Systems and methods for preparing a sample and performing a real-time assay of the sample
Systems and methods that facilitate the automatic (or substantially automatic) preparation of a sample of a product containing molecules for analysis and automatic (or substantially automatic) performance of an assay of that sample. Thus, the preparation and analysis can be performed substantially in-real time, or, in other words, much more quickly than presently allowed by conventional systems and methods.
1. A method comprising:
(a) moving a sample comprising molecules from a first vial to a sample loop;
(b) moving a volume of the sample from the sample loop to a first multi-port valve;
(c) moving the volume of the sample from the first multi-port valve to a second multi-port valve fluidly coupled to and arranged downstream of the first multi-port valve;
(d) when the second multi-port valve is in a first position, moving the volume of the sample from the second multi-port valve to a capture column;
(e) capturing the molecules in the sample in the capture column, thereby separating the molecules in the sample from a matrix of the sample;
(f) moving an elution buffer solution from a buffer source to the capture column, thereby eluting molecules captured by the capture column and moving an elution/molecule mixture comprising the elution buffer solution and the eluted molecules to a second vial arranged downstream of the capture column, the second vial comprising a flow-through vial; and
(g) moving the molecules in the second vial to an analytical device for analysis of the molecules.
2. The method of claim 1 , further comprising, after (f) and before (g), diluting the eluted molecules in the second vial to reduce a salt concentration of the molecules.
3. The method of claim 2 , further comprising, after (f) and before (g):
moving the second multi-port valve to a second position different from the first position; and
when the second multi-port valve is in the second position, moving the eluted molecules to a desalting column via the first multi-port valve and the second multi-port valve, and applying the eluted molecules to the desalting column, thereby reducing the salt concentration.
4. The method of claim 1 , further comprising, after (f) and before (g),
adding a quenching reagent to the eluted molecules in the second vial;
moving the quenching reagent and the eluted molecules from the second vial to a reaction coil;
incubating the eluted molecules with the quenching reagent in the reaction coil, thereby quenching the molecules; and
moving the quenched molecules from the reaction coil to the second vial.
5. The method of claim 1 , wherein (g) comprises:
adding a denaturing reagent and a reducing reagent to the second vial;
moving the eluted molecules, the denaturing reagent, and the reducing reagent from the second vial to a first reaction coil via the first multi-port valve;
incubating the eluted molecules with the denaturing reagent, and the reducing reagent in the first reaction coil, resulting in denatured and reduced molecules;
moving the denatured and reduced molecules to a third vial, the third vial also comprising a flow-through vial; and
moving the denatured and reduced molecules in the third vial to the analytical device.
6. The method of claim 5 , further comprising, prior to moving the denatured and reduced molecules in the third vial to the analytical device:
adding an alkylating reagent to the third vial;
moving the denatured and reduced molecules and the alkylating reagent from the third vial to the first reaction coil via the first multi-port valve;
incubating the denatured and reduced molecules with the alkylating reagent in the first reaction coil, thereby alkylating the denatured and reduced molecules;
moving the denatured, reduced, and alkylated molecules from the first reaction coil to the second vial;
moving the denatured, reduced, and alkylated molecules to a desalting column via the first multi-port valve and the second multi-port valve;
applying the denatured, reduced, and alkylated molecules to the desalting column, thereby resulting in desalted molecules;
moving the desalted molecules to the second vial;
combining the desalted molecules with an enzyme;
moving the desalted molecules and the enzyme to a second reaction coil;
incubating the desalted molecules and the enzyme in the second reaction coil, thereby digesting the molecules;
moving the digested molecules to the third vial; and
moving the digested molecules from the third vial to the analytical device for analysis of the digested molecules.
7. The method of claim 6 , wherein the incubating comprises maintaining the first reaction coil at a first predetermined incubation temperature and/or maintaining the second reaction coil at a second predetermined incubation temperature.
8. The method of claim 1 , wherein one or more of (a) through (g) are performed automatically using a controller.
9. The method of claim 1 , wherein (a) through (g) are performed in a closed system.
10. The method of claim 1 , wherein the molecules comprise polypeptides, wherein the capture column comprises a polypeptide binding column, and wherein (e) comprises binding the polypeptides in the sample to the polypeptide binding column, thereby separating the polypeptides in the sample from the matrix of the sample.
11. The method of claim 1 , wherein the molecules have a molecular weight of 900 Daltons or less, and wherein the capture column comprises a reverse phase column, or wherein the molecules comprise metabolites.
12. The method of claim 1 , wherein the analytical device comprises a mass spectrometer.
13. The method of claim 1 , wherein the flow-through vial comprises:
a base;
a gutter coupled to and extending outwardly from the base;
an inlet port formed in the base; and
an outlet port formed in the gutter.
14. The method of claim 13 , wherein the flow-through vial further comprises a lip arranged between the base and the gutter, the lip configured to reduce surface tension of fluid flowing through the flow-through vial.
15. A method comprising:
(a) moving a sample comprising polypeptides from a first vial to a sample loop;
(b) moving a volume of the sample from the sample loop to a first multi-port valve;
(c) moving the volume of the sample from the first multi-port valve to a second multi-port valve fluidly coupled to and arranged downstream of the first multi-port valve;
(d) moving the volume of the sample from the second multi-port valve to a polypeptide binding column;
(e) binding the polypeptides in the volume of the sample to the polypeptide binding column, thereby separating the polypeptides in the sample from a matrix of the sample;
(f) moving glycosidases to the polypeptide-binding column via the second multi-port valve to release glycans from the bound polypeptides;
(g) moving the released glycans out of the polypeptide-binding column and to a second vial arranged downstream of the polypeptide-binding column, the second vial comprising a flow-through vial;
(h) mixing the released glycans with a glycan-labeling reagent in the second vial;
(i) moving the mixture of the released glycans and the glycan-labeling reagent to a reaction coil via the first multi-port valve;
(j) incubating the mixture of the released glycans and the glycan-labeling reagent in the reaction coil, thereby labeling the glycans;
(k) moving the mixture from the reaction coil to a third vial, the third vial also comprising a flow-through vial; and
(l) moving the labeled glycans in the third vial to an analytical device for analysis of the labeled glycans.
16. A closed system comprising:
a first vial adapted to contain a sample comprising molecules;
a sample loop adapted to receive the sample from the first vial;
a first multi-port valve fluidly coupled to the sample loop and arranged to obtain a volume of the sample via a first port of the first multi-port valve;
a second multi-port valve fluidly coupled to and arranged downstream of the first multi-port valve;
a capture column arranged to be fluidly coupled to the second multi-port valve when the second multi-port valve is in a first position, the capture column configured to bind the molecules from the sample;
a second vial arranged downstream of the capture column; and
a buffer source fluidly coupled to the first multi-port valve and arranged to supply elution buffer solution to the second vial via the second multi-port valve and the capture column when the second multi-port valve is in the first position, such that the elution buffer solution is adapted to at least partially elute all of the molecules from the capture column,
wherein the second vial comprises a flow-through vial configured to filter the elution buffer solution from the elution/molecule mixture out of the second vial, thereby leaving only the eluted molecules in the second vial.
17. The closed system of claim 16 , further comprising a desalting column arranged to be fluidly coupled to the second multi-port valve when the second multi-port valve is in a second position different from the first position, wherein when the second multi-port valve is in the second position, the desalting column is arranged to receive the eluted molecules in the second vial and configured to reduce a salt concentration of the eluted molecules.
18. The closed system of claim 16 , wherein the flow-through vial comprises a base, a gutter coupled to and extending outwardly from the base, an inlet port formed in the base, and an outlet port formed in the gutter.