Methods for determining gene functions
Disclosed herein are methods, systems and compositions for determining substrate specificity of an enzyme. The disclosed methods, systems and compositions can be used for identifying enzymes capable of modifying substrates of interest and/or quantifying enzymatic activity.
1. A method for determining substrate specificity of an enzyme, comprising:
generating a barcoded enzyme by transcription and translation of a polynucleotide encoding a barcoded enzyme which comprises the enzyme cleavably fused to a predetermined peptide barcode, wherein the polynucleotide comprises a gene encoding the enzyme fused to a nucleotide sequence encoding the predetermined peptide barcode;
providing a sample comprising the barcoded enzyme;
incubating the barcoded enzyme with a protease capable of removing the predetermined peptide barcode from the barcoded enzyme and one or more candidate substrates to obtain one or more modified candidate substrates in one or more reactions, wherein the barcoded enzyme is incubated with the protease prior to or during being incubated with the one or more candidate substrates, and wherein the one or more candidate substrates differ from one another by at least one functional group;
generating a mass spectrum of each of the one or more reactions;
determining a substrate specificity of the enzyme with respect to each of the one or more candidate substrates based on
whether ions of each of the one or more modified candidate substrates are in the mass spectrum, and/or
the ratio of ions of each of the one or more candidate substrates and ions of a corresponding modified candidate substrate in the mass spectrum;
determining the identity of the barcoded enzyme in the sample by identifying ions of the predetermined peptide barcode in the mass spectrum; and
quantifying the barcoded enzyme based on ions of the predetermined peptide barcode in the mass spectrum.
2. The method of claim 1 , wherein incubating the barcoded enzyme with the protease and the one or more candidate substrates comprises adding the barcoded enzyme into a solution, wherein the solution comprises the protease and the one or more candidate substrates.
3. The method of claim 1 , wherein the barcoded enzyme is incubated with some or all of the one or more candidate substrates in one of the one or more reactions, or wherein the barcoded enzyme is incubated with one of the one or more candidate substrates in one of the one or more reactions.
4. The method of claim 1 , wherein volume of at least one of the one or more reactions is about 1 microliter, 1 nanoliter, 1 picoliter, or any combination thereof.
5. The method of claim 1 , wherein generating the barcoded enzyme comprises:
providing the polynucleotide encoding the barcoded enzyme; and
incubating the polynucleotide in a cell-free transcription/translation reaction to generate the barcoded enzyme.
6. The method of claim 5 , wherein the components of the cell-free transcription/translation reaction comprise a reaction buffer, a RNA polymerase, nucleotide triphosphates (NTPs), a ribonuclease inhibitor, a ribosome, aminoacyl-tRNA synthetase, tRNA, an amino acid mixture, an initiation factor, an elongation factor, a release factor, or a combination thereof.
7. The method of claim 1 , wherein generating the barcoded enzyme comprises generating the barcoded enzyme using a microfluidic device.
8. The method of claim 7 , wherein generating the barcoded enzyme comprises:
generating a first droplet comprising the polynucleotide encoding the barcoded enzyme using a first microchannel of the microfluidic device;
generating a second droplet comprising components of a cell-free transcription/translation reaction using a second microchannel of the microfluidic device;
generating a first combined droplet comprising the polynucleotide and the components of the cell-free transcription/translation reaction from the first droplet and the second droplet using an immiscible fluid; and
incubating the polynucleotide with the components of the cell-free transcription/translation reaction to generate the barcoded enzyme in the first combined droplet.
9. The method of claim 1 , wherein incubating the barcoded enzyme with the one or more candidate substrates comprises incubating the barcoded enzyme with the one or more candidate substrates using a microfluidic device.
10. The method of claim 9 , wherein incubating the barcoded enzyme with the one or more candidate substrates using the microfluidic device comprises:
generating a first droplet comprising the barcoded enzyme using a first microchannel of the microfluidic device;
generating a second droplet comprising the one or more candidate substrates using a second microchannel of the microfluidic device;
generating a first combined droplet comprising the barcoded enzyme and the one or more candidate substrates from the first droplet and the second droplet using an immiscible fluidic; and
incubating the barcoded enzyme with the one or more candidate substrates in the first combined droplet.
11. The method of claim 1 , wherein each of the one or more candidate substrates differ from its corresponding modified candidate substrate by at least one functional group.
12. The method of claim 1 , wherein the one or more candidate substrates comprise at least 10 substrates, 100 substrates, 1000 substrates, or any combination thereof.
13. The method of claim 1 , wherein the quantity of ions of the predetermined peptide barcode in the mass spectra has a linear relationship with the concentration of the barcoded enzyme in a reaction.
14. The method of claim 1 , wherein the barcoded enzyme further comprises a quantification barcode.
15. The method of claim 14 , further comprising quantifying the barcoded enzyme based on ions of the quantification barcode in the mass spectrum.
16. The method of claim 15 , wherein the quantity of ions of the quantification barcode in the mass spectra has a linear relationship with the concentration of the barcoded enzyme in a reaction.