US-12618038-B2 - Device and method for measuring in-cell protein folding

US12618038B2US 12618038 B2US12618038 B2US 12618038B2US-12618038-B2

Abstract

An apparatus, system, and method for studying protein folding in the native cellular environment is provided. The invention, termed pcIC-FPOP, combines pulse-chase experiments with in-cell protein footprinting coupled to mass spectrometry. This enables very high resolution information on the folding or mis-folding of proteins. The system ( 100 ) includes a multi-well plate ( 110 ), an incubator ( 120 ), a stage ( 130 ), a subsystem of reservoirs ( 172 ) and pumps ( 174 ), a laser source ( 140 ) and beam steering optics ( 150 ), and a computer system ( 160 ) programmed with a control module ( 162 ) to control the operation of the laser ( 140 ), optics ( 150 ), incubator ( 120 ), stage ( 130 ), or pumps ( 174 ), or some combination. This method permits studies that fill gaps in knowledge on protein folding and its role in disease.

Inventors

Lisa Jones
Anne Gershenson
Dante T. Johnson

Assignees

UNIVERSITY OF MARYLAND, BALTIMORE
THE UNIVERSITY OF MASSACHUSETTS

Dates

Publication Date: 20260505
Application Date: 20190328

Claims (11)

1 . A system comprising: a plate with a plurality of open wells; an incubator comprising: a frame defining a recess configured to receive the plate with the plurality of open wells and to removably accommodate the plate in the recess; a removeable cover for the recess, wherein the cover is transparent to laser light of a first wavelength and wherein the cover encloses the recess so that a controlled environment is established in the recess for preserving function of cells cultured in the plurality of wells, wherein the controlled environment encompasses the plurality of open wells and a region of the recess defined between the plate and the frame; an environmental supply coupled to the controlled environment and configured to supply heat and a gas mixture including carbon dioxide and water vapor to the controlled environment including the plurality of open wells and the region of the recess defined between the plate and the frame; and for each well of the plurality of wells, a plurality of perfusion tubes disposed to dispense fluid to or from a distal end at a position of each well, wherein each perfusion tube of the plurality of perfusion tubes is configured at a proximal end to be connected in fluid communication with one of a plurality of corresponding reservoirs outside the incubator; a laser source configured to emit a laser beam at the first wavelength; the environmental supply comprising; a thermal source configured to supply the heat to the controlled environment; and a gas source configured to supply the gas mixture; the plurality of corresponding reservoirs; a plurality of pumps in fluid communication with the plurality of corresponding reservoirs; at least one processor; and at least one memory including one or more sequences of instructions, the at least one memory and the one or more sequences of instructions configured to, with the at least one processor, cause the at least one processor to control on or more components of a group of components comprising the laser, the thermal source, the gas source, and the plurality of pumps; wherein the at least one memory and the one or more sequences of instructions are further configured to, with the at least one processor, cause the at least one processor to perform at least the steps of: a. for a first time interval, operate at least one first pump of the plurality of pumps to perfuse each well with fluid from a first reservoir of the plurality of corresponding reservoirs; b. for a second time interval after the first time interval operate at least one second pump of the plurality of pumps to perfuse each well with fluid from a different second reservoir of the plurality of corresponding reservoirs; c. for a third time interval after the second time interval operate at least one third pump of the plurality of pumps to perfuse a first well of the plurality of wells with fluid from a different third reservoir of the plurality of corresponding reservoirs; d. for a fourth time interval after the third time interval operate the laser source to illuminate the first well with the laser beam at the first wavelength; e. after the fourth time interval operate at least one fourth pump of the plurality of pumps to perfuse the first well with fluid from a different fourth reservoir of the plurality of corresponding reservoirs.
2 . The system as recited in claim 1 , the at least one memory and the one or more sequences of instructions are further configured to, with the at least one processor, cause the at least one processor to repeat steps c, d and e for each remaining well of the plurality of wells.
3 . The system as recited in claim 1 , wherein the first reservoir holds feeding media that includes at least one amino acid labeled with a mass spectrometry label; the second reservoir holds feeding media that does not include the mass spectrometry label; the third reservoir holds hydrogen peroxide; and the fourth reservoir holds a quench solution.
4 . The system as recited in claim 1 , further comprising: the plurality of perfusion tubes including four perfusion tubes connected at the distal end to each open well; wherein a first perfusion tube of the four perfusion tubes of each open well is connected between each open well and the first reservoir; wherein a second perfusion tube of the four perfusion tubes of each open well is connected between each open well and the different second reservoir; wherein a third perfusion tube of the four perfusion tubes of each open well is connected between each open well and the different third reservoir; wherein a fourth perfusion tube of the four perfusion tubes of each open well is connected between each open well and the different fourth reservoir.
5 . The system as recited in claim 4 , wherein the proximal end of each perfusion tube of the four perfusion tubes reaches outside the frame of the incubator to be in fluid communication with one of the plurality of pumps to move fluid to or from one of the plurality of corresponding reservoirs into each well.
6 . The system as recited in claim 4 , wherein the four perfusion tubes pass through openings defined by the frame of the incubator.
7 . The system as recited in claim 1 , wherein a mass of the incubator is less than 500 grams.
8 . The system as recited in claim 1 , wherein a maximum size dimension of the incubator is less than 150 millimeters.
9 . The system as recited in claim 1 , wherein the first wavelength is about 248 nanometers and the cover comprises fused quartz silica.
10 . The system as recited in claim 1 , wherein the thermal source is connected with the at least one processor by a communication line or a power line or both.
11 . The system as recited in claim 1 , wherein the gas source is connected with the at least one processor by a communication line or a power line or both.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a 371 national stage application of PCT Application No. PCT/US19/24691, filed Mar. 28, 2019, and claims benefit of Provisional Application No. 62/649,132, filed Mar. 28, 2018, the entire contents of which are hereby incorporated by reference as if fully set forth herein, under 35 U.S.C. § 119 (e). BACKGROUND 1. Field of the Invention The invention pertains to an apparatus and method for studying protein folding in the native cellular environment. The apparatus and method combine pulse-chase experiments with in-cell protein footprinting coupled to mass spectrometry. An embodiment of the apparatus includes multiple fluid conduits for application of fluids to different wells of a multi-well plate inside an incubator to accomplish the pulse-chase treatment of cells expressing a protein of interest. The method can be used to determine the intracellular folding pathways of proteins, including mis-folding pathways that lead to disease. 2. Background of the Invention Protein footprinting is a method that monitors protein conformation by selectively labeling/modifying or cleaving residues, where the selectivity is, in large part, a function of the residue's solvent accessibility as dictated by the conformation of the target protein. Therefore, this technique can be used to form an implicit picture of protein structure or conformation and to determine changes in conformation (i.e., tertiary and quaternary structure) that modify the accessibility of certain protein regions. Although the ways of modifying residues are diverse, and many have been in practice for over forty years, the advent of biomolecular mass spectrometry, employing electrospray and matrix-assisted laser desorption ionization (MALDI) and interfaced to liquid chromatography, makes possible highly specific, sensitive, and rapid analysis of modified peptides and proteins. See Fenn, et al., Science 246:64-71, 1989 and Karas et al., Anal. Chem. 57:2935-2939, 1985, the disclosures of which are hereby incorporated by reference in their entirety. In-cell protein folding has been monitored using temperature jumps and Förster resonance energy transfer (FRET) between fluorescent proteins at the N- and C-termini of a protein of interest, providing important data on how the environments of different organelles can affect protein folding. This and other fluorescence based methods have better time resolution. However, like other fluorescent based methods which only provide data on the local environment around the fluorophore, the FRET-based method only reports on changes in end-to-end distances, and as with in-cell NMR based methods that measure protein stability and folding, it only reports on fully synthesized chains. See, for example, Ebbinghaus et al., Nat. Methods 7:319-323, 2010 and Smith et al., Curr. Opin. Struct. Biol. 30:7-16, 2015. Protein oxidation by hydroxyl radicals is one class of footprinting methods; the various subclasses are differentiated by the means used to generate the hydroxyl radical (.OH). Hydroxyl radicals are useful for probing solvent accessibility because they have comparable size to solvent water molecules (and so proteins are accessible to hydroxyl radicals to a similar degree as water) and high reactivity with a significant fraction of amino acid side chains. The advantages of hydroxyl radical footprinting are twofold. First, the primary sequence of modified residues is preserved by virtue of the stable (irreversible) covalent modification that occurs even though a protein may be subjected to several hours and even days of handling and proteolysis following the chemical footprinting step. Second, the hydroxyl radical is a reactive reagent, modifying many amino acid residues and affording a higher coverage footprint than those covalent approaches that target specific residues (e.g., the acetylation of primary amines). Hydroxyl radicals are highly unstable and reactive oxidation agents which very quickly oxidize a large variety of groups such as those present in amino acid side groups, adding a hydroxyl group and thereby increasing mass, which is detectable by mass spectrometry. Fast photochemical oxidation of proteins (FPOP) is a mass spectrometry (MS)-based protein footprinting method. It uses a pulsed laser to photolyze hydrogen peroxide to generate OH radicals and modify proteins in a flow system on the microsecond timescale. The laser provides a spatially small, high flux of light, maximizing the exposure of a small volume of protein solution to hydroxyl radicals and ensures that the protein and hydrogen peroxide is irradiated only at the desired time and location. The FPOP method probes the solvent accessibility of amino acid side chains by allowing accessible side chains to react with the hydroxyl radicals and become modified thereby. The side chains that are accessible, and therefore become modified, change upon ligand binding or alterations in protein conformation. T