Dear Friends,
I am looking for a protocol to study the stability and half-life of a small peptide (6-8 amino acids) in human serum samples. Is there any simple method available for this? I appreciate your help on this.
Thank you.
Puru
LC-MS (liquid chromatography-mass spectrometry) is the best way. You begin by "crashing" the serum with organic solvent or acid, but you must use conditions in which the peptide remains in solution. Then separate the supernatant on LC and use MS to monitor the amount of the peptide remaining. To get help with this, talk to someone in a Pharmacology department, where this is a standard method for analyzing drug concentrations in blood samples.
If the peptide concentration is good (at least 2 mM), you can as well as use NMR spectroscopy to monitor it.
Dear Adam and Udaya, Thank you for your answers and suggestions.
Could please suggest what is maximum time point I should consider for peptide-serum incubation to design my experiments on this?
Next, what should the serum concentrations for these kind of experiments? I am planning to take 25% commercially available human serum! Do you think if it is OK to use this concentration for my initial experiments?
Thank you
Why not use 100% serum? That is the concentration in people.
In my experience, most in in vivo pharmacology experiments last for 24 hours, or sometimes only 8 hours. Perhaps this can guide your selection of time points.
Prof Dr Adam has given very relevant feedback,
Good luck Puroshottam !
I dealt with this exact problem while trying to spike plasma samples with a labeled peptide meant to be an internal standard. We did it with MALDI, after filtering through a tiny molecular weight cutoff filter to 'select' just the peptides for mass spec analysis. You could use LC/MS also, but my guess is MALDI should be easier -- particularly if you know the sequence of the peptide. In serum, you are virtually guaranteed to observe degradation, and you may be surprised how fast it is. In one case, we used a peptide that we literally never found intact - half-life was just seconds. I would strongly recommend switching to plasma if at all possible, and even then you may want to consider adding extra protease inhibitors. You must respect that serum is the result of extensive protease activity -- and it's definitely not the 'specific' activities that textbooks would have you believe (see last paper below).
These are papers we published on the subject, with our complete methods:
Article Investigation of Peptide Biomarker Stability in Plasma Sampl...
Article Intrinsic Peptidase Activity Causes a Sequential Multi-Step ...
Article Thrombin induces broad spectrum proteolysis in human serum samples
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