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Topic: Peptide acid hydrolysis  (Read 6757 times)

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Offline hplcnewuser1

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Peptide acid hydrolysis
« on: January 25, 2017, 11:11:51 AM »
Hi everyone, my product is a peptide 14aa long and I need to hydrolyze and show aminoacid content. Does anyone have an idea how to go about it as far as sample prep. What sort of glass tube to use? My peptide has only I K and E so no need for nitrogen since there's no tryptophan. I am thinking of using 6M HCL 20%tfa and placing the peptide in solution, then placing the glass tube in 110 celcius oven for 48hours. Does this seem right and do I need vacuum?
Also any help with HPLC analysis is appreciate.
Thanks

Offline AWK

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Re: Peptide acid hydrolysis
« Reply #1 on: January 25, 2017, 11:38:55 AM »
Many years ago I used 6M HCl,  sealed glass ampoule in boiling water (2-3 days).
AWK

Offline hplcnewuser1

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Re: Peptide acid hydrolysis
« Reply #2 on: January 25, 2017, 12:12:48 PM »
how much peptide powder did you use lets say for 1ml of 6M HCl. Do you let the ampule cool before breaking open at the end?

Offline Babcock_Hall

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Re: Peptide acid hydrolysis
« Reply #3 on: January 25, 2017, 01:43:10 PM »
Some time ago I performed this experiment on whole proteins.  Here are a couple of general thoughts:  One, constant boiling HCl (which is about 6.07 M) is preferred because it probably has fewer oxidizing impurities than off-the-shelf HCl.  Two, there are glass digestion vials that I bought from Pierce, but I imagine are sold by others as well.  They allow one to perform a few freeze pump thaw cycles to minimize the amount of oxygen that is present.  I don't remember too much about how much protein I used.  Early volumes in the series Methods in Enzymology might have some technical tips.

Offline hplcnewuser1

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Re: Peptide acid hydrolysis
« Reply #4 on: January 25, 2017, 07:33:39 PM »
what do you mean by freeze pump thaw. Also does the  mixture of peptide and HCl need to be constantly under vaccum while it is in the heating block? what keeps this soltion from just being sucked into the vacuum pump?
thanks

Offline Babcock_Hall

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Re: Peptide acid hydrolysis
« Reply #5 on: January 25, 2017, 07:43:28 PM »
I would freeze the protein/HCl solution in the tube; then open the digestion tube to a vacuum line and evacuate the tube; then I would close the valve and let the tube warm up until the frozen solution was a liquid again.  I am not sure how many times I did this, perhaps 2 or 3 cycles.  The heating is done with the valve closed.  The first link below is a general description of the freeze-pump-thaw cycle.  The second link is the tube.  The third link is a previous discussion here.
http://depts.washington.edu/eooptic/linkfiles/Freeze_Pump_Thaw.pdf
http://www.piercenet.com/product/vacuum-hydrolysis-tubes
http://www.chemicalforums.com/index.php?topic=77276.0

Offline hplcnewuser1

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Re: Peptide acid hydrolysis
« Reply #6 on: January 25, 2017, 08:05:11 PM »
Thank you babcock hall that is a good starting point for us.

Offline hplcnewuser1

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Re: Peptide acid hydrolysis
« Reply #7 on: January 31, 2017, 06:13:53 PM »
Hi guys
I added peptide to piecre hydrolysis tube suggested above, then 6M HCL and small amount of phenol.
Turned open slightly the tube cap and turned on the vacuum and notice initially  some liquid jumped up the tube but afterwards was staying in the bottom of the tube so was fine. vacuumed for 5 seconds at lowest reading on vacuum pump and closed the tube cap.
Placed tube on heating block and noticed that when temperature reached 70C the acid started to boil all the way up the tube!
I am not sure if I should have let the vacuum on for longer once the reading reached lowest setting?
Is HCL supposed to boil up the tube when placed in heating block ?
Thanks

Offline Babcock_Hall

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Re: Peptide acid hydrolysis
« Reply #8 on: January 31, 2017, 07:40:52 PM »
Did you do cycles of freeze-pump-thaw?

Offline hplcnewuser1

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Re: Peptide acid hydrolysis
« Reply #9 on: January 31, 2017, 08:20:32 PM »
no cycles, used 1ml acid in 6ml tube. acuracy is not critical in this case

Offline Babcock_Hall

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Re: Peptide acid hydrolysis
« Reply #10 on: February 01, 2017, 08:35:29 AM »
I have always done cycles.  The idea is to remove gases, especially oxygen.

Offline hplcnewuser1

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Re: Peptide acid hydrolysis
« Reply #11 on: February 21, 2017, 03:19:55 PM »
my peptide is not soluble in 6M HCl. Does the peptide need to be soluble in acid in order to perform acid hydrolysis?  thanks

Offline Babcock_Hall

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Re: Peptide acid hydrolysis
« Reply #12 on: February 21, 2017, 05:14:07 PM »
I am not sure.  Is the peptide soluble at 110 °C?  If it is, my prediction is that your hydrolysis will work.

Offline AWK

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Re: Peptide acid hydrolysis
« Reply #13 on: February 21, 2017, 07:13:28 PM »
Hydrolysis also will go with solid peptide (with limits of two phase reaction).
AWK

Offline hplcnewuser1

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Re: Peptide acid hydrolysis
« Reply #14 on: February 21, 2017, 07:36:45 PM »
the sample form precipitate, peptide seems to assemble in clusters, even at 110C. It looks cloudy same before and after hydrolysis in vacuum pierce tube. used 1.5ml of 6M HCl 1% peptide by weight

My question is wether it is OK for final hydrolyzate to look cloudy or should it be clear?
diluted 6X and neutralized w/ 3M NaOH the shot in HPLC. Good news is no peptide peak, but I was wandering if the peptide is stuck in the filter used to clean up sample.
thanks

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