Dear Mike,
Hope some of what follows answers some of your questions. I am of the grey
beard school ( yet shaved it off 5 years ago) who did in vacuo acid hydrolysis
20 years ago by sealing the hydrolysis tubes while under vacuum and using
constant boiling HCl overnight in a 100C oven. Today I use exclusively vapor
phase hydrolysis:
Q: Is this technique now widely accepted and used ?
A: I don't know the answer.
Q: What specialized manifold and sample vials are required ?
A: I use amino acid analysis (AAA or A(superscript: 3)) for determining
the concentration of peptide solutions, generally from HPLC
fractions, and rely on MS for characterization. So, I use long skinny
pipet tips to put 10 to 20 uL aliquots in 6x50 mm pyrex culture tubes, which
are generally about 100 to 1000 pmoles of peptide. After the samples are
dried in a Savant (about 20 min), these tubes are then put in a
125-mL Wheaton bottle, about 4 mLs of 6N HCl are put in the bottom of
the bottle, and a teflon-lined cap is tightly screwed on the bottle.
Q: What kind of block for heating works best ?
A: The bottle is put in a small crystallization dish so that it is tipped
at about a 45o angle, and then put in a 155C oven for 1 hour. When the
bottle has cooled down, the tubes are removed (using a 9-inch
dispo-Pasteur pipet inserted in tube to lift it out of the tipped
bottle) and dried again in the Savant for 5 minutes. The samples are dissolved
in 25 uL of 250 ug/mL K3EDTA, and 20 ul is spotted on the glass frits used
in a PE Biosystems Model 420 Amino Acid Analyzer (this is not a
comercial plug, so don't get bent out of shape)
Q: What hydrolysis time(s) should one try ?
A: The hydrolysis is performed for 1 hour in the 155C oven.
You might have noticed I do not take any steps to aid in recovery of Met, Trp or
Cys residues. I use the more stable residues for the final calculation of the
peptides and usually perform the analyses in triplicate.
Best of luck, and best regards.
Dick
Dr. Richard Noble
PE Biosystems
850 Lincoln Centre Drive
Foster City, CA 94404
Fax: 650-638-6311
Email: noblern@pebio.com
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