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. 2019 May 21;91(10):6577-6584.
doi: 10.1021/acs.analchem.9b00244. Epub 2019 May 7.

Illuminating Biological Interactions with in Vivo Protein Footprinting

Jessica A Espino  1 , Lisa M Jones  1
Affiliations

Illuminating Biological Interactions with in Vivo Protein Footprinting

Jessica A Espino et al. Anal Chem. .

Abstract

Protein footprinting coupled with mass spectrometry is being increasingly used for the study of protein interactions and conformations. The hydroxyl radical footprinting method, fast photochemical oxidation of proteins (FPOP), utilizes hydroxyl radicals to oxidatively modify solvent accessible amino acids. Here, we describe the further development of FPOP for protein structural analysis in vivo (IV-FPOP) with Caenorhabditis elegans. C. elegans, part of the nematode family, are used as model systems for many human diseases. The ability to perform structural studies in these worms would provide insight into the role of structure in disease pathogenesis. Many parameters were optimized for labeling within the worms including the microfluidic flow system and hydrogen peroxide concentration. IV-FPOP was able to modify several hundred proteins in various organs within the worms. The method successfully probed solvent accessibility similarily to in vitro FPOP, demonstrating its potential for use as a structural technique in a multiorgan system. The coupling of the method with mass spectrometry allows for amino-acid-residue-level structural information, a higher resolution than currently available in vivo methods.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
In vivo FPOP workflow. Worms are grown to the fourth larvae stage (L4) on nematode growth media plates. For IV-FPOP, worms are flowed through a 250 μm fused silica capillary in the presence of H2O2, and radicals are generated using a 248 nm wavelength excimer laser. Immediately after irradiation, excess H2O2 and radicals are quenched, worms are lysed, the protein extract is digested and prepared for mass spectrometry analysis, and the extent of FPOP modifications is calculated for proteins of interest.
Figure 2
Figure 2
Flow system adapted for in vivo labeling in C. elegans. (a) Schematic of IV-FPOP flow system. Worms are kept separated from H2O2 until just prior to labeling; the window for laser irradiation is in light blue. (b) Percent recovery of worms after the flow system for two biological replicates (BR) with fused silica of 250 (gray) and 150 (black) μm ID. Error bars are calculated from the standard deviation across technical triplicates.
Figure 3
Figure 3
C. elegans viability in the presence of H2O2. (a) Bright field (black and white) and fluorescent (red) images of C. elegans (∼1000) at various H2O2 concentrations after 30 s of incubation. Fluorescent images show dead worms stained with PI. (b) Percent viability of worms using nine different H2O2 concentrations for two biological replicates. Negative control worms are in the presence of 50% methanol, and positive control worms have no H2O2 added. Error bars are calculated from the standard deviation across technical triplicates.
Figure 4
Figure 4
IV-FPOP oxidatively modifies proteins within C. elegans. (a) Venn diagram of modified proteins at 20 and 50 Hz laser frequencies. (b) Pie chart of oxidatively modified proteins within different body systems.
Figure 5
Figure 5
Correlating IV-FPOP modification to solvent accessibility. (a) Myosin chaperon protein UNC-45 (gray) (PDB ID 4I2Z(46)) highlighting two modified peptides identified by LC/MS/MS analysis, 669–680 and 698–706 (green, left inset). UNC-45 is bound to the Hsp90 peptide fragment (blue). Oxidatively modified residues within this fragment are shown in sticks (red), and UNC-45 is rendered as a surface (right inset). (b]) Tandem MS spectra of UNC-45 peptide 669–680 (top) and 698–706 (bottom) showing b- and y-ions for the loss of CO2, an FPOP modification. (c) The calculated ln(PF) for the Hsp90 oxidatively modified residues, R697, M698, E699, and E700. (d) Tandem MS spectra for R697, M698, E699, and E700 showing a +16 FPOP modification.
Figure 6
Figure 6
Comparison of actin modification. Oxidation of actin correlates with solvent accessibility through IV-FPOP. (a) ln(PF) of peptides modified by IV-FPOP, IC-FPOP (adapted from Espino et al.), and in vitro HRPF (adapted from Guan et al.). (b) Peptides oxidatively modified with in vitro HRPF, IC-FPOP, and IV-FPOP (blue), IV-FPOP and in vitro HRFP only (yellow), in vitro HRFP and IC-FPOP only (green), and HRFP only (magenta).
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