Specification