Re: MassSpec: H-D exchange

Richard S. Johnson (rjohnson@immunex.com)
Fri, 27 Feb 98 13:36:10 -0800

Len:

I'm not an FTICR guru, but I might have something to say about HD
exchange and mass spectrometry. It sounds as if you are proposing to
perform D labeling in solution, and fragment the protein via a quick
digestion using an acid protease at 0 C, and measuring the deuterium
incorporation within each peptide (ala David Smith in Nebraska). I
think its important to note that in this experiment the protein is
labeled in a native state (neutral pH, salts, buffers present), the
labeling is quenched by dropping the pH and temperature, and then the
protein is dissected with a protease like pepsin. I assume that you
propose going further than measuring the deuterium incorporation in each
proteolytic fragment by performing msms on the peptides with the hope that
one could make residue by residue assignments of deuterium exchange rates
(as NMR can do). The question is whether the deuterium labeling of the
amide backbone is scrambled during the mass spectral analysis (ionization,
mass selection, CID and detection).
I think the short answer is "yes, the deuterium will very likely scramble".
There has been one report of two large helical peptides (J Am Soc Mass Spec 5,
425-433) where the peptides were labeled with D2O and analyzed by MS/MS on a
triple quad and yielded a fragmentation pattern where the deuterium
incorporation rates closely matched the solution phase deuteration rates. In
contrast, I once studied a tripeptide Formyl Met Leu Phe O-Me (J Mass Spec 30,
386-387) where an acidified solution of the peptide was mixed in a Tee at 0 C
with an excess of D2O (also acidified) and the mixture was then directed to an
ESI source of a triple quad. At low pH and low temp, the half life for amide HD
exchange is about one hour, and since this mixture was analyzed within a few
seconds the signal corresponding to M+D could only be due to the peptide plus a
deuteron charge. This M+D positive ion was subjected to MSMS on a triple
quad (millisecond time scale rather than seconds in FTICR, I think), and
the fragmentation patterns were best explained by assuming a complete
randomization of the deuteron. I think both reports are correct, and can
be explained by the observation that amides seem to form stronger H-bonds
with water than with other amides (JACS 116, 2149). Thus, it would seem
to me that when you take a peptide that is helical in solution and toss it
into a vacuum the H-bonding would become stronger. HD exchange is
dependent on H-bonding of the amide hydrogens, so it may be that for the
larger helical peptides, the helix is retained in the mass spec and
thereby prevents the randomizing that occurs with the smaller tripeptide.
It seems to me that such experiments might work in some cases (as in the
large helical peptides), but not in other, and perhaps most, situations.


Regards, Rich Johnson at Immunex, Seattle, WA
______________________________ Reply Separator _________________________________
Subject: MassSpec: H-D exchange
Author: Len Packman <lcp2@mole.bio.cam.ac.uk> at Internet
Date: 2/27/98 9:02 AM

A question for the FTICR mass spec gurus, based on a hypothetical
situation, but one of interest for future work.

Imagine you had succeeded in isolating, in an FTICR cell, a peptide (or a
mixture of peptides) which had undergone a partial exchange of some of its
hydrogens with deuterium as the result of a H-D exchange experiment (in an
intact protein which was then digested in seconds and introduced to the
spectrometer). The aim of the experiment would be to determine which
residues had undergone exchange. The sites of exchange could be any of the
amide backbone protons and, where susceptible, side chain sites too.

My question is this. If one attempted to analyse the structure of a peptide
in this state by collision-induced fragmentation, is it known whether the
protons and deuterons would move around the structure as part of the
fragmentation process and thus make interpretation impossible? I don't know
enough about the fragmentation mechanism to offer an opinion.

Thanks for any input.

Len

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Dr Len C. Packman
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