Click-PEGylation - A mobility shift approach to assess the redox state of cysteines in candidate proteins

Abstract

The redox state of cysteine thiols is critical for protein function. Whereas cysteines play an important role in the maintenance of protein structure through the formation of internal disulfides, their nucleophilic thiol groups can become oxidatively modified in response to diverse redox challenges and thereby function in signalling and antioxidant defences. These oxidative modifications occur in response to a range of agents and stimuli, and can lead to the existence of multiple redox states for a given protein. To assess the role(s) of a protein in redox signalling and antioxidant defence, it is thus vital to be able to assess which of the multiple thiol redox states are present and to investigate how these alter under different conditions. While this can be done by a range of mass spectrometric-based methods, these are time-consuming, costly, and best suited to study abundant proteins or to perform an unbiased proteomic screen. One approach that can facilitate a targeted assessment of candidate proteins, as well as proteins that are low in abundance or proteomically challenging, is by electrophoretic mobility shift assays. Redox-modified cysteine residues are selectively tagged with a large group, such as a polyethylene glycol (PEG) polymer, and then the proteins are separated by electrophoresis followed by immunoblotting, which allows the inference of redox changes based on band shifts. However, the applicability of this method has been impaired by the difficulty of cleanly modifying protein thiols by large PEG reagents. To establish a more robust method for redox-selective PEGylation, we have utilised a Click chemistry approach, where free thiol groups are first labelled with a reagent modified to contain an alkyne moiety, which is subsequently Click-reacted with a PEG molecule containing a complementary azide function. This strategy can be adapted to study reversibly reduced or oxidised cysteines. Separation of the thiol labelling step from the PEG conjugation greatly facilitates the fidelity and flexibility of this approach. Here we show how the Click-PEGylation technique can be used to interrogate the redox state of proteins.

Keywords: Click chemistry; Cysteine; Mobility shift; PEGylation; Redox; Thiol.

Graphical abstract

Fig. 1

Click-PEGylation schemes. (A) Principle of the Click-PEGylation reaction. A reduced thiol is alkylated with propargyl-maleimide, then conjugated with an azide-PEG of high molecular weight (e.g. 5 kDa) using copper-catalysed Click chemistry. One of two possible regioisomers is depicted. (B) Click-PEG_red reaction to label reduced thiols. A protein with two potentially reversibly oxidisable cysteine residues is shown. For Click-PEG_red, the sample is reacted with propargyl-maleimide to label reduced cysteines, which are subsequently derivatised with azide-PEG via Click chemistry. Optionally, oxidised cysteines can then be reduced in vitro and blocked with NEM before separation by electrophoresis. (C) Click-PEG_ox reaction to label oxidised thiols. Conversely, for ClickPEG_ox, the sample is first reacted with NEM to block any reduced cysteine residues. Next, previously oxidised thiols are reduced in vitro, allowing their reaction with propargyl-maleimide and derivatisation with azide-PEG. Finally, samples are separated by electrophoresis to determine the resulting redox mobility shifts.

Fig. 2

Assessment of Click-PEGylation using purified GAPDH in vitro. (A) Click-PEG_red of purified rabbit GAPDH, which contains 4 cysteine residues, and can therefore exist in 5 possible redox states from 0 to 4 labelled cysteine thiols. Coomassie-stained mobility shift gel of GAPDH under untreated (i.e. endogenous), reduced (10 mM TCEP), and oxidised (1 mM diamide) conditions, showing redox-dependent band shifting. Profile plots of the ‘+ catalyst’ lanes were performed in FIJI. (B) Quantification of GAPDH cysteine redox state distribution from (A). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±SEM of n=4 independent experiments. (C) Parallel Click-PEG_red and Click-PEG_ox of purified GAPDH under reduced (10 mM TCEP), and oxidised (1 mM diamide) conditions, detected by Western blotting, showing redox-dependent band shifting. Profile plots of the ‘+ catalyst’ lanes were performed in FIJI. (D) Quantification of GAPDH cysteine redox state distribution from (C). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane (n=1). (E) Effect of different azide-PEG sizes on the Click-PEG_red band shifting. Coomassie-stained mobility shift gel of purified GAPDH under reduced conditions (10 mM TCEP) derivatised with either 1, 2 or 5 kDa azide-PEG. Profile plots of the ‘+ catalyst’ lanes were performed in FIJI. (F) Quantification of band shifting distribution from (E). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane (n=1). (G) Assessment of sample recovery following the Click-PEG reaction. Quantification of total band intensity for the ‘+ catalyst’ lane of the Coomassie-stained gel in (A), assessed using FIJI and normalised to the ‘– catalyst’ lane as a loading control. Data are means±SEM of n=3 independent experiments. (H) Quantification of total band intensity for the ‘+ catalyst’ lane of the Western blot in (C), assessed using LiCor Image Studio software and normalised to the ‘– catalyst’ lane as a loading control. Data are means±SEM of n=3 independent experiments.

Fig. 3

Optimisation of the Click-PEGylation technique. (A) Effect of protein concentration on labelling efficiency during the Click-PEG reaction. Coomassie-stained mobility shift gels of purified GAPDH under untreated conditions reacted by Click-PEG_red, comparing starting concentrations of 1 and 0.1 mg protein/mL. Profile plots of the ‘+ catalyst’ lanes were performed in FIJI. (B) Effect of propargyl-maleimide concentration on Click-PEG labelling. Coomassie-stained mobility shift gels of purified GAPDH reacted by Click-PEG_red comparing 5 and 50 mM propargyl-maleimide, under untreated, reduced (10 mM TCEP) and oxidised (1 mM diamide) conditions. * indicates a higher molecular weight band. (C) Quantification of GAPDH thiol redox state distribution from (B). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means ±range of n=2 independent experiments. (D) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Coomassie-stained gel of purified GAPDH under reduced conditions (10 mM TCEP) reacted by Click-PEG_red, comparing incubation times of 10, 30 and 120 min. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (E) Quantification of GAPDH thiol redox state distribution from (D). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments. (F) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Time course as for (D), except that GAPDH redox state was assessed by Western blotting. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (G) Quantification of GAPDH thiol redox state distribution from (F). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments.

Fig. 4

Application of Click-PEGylation to purified catalase and to endogenous GAPDH in biological samples. (A) Click-PEG_red of purified catalase under untreated, reduced (10 mM TCEP) and oxidised (1 mM diamide) conditions, detected by Western blotting. Profile plots of the ‘+ catalyst’ lanes were performed in FIJI. (B) Effect of different azide-PEG sizes on the Click-PEG_red band shifting. Western blot of purified catalase under reduced conditions (10 mM TCEP) derivatised with either 1, 2 or 5 kDa azide-PEG. Profile plots of the ‘+ catalyst’ lanes were performed in FIJI. (C) Analysis of endogenous GAPDH from cell lysates (C2C12 mouse myoblast) by Click-PEG_ox and Click-PEG_red detected by Western blotting. In vitro treatment of cell lysates under reduced (10 mM TCEP) and oxidised (1 mM diamide) conditions, as well as in situ analysis of GAPDH redox status in cell culture (untreated). (D) Quantification of band shifting in (C). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane (n=1).