Protein Protocols Protein Protocols

708 Jensen and Wilm
Another method uses derivatization of free carboxyl groups, including the C-terminal
carboxyl group of peptides. The tryptic peptide mixture is split in two portions. The first
portion of the mixture is analyzed by nanoelectrospray tandem mass spectrometry and long
peptide sequences are generated through complete interpretation of tandem mass spectra
using the guidelines described in the preceding. The other portion of the peptide mixture is
O-methyl-esterified (17,35,36) and then analyzed. Every free carboxyl-group including the
C-terminus of peptides is esterified and therefore increase in mass by 14 Da. The number of
methyl-esters in a peptide can be determined by the mass shift of peptides which is predictable
from the previously interpreted tandem mass spectra of the native peptides. Because
all y–ion fragments produced from an esterified peptide contain the C-terminus they are all
shifted up in mass. Comparison of a set of tandem mass spectra obtained from a peptide and
the corresponding esterified peptide serve to confirm the amino acid sequence because the
y–ion series can unambiguosly be assigned. In addition, internal acidic residues, Asp and
Glu, are methylated as well and can easily be differentiated from their corresponding amide
residues, Asn and Gln, which otherwise differ in mass by only 1 Da.
2.12. Perspectives
Novel peptide sample preparation methods, mass analyzer configurations, and
peptide dissociation techniques are continuously developed to increase sensitivity, mass
accuracy, mass resolution, or sample throughput for peptide mass analysis and sequencing
by mass spectrometry. The combination of the MALDI source with a Q-TOF hybrid
instrument (37) and the development of electron capture dissociation (ECD) for
MS/MS sequencing of large peptides and small intact proteins (38) are just two recent
examples. Posttranslational modification of proteins leads to a change in the molecular
mass of the affected residues and mass spectrometry is, therefore, a versatile analytical
tool for structural characterization of modified peptides and proteins (39,40). MS-based
approaches to peptide and protein quantitation using stable isotope labeling are also
being pursued (41) and the use of multidimensional chromatography methods combined
with ESI-MS/MS is an alternative or complement to two-dimensional gel electrophoresis
for analysis of very complex protein mixtures (42–44). There is no doubt that
applications of mass spectrometry in biological research will expand substantially in
the future as the structure and function of all the gene products encoded in genomes of
model organisms, including humans, have to be characterized in molecular details.
References
1. Fenn, J. B., Mann, M., Meng, C. K., Wong, S. F., and Whitehouse, C. M. (1989) Electrospray
ionization for the mass spectrometry of large biomolecules. Science 246, 64 –71.
2. Wilm, M. S. and Mann, M. (1994) Electrospray and Taylor - cone theory, Dole’s beam of
macromolecules at last ? Int. J. Mass Spectrom. Ion Proces. 136, 167–180.
3. Wilm, M. and Mann, M. (1996) Analytical properties of the nano electrospray ion source.
Analyt. Chem. 66, 1–8.
4. Wilm, M. and Mann, M. (1996) Femtomole sequencing of proteins from polyacrylamide
gels by nano electrospray mass spectrometry. Nature 379, 466–469.
5. Shevchenko, A., Wilm, M., Vorm, O., and Mann, M. Mass spectrometric sequencing of
proteins from silver stained polyacrylamide gels. Analyt. Chem. 68, 850–858.
6. Roepstorff, P. and Fohlmann, J. (1984) Proposal for a common nomenclature for sequence
ions in mass spectra of peptides. Biomed. Mass Spectrom. 11, 601.