LC/MS Comparison


Fig. 4
a)

b)

It is sometimes desirable to find the similarities and differences between two LC/MS files (e.g. comparing digests of the phosphorylated and unphosphorylated forms of protein or, more generally, two digests of a protein that differ in their pretreatment in vitro or in vivo ). The program can be used to make such a comparison rapidly. Cytochrome C is an N-terminally blocked protein. In order to locate the N-terminal peptide the protein was first acetylated to block the lysine residues and then digested with chymotrypsin 1. Half of this digest was then re-acetylated.
a) The two contour plots of the digest with and without re-acetylation.
b) The ions found by the program to be present in both LC/MS runs were the same, plus one additional low level ion, as those found manually after several hours of inspection.

1 Thulin, C. D., & Walsh, K. A. in Techniques in Protein Chemistry ,Vol. VI, (Crabb, J. W., Ed.), Academic Press, Inc., 1995, 55-62.


CONCLUSION

We have designed a program, Sherpa, that provides a battery of searches to allow an investigator to interpret more efficiently LC/MS and MS/MS data from protein digests. As a result, the amount of manual interpretation needed is greatly reduced and focused. The flexible, nonlinear structure of Sherpa and its simple user interface allows hypotheses to be quickly tested under different settings and tolerances. Built-in rating systems for the results of searches draws attention to the most likely interpretations for further evaluation without excluding more remote possibilities from consideration.

REFERENCE
Taylor, J.A., Walsh, K.A., Johnson, R.S. Sherpa: A Macintosh-based expert system for the interpretation of ESI LC/MS and MS/MS of protein digests. Rapid Commun. Mass Spectrom. 10, 679-687 (1996).

ACKNOWLEDGEMENTS
JAT was supported in part by a Public Health Service National Research Service Award T32 GM07270 from the National Institute of General Medical Sciences. The cytochrome C data was kindly provided by Dr. Craig Thulin.

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Last Updated April 12, 1996
J. Alex Taylor (jataylor@hairyfatguy.com)