H₂O₂-Responsive Protein Labeling Reagent Hyp-Stamp^™ | Funakoshi

Hyp-Stamp^™ is an H₂O₂-responsive protein labeling reagent.
Hyp-Stamp^™ responds to cellular H₂O₂ and accomplishes fluorescein labeling of proteins localized nearby H₂O₂. Since Hyp-Stamp^™-mediated fluorescein-labeled proteins remain at the original site, and after cell or tissue fixation, users can observe the H₂O₂ localization in fixed cells or fixed tissues by fluorescent microscope. Further, users can analyze and identify individual proteins nearby H₂O₂.

Conventional H₂O₂-responsive fluorescent probes can observe H₂O₂ generation in living cells while they become undetectable in fixed cells because they are removed in the fixation process. On the other hand, Hyp-Stamp^™ can visualize the local site of H₂O₂ after fixation of the cells, while it cannot observe H₂O₂ localization in living cells before fixation because of the overwhelmed fluorescence derived from the excess unreacted Hyp-Stamp^™.

RAW264.7 cells were immunostimulated (treated with 1 ug/mL PMA) and incubated for 30 minutes. Afterward, 5 uM Hyp-Stamp^™ was added, and the cells were cultured for 30 minutes. Then, cells were fixed with cold methanol or 4% formaldehyde and immunostained with mitochondrial proteins Sdha and Hspa9. Confocal laser scanning microscope imaging shows the immunofluorescence against Sdha and Hspa9 partially merged with fluorescein-labeled vesicles.

Murine hippocampal slices were treated with mitochondrial Complex II inhibitor TTFA (1 mM, 30 min) to induce oxidative stress, then 5 uM Hyp-Stamp^™ was added and incubated for 1 hour. The slices were fixed with 4% formaldehyde and permeabilized with cold methanol, followed by fluorescent imaging with a confocal laser scanning microscope. Localization of TTFA-triggered H₂O₂ was observed in the hippocampal slices.

HeLa cells were treated with mitochondrial Complex II inhibitor TTFA (1 mM, 30 min) to induce oxidative stress, then 5 uM Hyp-Stamp^™ was added and incubated for 30 min. After preparing a cell lysate, samples are applied to an SDS-PAGE, imaged by Coomassie Brilliant Blue (CBB), and in-gel fluorescence. Results revealed the amount of fluorescein-labeled protein increased by TTFA treatment, while TTFA did not affect the amount of total protein.

5 uM Hyp-Stamp^™ was added to RAW264.7 cells in which immunostimulation (treated with 1 ug/mL PMA for 30 minutes) was given (+) or not (-), and the cells were cultured for 30 minutes. After preparing the cell lysates, immunoprecipitation was performed with an anti-fluorescein antibody to enrich fluorescein-labeled proteins. Proteins obtained by immunoprecipitation were applied to in-gel trypsin digestion to produce peptide solutions. After modifying the PMA(+) and PMA(-) samples with TMT heavy tag and TMT light tag, respectively, the two samples were mixed in a 1:1 ratio and subjected to LC-MS/MS analysis to investigate changes between samples comprehensively. Figures show the experimental scheme of proteomics analysis (left) and volcano plot of protein signal changes by PMA(+)/(-) (right). In the plot, proteins with log₂(PMA added/not added) > 1, and P value < 0.05 are shown in green dots. Mitochondrial proteins such as Sdha and many vesicle proteins were identified, suggesting that these proteins exited nearby H₂O₂ generated by immunostimulation.