Re: Protein Sequencing and MS
Ioannis Papayannopoulos (iap@iname.com)
Fri, 27 Feb 1998 15:03:36 -0500
Having done peptide and protein characterization pretty much exclusively by
mass spectrometry until about a year ago, when my lab acquired an Edman
sequencer as well, I thought I'd offer my opinion on the relative merits of
these techniques, especially since I now do small molecule work and,
therefore, I have no "conflicts of interest".
I think that all opinions expressed by various people on this topic are all
correct: one can do wonderful things by mass spectrometry, if one is
trained well, as many have described. I don't mean to imply that
experience is not critical for obtaining and interpreting Edman data, but I
think that the "look and feel" of such data has not changed as dramatically
in the past 5-10 years as that of MS/MS data, although sequencers have
certainly imroved; therefore the accumulated experience and expertise in
Edman sequencing has been easier to transfer and disseminate.
In my opinion, the most important result that one can obtain with an Edman
sequencer, and which is not readily obtainable by mass spectrometry, is the
N-terminal sequence of an intact protein. I know the argument that many
important proteins are blocked (I have made the same argument myself), but
in such cases mass spectrometry is not much help either (try to dissociate
and make sense of the MS/MS data from an intact 80 kDa protein with about
200 fmol of sample available). The same holds for C-terminal sequencers;
yes, I know one can use carboxypeptidases and mass spectrometry to get the
C-terminus sequence, but this is limited to peptides and relatively low MW
proteins, as well as to the whims of the enzymes themselves.
Personally I found it fascinating that I could load my sample on the
sequencer in the evening, go home, and in the morning have the unambiguous
determination of the first ten amino acids of my protein waiting for me on
the printer. I gather that this feeling of accomplishment has been an "old
hat" to many of the contributors to this forum.
Quantitation is another area where N-terminal sequencing provides
information not readily obtainable by mass spectrometry. For example, the
determination of the relative amounts of two amino acid sequences (e.g.
N-terminally truncated proteins) can often be obtained from Edman
sequencing data; this may not be of great interest to those involved in
discovering novel proteins, for example, but it is a critically important
issue in the development and characterization of protein drugs.
One can make the argument that the mass spectrometry instruments are much
more versatile, interesting and exciting than N-terminal sequencers, in
spite the chemistries and materials developed for the latter. As a mass
spectrometrist I find my ability to "play around" with the mass
spectrometer a more satisfying experience than loading my sample on the
sequencer. However, I would not attempt to use this as an argument when I
discuss the relative merits of the two techniques, which, as the bottom
line, are, indeed, quite complimentary.
Ioannis Papayannopoulos
CytoMed, Inc.
Cambridge, MA