Protein Protocols Protein Protocols

462 Ward
Fig. 1. Amino acid sequence of insulin showing position of disulfide bonds.
3. Blow nitrogen gas over the reaction mixture to expel any oxygen.
4. Seal the tubes with a screw cap.
5. Allow the reaction to proceed at 37°C for 30 min.
6. After this time, an aliquot of the reaction mixture may be taken and analyzed by mass
spectrometry. Bovine insulin is shown as an example. This protein contains two interchain
bridges and one intrachain bridge (Fig. 1). The matrix-assisted laser desorption ionization
(MALDI) mass spectrum of reduced and alkylated insulin shows molecular ions at m/z
2763 and m/z 3612 corresponding to the two fully alkylated peptide chains (Fig. 2) (see
Note 3). There are no other signals present, indicating that no side reactions have occurred.
7. The sample can now be loaded onto a hydrophobic column for either N-terminal sequence
analysis, using the HPG1000A protein sequencer, or in situ enzymatic or chemical
digestion.
4. Notes
1. The PTH-pyridylethylcysteine derivative can be readily assigned during Edman sequence
analysis. The relative elution position on the HPLC system of the Applied Biosystems
gas-phase sequencer is between PTH-valine and diphenylthiourea. On the Hewlett Packard
G1000A system, PTH-pyridylethylcysteine elutes at 14.2 min just before PTH-methionine.
2. All reagents should be of the highest quality available.
3. The pyridylethylation reaction as described allows for fast, effective derivatization of cysteine
residues. The reaction is efficient, giving a high yield of fully alkylated product with no
side products.
4. The concept of monitoring chemical reactions by mass spectrometry is not new, yet the
sensitivity and speed of MALDI provide a means of examining reaction products to enable
the controlled use of reagents that have previously proven troublesome. A recent publication
by Vestling et al. (3) describes the controlled use of BNPS Skatole, a reagent that to
date has been seldom used owing to side reactions. The reactivity of such a reagent may be
more widely used in the future now that the progress of the reaction can be easily monitored.
See Chapter 91 for further uses of MALDI.
References
1. Glazer, A. N., Delange, R. J., and Sigman, D. S. (1975) Chemical characterization of
proteins and their derivatives, in Chemical Modifications of Proteins (Work, T. S and
Work. E., eds.), North Holland, Amsterdam, 21–24.
2. Allen, G. (1989) Sequencing of proteins and peptides, in Laboratory Techniques in Biochemistry
and Molecular Biology (Burdon, R. H. and Van Knippenberg, P. H., eds.),
Elsevier, Amsterdam.
3. Vestling, M. M., Kelly, M. A., and Fenselau, C. (1994) Optimisation by mass spectrometry
of a tryptophan-specific protein cleavage reaction. Rapid Commun. Mass Spectrom.
8, 786–790.