Published: May 17th, 2021
A new static platform is used to characterize protein structure and interaction sites in the native cell environment utilizing a protein footprinting technique called in-cell fast photochemical oxidation of proteins (IC-FPOP).
Fast Photochemical Oxidation of proteins (FPOP) coupled with mass spectrometry (MS) has become an invaluable tool in structural proteomics to interrogate protein interactions, structure, and protein conformational dynamics as a function of solvent accessibility. In recent years, the scope of FPOP, a hydroxyl radical protein foot printing (HRPF) technique, has been expanded to protein labeling in live cell cultures, providing the means to study protein interactions in the convoluted cellular environment. In-cell protein modifications can provide insight into ligand induced structural changes or conformational changes accompanying protein complex formation, all within the cellular context. Protein footprinting has been accomplished employing a customary flow-based system and a 248 nm KrF excimer laser to yield hydroxyl radicals via photolysis of hydrogen peroxide, requiring 20 minutes of analysis for one cell sample.To facilitate time-resolved FPOP experiments, the use of a new 6-well plate-based IC-FPOP platform was pioneered. In the current system, a single laser pulse irradiates one entire well, which truncates the FPOP experimental time frame resulting in 20 seconds of analysis time, a 60-fold decrease. This greatly reduced analysis time makes it possible to research cellular mechanisms such as biochemical signaling cascades, protein folding, and differential experiments (i.e., drug-free vs. drug bound) in a time-dependent manner. This new instrumentation, entitled Platform Incubator with Movable XY Stage (PIXY), allows the user to perform cell culture and IC-FPOP directly on the optical bench using a platform incubator with temperature, CO2 and humidity control. The platform also includes a positioning stage, peristaltic pumps, and mirror optics for laser beam guidance. IC-FPOP conditions such as optics configuration, flow rates, transient transfections, and H2O2 concentration in PIXY have been optimized and peer-reviewed. Automation of all components of the system will reduce human manipulation and increase throughput.
Protein footprinting techniques can reveal profound information on the organization of proteins. These essential structural biology MS-based techniques are a component of the mass spectrometry toolbox. These methods probe protein higher order structure (HOS) and synergy via covalent labeling1,2,3,4. Fast photochemical oxidation of proteins (FPOP) employs hydroxyl radicals to oxidatively modify solvent accessible side chains of amino acids5,6 (Table 1). The method utilizes an excimer laser at 248 nm for photolysis of hydrogen peroxide (H2O2) to generate hydroxyl radicals. Theoretically, 19 of the 20 amino acids can be oxidatively modified with Gly being the lone exception. However, owing to the varying reactivity rates of amino acids with hydroxyl radicals, modification of only a subset of these has been observed experimentally. Still, the method does have the potential for analysis over the length of a protein sequence5. FPOP modifies proteins on the microsecond timescale, making it useful in studying weak interactions with fast off rates. Solvent accessibility changes upon ligand-binding or a change in protein conformation, thus, the power of the method lies in the comparison of the labeling pattern of a protein in multiple states (i.e., ligand-free compared to ligand-bound). As a result, FPOP has been successful in identifying protein-protein and protein-ligand interaction sites and regions of conformational change7,8,9,10. The FPOP method has been extended from the study of purified protein systems to in-cell analysis. In-cell FPOP (IC-FPOP) can oxidatively modify over a thousand proteins in cells to provide structural information across the proteome11,12. The conventional IC-FPOP platform utilizes a flow system to flow cells single file past the laser beam. The development of this system allowed individual cells to have equal exposure to laser irradiation. This led to a 13-fold rise in the number of oxidatively labeled proteins12. However, a limitation of the flow system is the length of a single sample experiment consisting of a 10-minute irradiation interval during which modification takes place and an additional 10-minute wash cycle. The time constraints of IC-FPOP makes it unsuitable for studying short lived protein folding intermediates or changes that exist among interaction networks in biochemical signaling cascades. This temporal limitation inspired the design of a new IC-FPOP platform equipped with higher throughput.
To accurately measure protein higher order structure in the native cell environment, the new design allows cell culture to be accomplished directly at the laser platform, which enables IC-FPOP to be high throughput. This setup also allows minimized perturbations to the cellular environment, in contrast to IC-FPOP using flow where adherent cells must be removed from the substrate. The new platform permits IC-FPOP to occur in a sterile incubation system using a CO2 and temperature-controlled stage top chamber while utilizing configured mirror optics for laser beam guidance, a positioning system for XY motion, and peristaltic pumps for chemical exchange. The new platform for conducting IC-FPOP is entitled Platform Incubator with Movable XY Stage (PIXY) (Figure 1). In PIXY, IC-FPOP is carried out on human cells grown in six-well plates within the platform incubator chamber. For this configuration, the laser beam is reflected downward onto the plate using beam compatible mirrors as a positioning stage that holds the incubator is moved, in the XY-plane, so the laser beam is strategically aligned to only irradiate one well at a time. Validation studies show that IC-FPOP can be performed faster in PIXY than in the flow system and leads to increased amino acid modifications per protein. The development of this new IC-FPOP platform will expound upon the knowledge that can be gained from cellular experiments13.
1. Assembly of Platform Incubator with Movable XY stage
NOTE: The new platform includes the incubation system, the positioning stage and controllers, the peristaltic pumps, the 248 nm KrF excimer laser, and the optical mirrors assembled on an Imperial optical breadboard.
2. Synchronization and initial automation of system via integration software
3. Grow cells in the platform incubator
NOTE: Cells must be placed in the platform incubator under sterile conditions in a cell culture hood the day before experimentation.
4. Make quench buffer and H2O2
5. Set up the platform incubator for IC-FPOP
NOTE: The platform incubator must be assembled under sterile conditions. Assemble the incubator in a sterile cell culture hood.
6. Performing IC-FPOP in the platform incubator
7. Protein extraction, purification, and proteolysis
8. High performance Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)
9. Proteome discoverer/data processing
To confirm that the platform incubator conditions are sufficient for cell culture at the laser platform, GCaMP2 was transiently transfected into HEK293T and transfection efficiency for both plates was assessed via fluorescence imaging (Figure 4A). GCaMP2 is a calcium sensing fluorescent protein used as a genetically encoded intracellular calcium indicator. It is a fusion of green fluorescent protein (GFP) and the calcium-binding protein, calmodulin. A luciferase assay was performed on HEK 293 cells transfected with plasmid prl-TK in order to quantify transfection efficiency (Figure 4B). These results show that the platform incubator exceeded the performance of the standard incubator, with a 1.13-fold increase in transfection efficiency, providing a quantitative benchmark for optimal cell culture environment.
FPOP modifications in HEK293T cells labeled in the flow system were compared to those labeled in the platform incubator and showed that the platform incubator outperforms the flow system both in the number of proteins modified (Figure 5) and the total FPOP coverage in those proteins. The number of FPOP modified proteins acquired in the platform incubator was approximately 1051, 2.2- fold more than those acquired in a typical experiment. Modifications were combined between two biological replicates for each experiment. Furthermore, PIXY provides higher throughput.
To demonstrate the advantage of higher modification coverage across a protein, IC-FPOP modifications were localized on the peptide level and the extent of modification was quantified to distinguish differences in outcomes between the systems for actin, a 375 amino acid protein. In the flow system, two modified peptides were detected, providing limited structural information (Figure 6A). However, five modified peptides spanning the actin sequence were detected in the platform incubator. Tandem mass spectra indicate that residue Pro322 was both modified and detected in each experiment (Figure 6B). The five peptides modified in the platform incubator samples contained twelve modified residues, while only four residues were modified with the flow system (Figure 6C). The increase in oxidation coverage provides more structural information across the protein.
Espino et al. demonstrated the capacity of FPOP to be performed in vivo (IV-FPOP) within C. elegans, a worm model for human disease states17.While IV-FPOP is also performed via a flow system, the PIXY system was tested for compatibility with the worms. Approximately 10,000 worms were incubated in each well in the platform incubator at 20 °C. LC−MS/MS analysis revealed that 792 proteins were modified by IV-FPOP in the platform incubator compared to the 545 proteins modified with the flow system (Figure 7). These results demonstrate that in addition to 2D cell culture, this new methodology is also compatible with the study of other biological systems such as C. elegans.
Figure 1. Schematic of PIXY System. System components: (A) stage-top incubator, (B) positioning system , (C) peristaltic pumps, and (D) perfusion lines. Cell culture media is removed from each well via pumps before H2O2 and quench solutions are infused at calculated timepoints. Laser path for irradiation showcased in white. Reprinted with permission from Johnson, D. T., Punshon-Smith, B., Espino, J. A., Gershenson, A., Jones, L. M., Implementing In-Cell Fast Photochemical Oxidation of Proteins in a Platform Incubator with a Movable XY Stage. Analytical Chemistry, 92(2), 1691-1696 2019. Copyright 2020 American Chemical Society. Please click here to view a larger version of this figure.
Figure 2. Fully Assembled PIXY system. (A) Touch monitoring system, (B) carbon dioxide unit, (C) temperature unit, (D) air pump, (E) humidifier, (F) optical mirrors, (G) platform incubator, (H) positioning stage, (I) 248nm KrF excimer laser, and (J) peristaltic pumps. Please click here to view a larger version of this figure.
Figure 3. Automation of Peristaltic Pumps. (A) Example command script in LABVIEW. Command options include volume, flow rate, pauses, flow direction. Speed, stage distance, and location are currently being automated. (B) Script reader in LABVIEW. Here, command scripts are uploaded then Run Sequence and START are pressed to initiate pumps. Please click here to view a larger version of this figure.
Figure 4. HEK cell transfection efficiency. (A) Mean fluorescent intensity of GCaMP2 transfection comparison between standard incubator (Control) and stage-top incubator (PIXY). Dots and squares represent each point in a well where a measure was taken. (B) Transfection efficiency quantitated and validated with a different vector plasmid, pRL-TK. P-value< 0.005. Reprinted with permission from Johnson, D. T., Punshon-Smith, B., Espino, J. A., Gershenson, A., Jones, L. M., Implementing In-Cell Fast Photochemical Oxidation of Proteins in a Platform Incubator with a Movable XY Stage. Analytical Chemistry, 92(2), 1691-1696 2019. Copyright 2020 American Chemical Society. Please click here to view a larger version of this figure.
Figure 5. Comparison of proteins modified in the single cell flow system and PIXY. Venn diagram of proteins modified using in the flow system (purple) and in PIXY (green). Reprinted with permission from Johnson, D. T., Punshon-Smith, B., Espino, J. A., Gershenson, A., Jones, L. M., Implementing In-Cell Fast Photochemical Oxidation of Proteins in a Platform Incubator with a Movable XY Stage. Analytical Chemistry, 92(2), 1691-1696 2019. Copyright 2020 American Chemical Society. Please click here to view a larger version of this figure.
Figure 6. Localization of IC-FPOP modifications. Comparison of IC-FPOP modifications between systems. (A) Bar graph of oxidatively modified peptides within actin from the flow system (purple) vs platform incubator (green). (B) Tandem MS spectra of actin (peptide 316-326) with modified proline in both systems and unmodified actin peptide (C) FPOP modified residues of actin (PDB: 6ZXJ, chain A 11 modified residues in platform incubator (green), 3 modified residues in the flow system (purple), 1 overlapping modified residue (yellow). Reprinted with permission from Johnson, D. T., Punshon-Smith, B., Espino, J. A., Gershenson, A., Jones, L. M., Implementing In-Cell Fast Photochemical Oxidation of Proteins in a Platform Incubator with a Movable XY Stage. Analytical Chemistry, 92(2), 1691-1696 2019. Copyright 2020 American Chemical Society. Please click here to view a larger version of this figure.
Figure 7. Comparison of FPOP modified proteins in C. elegans by flow vs PIXY. There is a 1.5-fold increase in oxidatively modified proteins using PIXY when compared to the flow system. Reprinted with permission from Johnson, D. T., Punshon-Smith, B., Espino, J. A., Gershenson, A., Jones, L. M., Implementing In-Cell Fast Photochemical Oxidation of Proteins in a Platform Incubator with a Movable XY Stage. Analytical Chemistry, 92(2), 1691-1696 2019. Copyright 2020 American Chemical Society. Please click here to view a larger version of this figure.
Table 1. Workflow modification distribution and mass shifts (Da). Please click here to download this table.
Proteins perform much of the work in living cells. Given this importance, detailed data on protein function and higher order structure (HOS) in the cellular environment is needed to deepen understanding of the intricacies in larger complexes and enzymatic reactions in cells as opposed to purified systems. To do this, a hydroxyl radical protein foot printing (HRFP) method was adopted entitled In-Cell Fast Photochemical Oxidation of Proteins (IC-FPOP). Most FPOP studies have been done in vitro in relatively pure protein systems, which markedly contrasts with the crowded molecular environment which affects binding interactions and protein conformational dynamics. As a result, there is a chasm between the findings from in vitro experiments18 and those that would be obtained in an actual cellular environment. To bridge the gap between the idealized conditions of an in vitro FPOP experiment and the complex nature of the cell, a new automated six-well plate-based in cell-FPOP platform has been developed. This novel FPOP technology is capable of identifying and characterizing these molecular species and tracing their dynamic molecular interactions in both healthy and diseased states. This new platform is called Platform Incubator with Movable XY stage (PIXY).
FPOP has been successfully used to characterize the structural information within the proteome. However, every biological technique has certain limitations that require further improvement. Specific reagents are required during laser photolysis and to efficiently quench unreacted hydroxyl radicals. Separation of digested peptides can require large amounts of time to maximize structural information.This wealth of information can also require extensive quantitation during post-MS data analysis1. The platform incubator, including the peripheral machinery needed for cell culture and IC-FPOP at the laser platform, comes with a large cost that may not be feasible for some labs. As progress continues to be made, robust software and analysis tools should advance the technique further; some of which is showcased in this study. Current studies in this platform incubator have been performed on HEK293T cells and in C. elegans. The IC-FPOP method has been shown to be compatible with a wide variety of cell lines including Chinese hamster ovary (CHO), Vero, MCF-7, and MCF10-A cells19. Since the general IC-FPOP method is translatable to this static platform, these cell lines should be amenable for study using PIXY as well.
IC-FPOP utilizes H2O2 to oxidatively modify solvent accessible side chains of amino acids, to then further discern protein interactions, structure, and metabolic effects within viable cells which is significant in providing biological context. It is essential before an IC-FPOP experiment to confirm that the cells are viable after H2O2 addition. Cell viability studies demonstrated that the cells were viable in the presence of H2O2 concentrations up to 200 mM 13. It is also important to make sure H2O2 is infused at a final concentration of 200 mM directly on cells after media is removed. Failure to completely remove cell culture media will cause varying concentrations of H2O2. Compared to standard conditions, increasing the incubation time to 10 seconds along with increasing the H2O2 concentration led to a higher number of proteins modified by IC-FPOP in the platform incubator. It is imperative to prime peristaltic pumps before use to ensure pumps are working properly and liquid is being dispersed. Failure to do so may cause air bubbles in the tubing, insufficient volume of H2O2 to immerse cells, and/or insufficient volume of quench solution.
Another issue that may arise is unwanted delays in the system. An example of this is the process of verifying received commands for the pump systems which adds significant delays on the order of 1000 or more milliseconds using the integration software. This problem can be fixed by minimizing the communication with the pumps during the experiment and using pre-set commands ahead of time as much as possible.
In the future, the goal for PIXY is producing a fully automated and integrated system. In addition to the peristaltic pumps, the triggering of the laser pulse will be automated. A new positioning system will also be utilized for the rapid movement of the platform incubator to enhance speed and accuracy. All components of the system will continue to be programmed using the integration software to further increase throughput.
The authors declare no competing financial interest.
The research presented herein is associated with the below referenced patent application:
U.S. Non-Provisional Patent Application Number: 17/042,565
Title: "Device and Method for Determining Protein Folding"
UMB Docket Number: LJ-2018-104 UMass Ref: UMA 18-059.
This work was supported by a grant from the NIH R01 GM128983-01.
|Ismatec Reglo ICC Peristaltic Pumps
|Kinematic Mirror Mount for Ø2" Optics
|50.0mm 248nm 45°, Excimer Laser Line Mirror
|Acetone, HPLC Grade
|Acetonitrile with 0.1% Formic Acid (v/v), LC/MS Grade
|ACQUITY UPLC M-Class Symmetry C18 Trap Column, 100Å, 5 µm, 180 µm x 20 mm, 2G, V/M, 1/pkg
|other high resolution instruments (e.g. Q exactive Orbitrap or Orbitrap Fusion) can be used
|ACQUITY UPLC M-Class System
|Afinia H480 3D Printer
|(Innovation Space University of Maryland Baltimore Campus Library)
|Aqua 5 µm C18 125 Å packing material
|carbon dioxide unit
|connectors (Y PP 1/16” and 1/16x1/8”)
|Delicate Task Wipers
|Dulbecco’s phosphate-buffered saline (DPBS)
|EX350 excimer laser
|Fetal bovine serum (FBS)
|Formic Acid, LC/MS Grade
|4 L quantity is not necessary
|Paul Shapiro Lab (University of Maryland Baltimore)
|LABVIEW Professional 2018
|MadMotor Positioning system and controllers
|Mad City Labs
|Methanol, LC/MS Grade
|any brand is sufficient
|Mad City Labs
|OKO Touch Monitoring System
|Orbitrap Fusion Lumos Tribrid Mass Spectrometer
|PE50-C pyroelectric energy meter
|Pierce Quantitative Colorimetric Peptide Assay
|Pierce Rapid Gold BCA Protein Assay Kit
|Pierce Trypsin Protease, MS Grade
|Pierce Universal Nuclease for Cell Lysis
|Six 33 mm PLA filament rings
|(Innovation Space University of Maryland Baltimore Campus Library)
|Thermo Scientific Pierce RIPA Buffer
|(Innovation Space University of Maryland Baltimore Campus Library)
|any 30% hydrogen peroxide is sufficient
|tubing (Tygon 3.18 and 1.59 ID)
|Water with 0.1% Formic Acid (v/v), LC/MS Grade
|4 L quantity is not necessary
|Water, LC/MS Grade
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