Protocol for reconstituting peptides/peptidomimetics from DMSO to aqueous buffers for circular dichroism analyses

Summary

Circular dichroism (CD) spectrometry is a rapid technique for detecting protein secondary structure, particularly helicity. DMSO is used to ensure optimal solubility of peptides/peptidomimetics; however, its background absorbance hinders effective CD analysis. Here, we present a protocol for reconstituting peptides/peptidomimetics from DMSO to aqueous buffers for CD analyses. We describe steps for identifying chemicals that induce DMSO evaporation, extracting peptides/peptidomimetics from DMSO, and CD spectrometer setup and analysis. We then detail procedures for secondary structure analyses of reconstituted peptides/peptidomimetics.

For complete details on the use and execution of this protocol, please refer to Gao et al. (2023).¹

Graphical abstract

Highlights

• •CD spectrometer can detect structural helicity in peptidomimetics
• •DMSO hinders CD detection when used to solubilize peptidomimetics
• •Instructions for removing DMSO from peptidomimetics using an evaporation assay
• •Reconstitute peptidomimetics in aqueous solutions for optimal CD detection

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Circular dichroism (CD) spectrometry is a rapid technique for detecting protein secondary structure, particularly helicity. DMSO is used to ensure optimal solubility of peptides/peptidomimetics; however, its background absorbance hinders effective CD analysis. Here, we present a protocol for reconstituting peptides/peptidomimetics from DMSO to aqueous buffers for CD analyses. We describe steps for identifying chemicals that induce DMSO evaporation, extracting peptides/peptidomimetics from DMSO, and CD spectrometer setup and analysis. We then detail procedures for secondary structure analyses of reconstituted peptides/peptidomimetics.

Before you begin

Peptides/peptidomimetics represent an essential category of candidates and chemical forms of drugs in the field of drug development, particularly when targeting specific helical regions within proteins for potential therapeutic benefits. Researchers often solubilize the peptides in dimethyl sulfoxide (DMSO) for precise measurements and assessments in various experimental setups. While DMSO is often considered as an excellent solvent because of its aprotic nature, inertness, low toxicity, and stability, its significant far-UV absorbance results in inaccurate circular dichroism (CD) analysis. While a helical protein in aqueous buffer exhibits a characteristic helical CD profile² (Figure 1A), CD spectrometry is unable to assess the helicity for a helical peptide in DMSO due to its high background reflected by the high tension (HT) voltage¹ (Figures 1B and 1C). Since peptide helicity is essential for their biological functions, the incorporation of DMSO poses a critical limitation in characterizing peptide drugs.

Figure 1.

DMSO exhibits a high background absorbance in CD analysis

(A) The upper panel shows the CD spectra of a helical-bundle protein R7R8 and a non-helical peptide B1KLE. CD spectra are used to characterize the helical configuration of peptides or proteins. R7R8 serves as a positive control, exhibiting a standard α-helix CD spectrum. The CD spectra of two concentrations (0.44 mM in yellow, and 0.88 mM in gray) of R7R8 are shown. The non-helical B1KLE peptide shows the CD signals of a random coil (blue) and is used as a negative control. The HT voltage of each sample is presented in the lower panel.

(B) The upper panel shows the CD spectrum of 10 mM S-TBS peptide dissolved in DMSO. The HT voltage, with a maximum HT of 900 V, is shown in the lower panel.

(C) The upper panel shows the CD spectrum of 10 mM S-TBS peptide dissolved in 1% DMSO. The HT voltage, with a maximum HT of 900 V, is shown in the lower panel.

To address this issue, we developed a protocol to rapidly extract peptides/peptidomimetics from DMSO without the need of additional equipment, allowing their reconstitution in aqueous buffers for CD analysis. We first designed a DMSO evaporation assay to identify suitable chemicals that expedite DMSO evaporation. Using the identified chemical solution, we are able to extract peptides and peptidomimetics under investigation from DMSO and reconstitute them in aqueous solutions for CD analyses. The reconstituted peptides/peptidomimetics exhibit characteristic helical CD profile as expected. The recently developed stapling modifications of peptides have been widely used in drug development to enhance peptide stability, helicity, and membrane permeability. This protocol allows us to take advantage of the potential therapeutic benefits of stapled peptides derived from helical regions of proteins. In addition, using DMSO as a solvent for small-molecule compounds or other reagents affects various biochemical, biological, or pharmacological processes, including protein crystallization, cell-based functional analyses, and chemical synthesis. This protocol also offers a general method for exchanging DMSO to other desired solvents. Thus, this protocol significantly improves the quality of our CD data with a few simple preparation steps, and also helps advance the development of new therapeutic agents by overcoming the challenges of using DMSO as the solvent.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Chemicals, peptides, and recombinant proteins
Ammonium nitrate (NH4NO3)	Fisher	A676-500
Potassium nitrate (KNO3)	Sigma	P8394-500G
Dimethyl sulfoxide (DMSO)	Fisher	BP231-1
B1KLE (peptide) (sequence: WKLEENPIYK)	Genemed (sequence: WKLEENPIYK)	LOT#: 126798
R7R8: Talin-1 (UniProt ID: P26039) R7R8 domains, residues 1357–1657	Purified recombinant protein	N/A
S-TBS: a stapled peptide derived from the Talin-binding site in RIAM (sequence: NEDIDQMFS TLLGE(S5)Dll(S5)TQS)3	New England Peptide	Lot# LB7720
S-4E: S-TBS peptidomimetic bearing four Glu substitutions (sequence: NEDIDQEES TEEGE(S5)Dll(S5)TQS)1	Biosynth	Lot# BU18769
Software and algorithms
Jasco Spectra Manager	Jasco	Version: 2.15.03
Other
CD spectrometer	Jasco	Model J-1100
24-well Hampton Research VDX plate	Fisher	NC2211685
Siliconized glass circle cover slides (22 mm)	Hampton Research	HR3-231
100% Pure petroleum jelly	Vaseline	N/A

Materials and equipment

Solutions tested for peptide reconstitution

Ammonium Nitrate (NH₃NO₃) Solutions

Powder	Dissolved in DMSO and make the final volume to	Final concentration
0.6 g	1 mL	60%
0.5 g	1 mL	50%
0.4 g	1 mL	40%
0.3 g	1 mL	30%
0.2 g	1 mL	20%
0.1 g	1 mL	10%

Note: Store at 23°C–27°C. Storage time is 1 month.

Potassium Nitrate (KNO₃) Solutions

Powder	Dissolved in DMSO and make the final volume to	Final concentration
0.20 g	1 mL	20%
0.17 g	1 mL	17%
0.14 g	1 mL	14%
0.11 g	1 mL	11%
0.08 g	1 mL	8%
0.05 g	1 mL	5%

Note: Store at 23°C–27°C. Storage time is 1 month.

Reconstitution Buffer

Reagent	Final concentration	Amount
1 M Tris 7.5	20 mM	10 mL
5 M NaCl	100 mM	10 mL
DI Water	N/A	980 mL
Total	N/A	1 L

Note: Store at 4°C. Preferably make fresh for each experiment.

Step-by-step method details

Identification of chemicals that induce DMSO evaporation

Timing: 1 day (this step is not required for the protocol)

This section describes the process of identifying and verifying candidate chemicals capable of inducing DMSO evaporation, allowing the reconstitution of peptides/peptidomimetics in aqueous buffers. Attempting to evaporate DMSO by natural air drying is unsuccessful, likely to due to its high boiling point and strong intermolecular forces. To facilitate DMSO evaporation, we employed a vapor diffusion method commonly used in protein crystallization. We then screened for chemicals highly soluble in DMSO and assessed their ability in inducing DMSO evaporation. The 60% NH₄NO₃ solution induces complete DMSO evaporation within 16 h in a 24-well VDX plate at 25°C.

• 1.
  Prepare a 24-well crystal screening plate.

  • a.
    Apply Vaseline to the edge of each well in the plate to seal it when a glass cover slide is placed on the well.
• 2.Select chemicals based on a screening of their solubility in DMSO.

Note: Scholarly literature and online research indicate that Nitrate salts have high solubility in DMSO.

Note: Screening of the inventory of nitrate chemicals revealed that Ammonium Nitrate and Potassium Nitrate exhibit the highest solubility in DMSO.

• 3.
  Prepare well solutions using NH₄NO₃ and KNO₃ to assess DMSO evaporation in the crystallization plate.

  • a.
    Prepare 60%, 50%, 40%, 30%, 20%, and 10% ammonium nitrate (NH₄NO₃) solutions in DMSO.
  • b.
    Prepare 20%, 17%, 14%, 11%, 8% and 5% potassium nitrate (KNO₃) solutions in DMSO.
• 4.
  Assess NH₄NO₃ and KNO₃ solutions for inducing DMSO evaporation.

  • a.
    Incubate 1 mL well solutions containing a gradient of nitrate salt concentrations with 4 μL of 100% DMSO, placed on the glass cover slides, within a 24-well crystallization plate (Figure 2A).
  • b.
    Evaluate DMSO evaporation by visually inspecting the size of DMSO droplets for up to 16 h (Figure S1).
  • c.
    DMSO evaporates faster when incubated with higher concentration of NH₄NO₃ solution. DMSO incubated with a 60% NH₄NO₃ solution is completely evaporated after 16 h (Figure 2B).

Figure 2.

DMSO evaporation assay

(A) A schematic of a 24-well VDX plate illustrates the setup of the evaporation assay.

(B) NH₄NO₃ of 10%–60% is used as well solutions for DMSO evaporation. Images of the drops in the 10% and 60%. NH₄NO₃ wells are taken right after the set up (time 0 h) and after 16 h of incubation (time 16 h).

Extract peptides/peptidomimetics from DMSO using the NH₄NO₃ solution

Timing: 16 h

This is the critical step that extracts DMSO from the peptide solution. A droplet of peptide solution is incubated with a 60% NH₄NO₃ solution for up to 16 h to thoroughly remove DMSO. The dried peptide can subsequently be reconstituted in an aqueous solution for CD analysis.

• 5.Add 1 mL 60% NH₄NO₃ solution to a VDX plate well.
• 6.Place 2–4 μL DMSO-solubilized peptides/peptidomimetics droplets on the center of a glass cover slip.
• 7.Flip the cover slip and place it on top of a plate well.

Note: Align and seal the greased edges by gently pressing and rotating the cover slip.

• 8.Incubate the droplets of peptides/peptidomimetics solution with 1 mL of the 60% NH₄NO₃ well solution for up to 16 h at 25°C.
• 9.After DMSO is completely evaporated, flip over the glass cover slip and place 4 μL of reconstitution buffer on the dried peptides/peptidomimetics.

Note: Pipetting the buffer vigorously helps the reconstitution of the sample.

CD spectrometer setup

Timing: 15 min + 1 min/sample

This step describes the procedure for setting up the CD spectrometer for data collection.

• 10.Open the Nitrogen tank and set the pressure for the spectrometer within the range of 20–35 ftˆ3/h. Maintain the pressure during the experiment.
• 11.Open Jasco Spectra Manager (Version 2.15.03.).
• 12.Turn on CD spectrometer.
• 13.Turn on Thermal controller.
• 14.
  Open Spectra Measurement and allow system to Purge for 10 min.

  • a.
    In Spectra Measurement general parameters are set to:

    • i.
      Channels Num: 3.
    • ii.
      Ch1: CD.
    • iii.
      Ch2: HT.
    • iv.
      CH3: Abs.
    • v.
      Start: 250 nm.
    • vi.
      End: 190 nm.
    • vii.
      Data Pitch 0.1 nm.
    • viii.
      Start Mode: immediately.
    • ix.
      Scanning Mode: Continuous.
    • x.
      Scan Speed: 100 nm/min.
    • xi.
      Accumulations [Check Box]; 3 accumulations.
  • b.
    Spectra Measurement control parameters are set to.

    • i.
      Corrections: None.

Note: Since the buffers are scanned separately for subtraction from the corresponding samples, there is no need to configure automatic subtraction.

• 15.Load 2 μL of reconstitution buffer to a 2 μL cuvette and settle the cuvette properly.
• 16.
  Collect CD data of the buffer using the Spectra Measurement window. The buffer spectra will be used in the subtraction tool of the Spectra Manager’s Spectra Analysis to subtract the signal of the buffer from the sample.

  • a.
    Go to Sample Measurement.

    • i.
      Input sample information in pop-up window.

Note: Critical information includes name, concentration, and buffer used.

CD spectrometry analysis

Timing: 15 min

This step describes the process of collecting CD spectrometry data from the reconstituted peptides/peptidomimetics solution and data processing using Spectra Manager.

• 17.Apply 2 μL reconstituted sample to the 2 μL cuvette and ensure proper placement of the cuvette on the base.
• 18.
  Collect CD spectra for the reconstituted sample on the CD spectrometer.

  • a.
    Go to Sample Measurement.

    • i.
      Include sample information in pop-up window.

Note: Fill in name, concentration, and buffer used.

• 19.
  Subtract the buffer spectra from the sample spectra.

  • a.
    Go to Spectra Managers, Spectra Analysis, select samples spectra alongside the previously saved buffer spectra -> Processing -> Subtraction.
  • b.
    Set the Subtraction window as [Sample] – [Buffer] = [Adjusted Result].

Note: The reconstitution buffer exhibits only minimal UV absorption and low background CD signals.

• 20.Perform baseline correction by selecting the adjusted spectra -> processing -> corrections -> baseline.

Secondary structure analyses of reconstituted peptides/peptidomimetics

Timing: 1 h (this step is not required for the protocol)

This step describes the helical structure characterization of a set of experimental peptides and verifies that the reconstitution process does not result in false helical CD spectra of non-helical peptides. Helicity of the samples can be identified by negative peaks a 208 nm and 222 nm on the CD spectra.

• 21.Reconstitute the S-TBS peptide from DMSO into the reconstitution buffer following the extraction protocol using NH₄NO₃ and subject to CD analysis.

Note: The reconstituted peptide exhibits a CD spectrum of an α-helical peptide and (Figure 3A) with optimal HT values.

• 22.Reconstitute the S-4E peptide from DMSO into the reconstitution buffer following the extraction protocol using NH₄NO₃ and subject to CD analysis.

Note: Reconstituted S-4E peptide from DMSO exhibits a helical-type CD spectrum (Figure 3B). However, S-4E peptide dissolved in 90% DMSO is not suitable for CD analysis (Figure S2). It is worth noting that S-4E exhibits a ratio of 0.4 for CD signals at 222 nm and 208 nm, suggesting a 3₁₀-helix configuration.⁴

• 23.
  Reconstitute the B1KLE peptide from DMSO into the reconstitution buffer following the extraction protocol using NH₄NO₃ and subject to CD analysis.

  • a.
    The B1KLE peptide directly dissolved in the reconstitution buffer exhibits CD spectra of a non-helical configuration (Figure 1A).

    Note: B1KLE is a non-helical peptide derived from the talin-binding site in integrin β1.⁵

  • b.
    The DMSO-solubilized B1KLE generates high absorbance background and is inapplicable for CD analysis as expected (Figure 4A).
  • c.
    The reconstituted B1KLE exhibits a CD spectrum consistent with that of a non-helical peptide with an optimized absorbance background (Figure 4B).

    Note: This step confirms that reconstitution of peptides from DMSO does not impact their secondary structure characteristics.

Figure 3.

The CD spectra of reconstituted S-TBS and S-4E peptides

(A) The CD spectrum of reconstituted S-TBS peptide at 25 mM. The HT voltage is presented in the lower panel.

(B) The CD spectrum of 20 mM reconstituted S-4E at 20 mM. The HT voltage is presented in the lower panel.

Figure 4.

Reconstitution of non-helical peptide yields no helical CD signal

(A) The upper panel shows the CD spectrum of DMSO-solubilized B1KLE. The HT voltage, with a maximum HT of 900 V, is shown in the lower panel.

(B) The upper panel shows the CD spectrum of the reconstituted B1KLE. The HT voltage for each sample is shown in the lower panel in respective colors.

Expected outcomes

Peptides dissolved in DMSO or a buffer containing DMSO exhibit CD spectra with significant background absorbance that is unsuitable for assessing helicity (Figures 1B and 1C). To resolubilize these peptides in aqueous buffers, peptides initially dissolved in DMSO are set up on a coverslip over the well solutions containing NH4NO3, which facilitates the DMSO evaporation, and the peptides are extracted as a result. After the peptides are totally “dried”, they are reconstituted in an aqueous buffer (20 mM Tris 7.5, 100 mM NaCl). The reconstituted peptides are expected to yield optimal CD spectra for helicity validation. Helical peptides typically exhibit a positive peak at 193 nm, and two negative peaks at 208 nm and 222 nm, respectively (Figures 1A, 3A, and 3B). We have previously verified the helicity of the stapled peptide S-TBS by crystallography.¹ Our CD spectrum data indicate that the C8-stapling modification of the peptide does not affect its helical CD spectrum characteristics. Our results offer robust experimental support for future structural analyses of stapled peptides using CD. Moreover, this protocol can also be employed to perform solvent exchange for DMSO-solubilized small-molecule compounds or other substances for optimal biochemical and pharmacological assessments.

Limitations

The limitations of this protocol include structural instability of the peptides in solution at 25°C, the inherent low aqueous solubility of the peptides, and incomplete reconstitution of the dry peptides from cover slips. These intrinsic properties of peptides pose significant challenges. Nevertheless, both the evaporation of DMSO and CD measurement can be performed at a lower temperature, such as 4°C, to help maintain the stability of the peptides. A Highly sensitive CD spectrometer may also be used for peptides with low aqueous solubility or incomplete reconstitution to assess helicity.

Troubleshooting

Problem 1

If the concentration of the sample droplet is excessively high, it will result in unreadable CD spectra.

Potential solution

To address this problem, the reconstituted droplet may be further diluted before applied for CD analysis. The extent of dilution depends on the initial sample concentration and the limit of detection (LoD) of the CD spectrometer.

Problem 2

The droplet does not “dry” after 16 h.

Potential solution

To address this problem, the evaporation time can be extended up to 48 h to ensure complete evaporation of DMSO. The temperature should be maintained at 25°C ± 2°C.

Problem 3

Buffer exhibits CD spectra with α-helix characteristics.

Potential solution

This problem is often attributed to residual samples from previous experiments. It is critical to thoroughly clean the cuvette and glass slides that hold the sample in the CD spectrometer using acetone and lens paper to remove all residual samples and solvents. In some cases, these residues can be difficult to remove if they have dried in the cuvette for an extended period. It is therefore crucial to perform a thorough cleaning of the instrument after each experiment and verify the background CD signals during buffer equilibration.

Problem 4

Intrinsic low aqueous solubility of peptides/peptidomimetics.

Potential solution

Peptides/peptidomimetics can often be solubilized in DMSO at a high concentration up to 20–50 mM range. In contrast, the CD spectrometer can detect the helicity of the peptide at 10–100 μM range, which can be achieved for most peptides/peptidomimetics with limited aqueous solubility. The CD signal can also be acquired at elevated temperatures (below the denaturation temperature) that enhance the aqueous solubility of peptides/peptidomimetics.

Problem 5

The well solution may dry out if not sealed properly.

Potential solution

The well solution can always be reused for evaporating DMSO in different peptides. However, if the well is not properly sealed by the cover slip, the well solution may dry out after a few days. To prevent this, ensure the well is properly sealed by applying Vaseline along the edge and firmly attaching the cover slip. If the solution does dry out, the powder in the well may be reconstituted with the appropriate amount of DMSO for reuse.

Problem 6

DMSO does not evaporate at a low temperature.

Potential solution

DMSO has a freezing point of 19°C. If the VDX plate is incubated at or below this temperature, DMSO may “freeze”. This would significantly slow down its evaporation. Ensure the plate is incubated in a temperature-controlled area that is at 25°C ± 2°C.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Jinhua Wu (jinhua.wu@fccc.edu).

Technical contact

Technical questions on executing this protocol should be directed to the technical contact, Jinhua Wu (jinhua.wu@fccc.edu).

Materials availability

This study did not generate new materials and new unique reagents.

Data and code availability

All data reported in this paper will be shared by the lead contact upon request. This paper did not generate original code.