I have immunoprecipitated my protein of interest, along with putative interacting proteins. This mixture was resolved by SDS-PAGE, and proteins were in-gel digested by trypsin. Resulting peptides were then recovered, suspended in 5% formic acid, and placed at 4 degrees C.
All solvents used were HPLC MS grade.
My question is, How long can I expect these peptides to remain stable? Assuming they are in closed autosampler vials under refrigeration, can I expect the peptide mixture to be stable indefinitely? Or are they susceptible to some kind of degradation?
Peptides in general should be stable under refrigeration depending on the nature of these peptides. As peptides are the end products of digestion, they are not further digested by the trypsin. But they may be still susceptible to non enzymatic changes. Sometimes you may loose information about post translation modification like phosphorylation with time.If you want to store them for longer periods of time store them at -20 or -80 C.
Peptides in general should be stable under refrigeration depending on the nature of these peptides. As peptides are the end products of digestion, they are not further digested by the trypsin. But they may be still susceptible to non enzymatic changes. Sometimes you may loose information about post translation modification like phosphorylation with time.If you want to store them for longer periods of time store them at -20 or -80 C.
I agree with B. Packialakshmi, the peptides are dependent on the protein from which they came. Most will be very stable, but post-translational modifications may be lost. Typically, we store our peptides in a lyophilized state at 4oC until we are ready to run them for LC-MS/MS. Once we are ready to run the samples, we then resuspend them. The length of storage could also impact your results and if you are planning on repeating the experiment, you would want to be sure to note how long the samples are stored before being analyzed. Future experiments that do not have the same storage time may give very different results. Hope this helps -
Peptides are not very stable in aqueous buffers and can be very unstable depending on the amino acid composition and buffer pH which can have a dramatic effect on stability. Have first hand experience with this issue. If you have to store them in an aqueous buffer, aliquot and snap freeze in LN2 and store at -20-80oC. But best to keep as a dry powder protected from light and moisture at -20oC until needed and then re-suspend and use immediately. See the below on peptide stability factors.
From Genscript website (http://www.genscript.com/peptide_storage_and_handling.html):
The stability of each peptide is unique and is dependent on its sequence. Lyophilized peptides should be stored at -20℃, away from bright light. Most lyophilized peptides are stable for several years under these conditions, however, certain amino acid residues in peptide sequences can thwart long-term stability. Peptides containing Cys, Met, or Trp residues are prone to oxidation and require storage under anaerobic conditions to maintain stability. Peptides containing Asp, Glu, Lys, Arg, or His are prone to moisture absorption from the air, called deliquescence. These peptides should be stored in a dessicator in a tightly capped vial.
In addition, peptides that undergo freeze-thaw cycles are susceptible to degradation. Whenever possible, the amount of peptide required for each experimental set should be pre-determined, and peptides should be aliquoted into separate vials accordingly. Aliquoting reduces the number of freeze-thaw cycles, and reduces the amount of air exposure to the peptide.
Storage Guidelines for Peptides in Solution:
The shelf-life of peptides in solution is very limited, much shorter than that of lyophilized peptides. This is especially true for peptides whose sequences contain Cys, Met,Trp, Asp, Gln, and N-terminal Glu. In addition, peptides stored in solution are susceptible to bacterial degradation, therefore GenScript does not recommend storing peptides in solution. However, if storage of your peptide in solution is unavoidable, use sterile buffers at pH 5-6 to dissolve your peptide, divide the peptide solution into aliquots, and store the aliquots at -20℃. Avoid freeze-thaw cycles of the aliquots. You may choose to pass your peptide solution through a 0.2 µm filter to remove potential bacterial contamination from your peptide solution. If you dissolve too much of your peptide, re-lyophilization of the peptide solution will help you to maintain the stability of the excess peptide.
Preparing Peptides for Use:
When preparing your peptides for use in an experiment or assay, follow the guidelines below:
Warm the peptide vial to room temperature before opening.
Weigh out the desired quantity of peptide quickly.
Reseal the vial tightly.
Store the remaining peptide in a freezer, preferably below -20℃, under dry conditions.
Peptide Stability Guidelines Quick Chart
For all peptides
DO avoid repeated freeze-thaw cycles
DO aliquot lyophilized peptide according to daily experimental needs (Try GenScript’s free aliquoting service)
DON’T store peptides long-term in solution
DON’T repeatedly open the stock peptide vial
For peptides containing Cys, Met, or Trp residues
DO limit peptide exposure to air
DO purge assay buffers with argon or nitrogen gas
DO store peptides in tightly capped vials
For peptides containing Asp, Glu, Lys, Arg, or His
DO limit peptide exposure to air
DO store lyophilized peptides in a dessicator
DO store peptides in tightly capped vials
For peptides that must be stored in solution
DO avoid repeated freeze-thaw cycles
DO aliquot your peptide solution according to daily experimental needs
DO use sterile buffers to dissolve your peptide
DO filter your peptide using a 0.2 µm filter to remove bacterial contamination
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