A reference guide for solution preparation
Choosing a reconstitution solvent for peptides.
The reconstitution solvent for a laboratory stock solution is chosen from two things: the peptide’s sequence and the method that will use the solution. This guide starts from water, uses the charges in the sequence to choose an acidic, basic or organic first solvent, and explains how salts, co-solvents and the choice of acid shape an assay or an HPLC or LC-MS run.
For laboratory research use only. Not for human or veterinary use.
In this guide
Why start with water, and which grade?
Water is the first solvent to try. An acid, a base or an organic solvent comes in only when water alone does not dissolve the peptide.
GenScript’s solubility guide tries water first. Hoofnagle and colleagues, writing on peptide standards for mass spectrometry, also dissolve first in water, adjust the pH from the sequence and add a small amount of organic solvent only to help. Peptides of fewer than about six residues generally dissolve in water alone.
The grade of water matters as much as the decision to start with it. Match it to the method that will use the solution.
- Type I (ultrapure) water
- Resistivity of 18.2 MΩ·cm and total organic carbon below about 10 µg/L. The grade for HPLC, cell and tissue culture and ICP-MS.
- HPLC-grade water
- Sold bottled for mobile phases, filtered through a 0.2 µm membrane, with no more than 1 mg/L of residue after evaporation.
- Standards
- ASTM D1193 and ISO 3696 define the laboratory water grades.
Neither the Type I definition nor the HPLC-grade specification says the water is sterile. For cell-based work, use water sold sterile, or filter it: Sigma-Aldrich names sterile distilled water, or sterile 0.1% acetic acid where applicable, for stock solutions. The main guide in this series covers filtration and what “sterile” can and cannot show.
How does a peptide’s charge point to a first reconstitution solvent?
For an analytical method or an assay, the first reconstitution solvent follows from counting the charges in the sequence.
The common rule scores each group like this:
- Basic groups: +1 for each lysine (K) and arginine (R), and +1 for a free N-terminal amine.
- Acidic groups: −1 for each aspartic acid (D) and glutamic acid (E), and −1 for a free C-terminal carboxyl.
- Histidine (H): sources differ. Sigma-Aldrich counts +1 only below pH 6 and 0 above it; JPT and GenScript count +1 at any pH.
The ends count. Bachem’s guide says the N- and C-terminal groups must be taken into consideration, and an acetylated N-terminus or an amidated C-terminus carries no terminal charge. Read the end groups on the certificate before you count.
A positive sum marks a basic peptide, a negative sum an acidic one, and zero a neutral one. After water, each class has its usual first solvent: dilute acetic acid for a basic peptide, dilute ammonium bicarbonate or ammonia for an acidic one, and a small volume of organic solvent for a neutral or very hydrophobic one.
Sigma-Aldrich also weighs the share of charged residues. When fewer than 10% of the residues are charged, or more than 50% are hydrophobic, it goes straight to an organic solvent such as acetonitrile, DMSO or DMF.
Peptides generally carry more charges at pH 6 to 8 than at pH 2 to 6, which is why many dissolve best near neutral pH. Very hydrophobic sequences and those that tend to aggregate are the exceptions. Bachem adds that trifluoroacetate salts rich in arginine and lysine tend to dissolve at neutral pH.
The rule is a starting point, not a prediction. Choosing a solvent can take trial and error, so confirm the choice with a solubility test on a small portion before dissolving the whole sample.
- Method limits firstFor cell assays, check the ≤1% DMSO limit; for LC-MS, avoid non-volatile salts. Test a small portion.
- Read the sequence and certificateCheck salt form and terminal groups; test a small portion.
- Short sequenceFor fewer than about six residues, try Type I water.
- Count chargeInclude free terminal groups, K, R, D and E; His is positive below pH 6.
- Positive chargeTry water, then a small volume of 10–30% acetic acid if needed.
- Negative chargeCheck free cysteine. Otherwise try water, then dilute base; keep pH below 8.
- Free cysteineAvoid ammonia and bicarbonate; use a degassed, slightly acidic solvent or DMF.
- Neutral or hydrophobicUse the smallest volume of acetonitrile, DMSO or DMF, then add to water or buffer.
- Negative charge without free cysteineTry water, then a small volume of 0.1 M ammonium bicarbonate or dilute ammonia; check pH near 7 and stay below 8.
- Bath and clarity checkUse a short bath sonication; warm to no more than 40 °C if needed. Gel, haze or particles mean suspended material.
- If still unclearIf the first solvent can be removed, freeze-dry the test portion and try another. Otherwise stop and record each solvent tried.
- When dissolvedDilute into buffer with salts last; record the final solvent composition.
When is dilute acetic acid the right first solvent?
When the charge count is positive and water alone has not dissolved the peptide, suppliers add a small volume of acetic acid, then dilute with water to the concentration the method needs.
Acetic acid is volatile. A test portion that does not dissolve can be dried by lyophilization and tried in the next solvent, which is why Sigma-Aldrich advises a first solvent that lyophilization can remove.
For a general stock, Sigma-Aldrich uses much weaker acid, 0.1% acetic acid. Its method for sets of peptides raises the acid to 10% (v/v) only for peptides that stay undissolved.
A figure of 0.6% acetic acid circulates on vendor websites, but none of the supplier or peer-reviewed documents reviewed for this guide gives it.
The supplier guides agree on the approach and differ on the strength. The figures below are each source’s own.
- Hoofnagle et al., 2016
- For mass spectrometry standards: acid up to 1%, as formic acid or TFA
For scale, by our arithmetic from the density and molar mass of acetic acid: 25% (v/v) is about 4.4 M, 10 to 30% about 1.7 to 5.2 M, and 0.1% about 17 mM.
Which base for an acidic peptide, and what about cysteine?
For an acidic peptide that water has not dissolved, suppliers add a small volume of a volatile base, dilute with water and check the pH.
The strengths they give:
- Sigma-Aldrich: 0.1 M ammonium bicarbonate, then water, making sure the final pH is about 7 and adjusting it in small steps if needed.
- Bachem: 0.1% aqueous ammonia.
- GenScript: a small volume of ammonium hydroxide.
- AAPPTec: 10% ammonium bicarbonate.
- Hoofnagle and colleagues: 1% ammonium bicarbonate, or ammonium hydroxide.
A free cysteine changes the choice. Its thiol oxidizes rapidly to a disulfide above pH 7, so Bachem dissolves such peptides in a carefully degassed acidic solvent, and GenScript rules out ammonium hydroxide and other basic solutions for them.
Sigma-Aldrich adds that cysteine oxidation speeds up as the pH rises: avoid pH above 8, and chill the solution if a higher pH cannot be avoided. Thermo Fisher raises pH only with very weak bases, because a strong base can racemize a peptide.
Peptides containing cysteine, methionine or tryptophan also call for oxygen-free solvents, and GenScript purges assay buffers with argon or nitrogen. A reducing agent such as DTT protects a free thiol, but DTT and TCEP also open disulfide bonds, so leave them out when a disulfide is part of the peptide’s structure.
How are DMSO, DMF and acetonitrile used as co-solvents?
A neutral or very hydrophobic peptide starts in a small volume of organic solvent. The rule is to use as little as dissolves the peptide, then dilute.
Dissolve fully, then dilute slowly
Dissolve the peptide completely in the smallest practical volume of acetonitrile, DMSO or DMF; Thermo Fisher uses a 50% (v/v) mixture of DMSO and water. Then add the organic solution dropwise into the aqueous buffer with gentle, constant agitation. If DMSO interferes with the experiment, DMF or acetonitrile can replace it. If the peptide comes back out of solution, Thermo Fisher dries it again and restarts with slightly more 50% DMSO.
DMSO and DMF cannot be dried off
Water, acetic acid and acetonitrile can be removed by lyophilization; DMSO and DMF cannot. Commit a sample to them only when the method tolerates them. Hoofnagle and colleagues also note that peptides stored in neat DMSO may precipitate when added to an aqueous solution, and that DMSO solutions need dilution before LC-MS to avoid poor chromatography.
DMSO is a mild oxidant
DMSO oxidizes free thiols to disulfides; in aqueous buffer, at about pH 3 to 8, it is used on purpose to form disulfide bonds in peptides. A cysteine peptide in a DMSO stock can therefore pair up or cyclize, which is why JPT and GenScript switch to DMF. For methionine the evidence is narrower: the method’s inventor reported no adverse reactions with methionine under those aqueous conditions, while Hoofnagle and colleagues note that methionine oxidation may occur in stocks stored in neat DMSO. Treat it as a storage precaution.
Keep the final DMSO low
In cell-based methods, the final DMSO concentration is commonly kept at or below 1% (v/v); JPT and STEMCELL Technologies both give that ceiling. Many cell methods use less, so follow the method. Plan the stock backwards from the limit: a neat DMSO stock diluted 1:100 into the assay gives 1% DMSO, and 1:1000 gives 0.1%. Bachem cautions that high concentrations of organic solvents are incompatible with cells.
Why does PBS go in after the peptide has dissolved?
Salts hinder solubility, so a buffer such as PBS is not the first solvent for a concentrated stock. Salts go in once the peptide has fully dissolved.
Hoofnagle and colleagues give this order for peptide standards. Sigma-Aldrich adds a practical reason: if a peptide fails to dissolve in an assay buffer that contains non-volatile salts, getting it back free of those salts is difficult.
The order is not absolute. Bachem recommends PBS at pH 7.0 to 7.4 when a concentration of 1 mg/mL or less is enough, and Thermo Fisher lists sterile water or a pH 7 buffer such as PBS, Tris or phosphate for its standard peptides.
For a concentrated stock, or a sequence whose behaviour is unknown, water or a volatile first solvent comes first and buffer second. Anything headed for LC-MS needs volatile buffers instead of PBS; the section on the analysis, below, explains why.
Preserved diluents
Water preserved with benzyl alcohol (sold as bacteriostatic water) is a preserved diluent, not a laboratory solvent choice.
It is sterile water with benzyl alcohol added as a preservative, typically at 0.9%, or 9 mg/mL, with a pH between 4.5 and 7.0. The preservative is there so that one container can be opened and sampled repeatedly. In Canada it is an authorized drug (DIN 00038202), and none of the supplier solubility guides reviewed for this page lists it as a peptide solvent.
Benzyl alcohol is cytotoxic to cultured cells. In a published study on cultured cells, it was toxic within 5 minutes at 9 mg/mL, and at 0.225 mg/mL it caused damage and impaired cell function at 2 hours; 0.0225 mg/mL did not harm the cells.
Preservatives of this kind can also drive aggregation. In a protein formulation study, dissolving a freeze-dried protein in 0.9% benzyl alcohol produced more aggregation than dissolving it in water, although benzyl alcohol did not speed up aggregation during later storage at room temperature.
Dilution does not remove it. Diluted 1:100 into an assay, 9 mg/mL of benzyl alcohol still leaves 0.09 mg/mL: four times the level that did not harm the cells in that study, and 40% of the level that damaged them at 2 hours. That concentration was not itself tested, but reaching 0.0225 mg/mL would take a 1:400 dilution.
A laboratory gets the protection a preservative offers from single-use aliquots and clean technique, without adding a cytotoxic, aggregation-prone component to the assay. Laboratory methods therefore generally choose unpreserved solvents: water of the grade the method needs, or the acid, base or organic solvent the charge rule points to.
Sources: the composition is from a US product label, and the Canadian status from Health Canada’s Drug Product Database. The cell findings are from Chang et al., 2008, and the aggregation findings from Roy et al., 2005.
How does the solvent affect an HPLC or LC-MS analysis?
A reconstitution solvent that suits the assay can still spoil an HPLC or LC-MS analysis. Check what the solution will carry into the instrument before you commit the sample.
DMSO and DMF absorb where peptides are read
A solvent’s UV cut-off is the wavelength at which a 1 cm path reads an absorbance of 1 (AU) against water. Water and acetonitrile cut off at 190 nm, methanol at 205 nm and TFA at 210 nm. DMSO and DMF both cut off at 268 nm, so they absorb strongly in the low-UV range where peptides are detected.
The acids absorb too
As mobile-phase additives, TFA absorbs significantly below 220 nm, and formic and acetic acids below 240 nm.
Non-volatile salts foul the instrument
Inorganic buffer salts such as PBS soon foul a mass spectrometer. LC-MS uses volatile buffers such as ammonium acetate or ammonium formate, with formic or acetic acid to control the pH and help ionization.
Sharp peaks or a strong signal
TFA is a very strong ion-pairing agent and gives sharp peptide peaks, but it suppresses the electrospray signal through ion pairing and changes in surface tension. Formic acid does not ion-pair; peptides still separate well, but the peaks can be broader. In some cases a little TFA, about 0.03%, is as effective as a mixture of 0.1% formic acid and 0.05% TFA, under conditions compatible with MS detection.
One published starting point
For peptide standards measured by mass spectrometry, Hoofnagle and colleagues formulate peptides in 5 to 30% acetonitrile with 0.1% formic acid at 0.5 to 1 mg/mL, and suggest 5% acetonitrile with 0.1 to 1% formic acid as a general starting solution.
Sources: the Burdick & Jackson UV cut-off table; Sigma-Aldrich on HPLC mobile-phase preparation; Advanced Materials Technology on mobile-phase additives for LC-MS; Apffel et al., 1995; Hoofnagle et al., 2016.
Common questions about peptide solvents.
Should a peptide go into water or buffer first?
Water first. Supplier guides and Hoofnagle and colleagues dissolve a peptide in water, or in a small volume of a volatile acid, base or organic solvent, and add buffer only once it has dissolved: salts hinder solubility, and a peptide that fails in buffer is hard to get back free of salts. For analytical work, use Type I water or bottled HPLC-grade water; for cell-based work, use water sold sterile, or filter it. Bachem recommends PBS at pH 7.0 to 7.4 when 1 mg/mL or less is enough.
What strength of acetic acid do suppliers use for basic peptides?
When the charge count is positive and water alone has not dissolved the peptide, suppliers add a small volume of acetic acid and then dilute with water: Sigma-Aldrich uses 25%, GenScript 10 to 30% and AAPPTec 30%. For a general stock, Sigma-Aldrich uses 0.1% acetic acid. Because acetic acid is volatile, a test portion that fails can be dried and tried again in another solvent.
Ammonium bicarbonate or ammonium hydroxide for an acidic peptide?
Either, in a small volume, followed by dilution with water. Sigma-Aldrich uses 0.1 M ammonium bicarbonate and checks that the final pH is about 7; Bachem uses 0.1% aqueous ammonia, and GenScript a small volume of ammonium hydroxide. Neither suits a peptide with a free cysteine, which oxidizes rapidly above pH 7. Avoid pH above 8, chill the solution if you cannot, and raise pH only with weak bases.
What final DMSO concentration do cell-based methods allow?
It is commonly kept at or below 1% (v/v), the ceiling JPT and STEMCELL Technologies both give, and many cell methods use less. A neat DMSO stock diluted 1:100 gives 1%, and 1:1000 gives 0.1%. DMSO cannot be removed afterwards by lyophilization.
Why avoid DMSO with cysteine or methionine?
DMSO oxidizes free thiols to disulfides and is used on purpose to form disulfide bonds in peptides, so a cysteine peptide in a DMSO stock can pair up or cyclize; JPT and GenScript use DMF instead. For methionine, Hoofnagle and colleagues note that oxidation may occur in stocks stored in neat DMSO, although the inventor of the DMSO method reported no adverse reactions with methionine, so treat it as a storage precaution.
What solvent suits a peptide pool?
No single solvent suits every pool. A common approach dissolves the pool in a small volume of DMSO and dilutes it with water: STEMCELL Technologies’ example uses 40 µL of DMSO and 210 µL of water for a stock at 100 µg/mL per peptide, and keeps the final DMSO in the cell assay below 1% (v/v). Sigma-Aldrich’s method for sets of peptides with mixed properties works through 0.1% acetic acid, 10% acetic acid and 20% acetonitrile before turning to DMF or DMSO.
What do urea, guanidine or arginine do to an assay?
Chaotropes break up hydrophobic aggregation and gels. GenScript uses 6 M guanidine hydrochloride or 8 M urea, and AAPPTec 6 M urea, with or without 20% acetic acid, or 6 M guanidine hydrochloride. They interfere with most biological systems, so the assay’s tolerance sets the limit, and urea also slowly carbamylates N-termini and lysines, faster with heat and at alkaline pH, which hampers identification by MS. Arginine suppresses aggregation in protein work at 0.1 to 2 M, but none of the peptide-supplier guides reviewed here names it.