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in a boiling bath, and then mixed with 97 atom % 1SOlabeled water, nonionic detergent (Triton X-100), and an appropriate amount of PNGase-F. The mixture is incubated for 24 hr at 37°C (Tarentino et al., 1985). (b) Digestion of the deglycosylated protein with a proteolytic enzyme(s) in a buffer prepared with normal water. (c) Separation of constituent peptides by reversed-phase HPLC. (d) Detection of ~sO-labeled peptides in a digest by FAB mass spectrometric analysis. (e) Sequence analysis of the 1sO-labeled peptides by CID-MS or CID-MS/MS. Since PNGase-F cleaves quantitatively at the fl-aspartylglycosylamine linkage of carbohydrate-linked Asn to yield Asp, all the converted Asp residues specifically incorporate ~SO at the fl-carboxyl groups. As the ~80-labeling at the fl-carboxyl groups of the converted Asp residues are retained during proteolytic digestion, FAB mass spectra of ~80-labeled peptides show doublet signals (MH ÷ and MH ÷ + 2). It permits facile identification of the peptides containing the converted Asp residues originated from carbohydrate-linked Asn. Furthermore, CID-MS of those peptides reveals the specific positions for N-glcosylation together with their amino acid sequences, because all the N-terminal and C-terminal sequence ions that contain the converted Asp show the doublet signals, and after the loss of the converted Asp the isotopic ion distributions of the sequence ions change into normal ones. Even though both the converted Asp and original Asp are present in a peptide, the converted Asp can be differentiated from the original one based on the isotopic ion distributions of the sequence ions. More precise and reliable identification of N-glycosylation sites in a glycoprotein could be achieved by using CID-MS/MS. We demonstrate that the method can be used for identication of N-glycosylation sites in an unknown glycoprotein by applying it to analysis of the N-glycosylation sites in the recombinant Bacillus licheniformis a-anylase produced in Pichia pastoris. The method should be efficient for analysis of both N-glycosylation sites and their included amino acid sequences, information about which is indispensable for understanding the biological functions of glycoproteins. References

Takao, T., Hori, H., Okamoto, K., Kamachi, M., Shimonishi, Y. (1991). Rapid Commun. Mass Spectrom. 5, 312-315. Takao, T., Gonzalez, J., Hori, H., Yoshidome, K., Sato, K., Kammei, Y., and Shimonishi, Y. (1992). 40th ASMS Conference on Mass Spectrometryand Allied Topics (Washington, D.C.). Tarentino, A. L., Gomez, C. M., and Plummer, T. H., Jr. (1985). BiochemisoT 24, 4665-4671.

R. G. Cooks, K. A. Cox, and J. D. Williams. High-Performance Mass Spectrometry with the Ion Trap Mass Spectrometer. (Department of Chemistry, Purdue University, West Lafayette, Indiana 47907 U.S.A.)

M P S A Short Communications

The ion trap mass spectrometer can be used to trap ions generated by desorption ionization, and the trapped ions can be ejected and mass-analyzed by causing resonance between their motion and an external electric dipole field. This procedure allows molecular weights to be determined for small peptides (to ~3000 D). By slowly scanning the magnitude of the voltage which establishes the trapping field, it is possible to improve the mass resolution in this experiment to better than unit resolution for these peptides. In addition, much higher resolution is achievable for other types of ions such as salt dusters. Individual peptide ions can be isolated in the ion trap and subsequently dissociated by increasing their kinetic energy, again through resonance with an external dipole field. This allows tandem mass spectrometry to be performed and sequence information to be obtained. Examples are given of work on model peptides and attention is also directed to new methods of increasing the efficiency of dissociation through use of heavy target gases and collisions with surfaces. Methods which use internal standards to improve mass measurement accuracy are also introduced. The quadrupole ion trap was invented by Paul (Paul et al., 1953) and developed as a means of performing gas chromatography/mass spectrometry by Stafford (Stafford et al., 1984), who introduced a mass selective method of ion ejection providing a convenient method of scanning a mass spectrum. This instrument, introduced commercially in 1983, utilized electron ionization to convert gaseous samples into ions and had a maximum mass/charge range of 650 D / charge. Subsequently, a number of investigators (Kaiser et al., 1991; Louris et al., 1990; Johnson et al., 1990) have extended the capabilities of the instrument. The additional capabilities include the following: (a) extension of the mass/ charge range of the instrument; (b) implementation of desorption ionization methods; and (c) capabilities for tandem mass spectrometry. Each of these is necessary in order to obtain structural information on biological compounds. In particular, desorption ionization, which is typically performed in an external ion source using Cs + ion bombardment, is a method well-suited to ionizing peptides. The resulting peptide ions are accelerated into the ion trap and are contained through collisions with helium gas. Once trapped they can be mass-analyzed using the resonance ejection method which extends the mass-to-charge range by resonantly exciting ions with an external dipole field causing them to become unstable, and hence be ejected from the trap and detected. The ability to resonantly excite ions in a mass-selective fashion also forms the basis of collisioninduced dissociation, an experiment in which ions gain enough energy to dissociate rather than to be ejected and the product ions are detected. The result is a tandem mass spectrometry experiment which yields characteristic peptide fragment ions. It has been shown that sequence information is available from these fragments (Cooks et al., 1991), Although considerable progress has been made in the transformation of the quadrupole ion trap into a

M P S A Short Communications

high-performance mass spectrometer, much remains to be done. The principle issues at hand are (a) mass measurement accuracy; (b) internal energy deposition; (c) improvements in performance so as to obtain similar resolution, mass range, and dissociation efficiency with peptides as are obtained with model ions. The problem of mass measurement accuracy results from the fact that the field experienced by the ions is dependent on the ion population in the trap as well as on other experimental variables. This causes mass shifts which have complex origins. Results are given for experiments in which this problem is alleviated by simultaneously injecting the peptide of interest, as well as a model calibration compound, into the trap. This is achieved using a split probe and simultaneous irradiation of both samples. In addition, results are presented of simulations of ion motion (Julian et al., 1992) which provide information on the origins of the mass shifts. In order to maximize the information on peptide sequences obtained by tandem mass spectrometry using the ion trap, it is desirable to have control over the internal energy transferred in the collision process. This is achievable through control of the period of resonance irradiation and the amplitude of the resonance field. However, the fact that collisions occur with a light gas (helium) is a limitation. Experiments are described in which three alternatives are explored: (i) multiple stage experiments, commonly known as MS" experiments, in which the products of one dissociation step are the precursors of the next; (ii) collisions with an admixed heavy gas (xenon); and (iii) collisions with the walls of the ion trap, a process known as surface-induced dissociation (SID). Results of the applications of these new methods to peptide sequencing are given and data are also given for model compounds which allow the total internal energy deposited and the total yield of product ions to be quantified. The factors which limit the performance of the ion trap when it is used to study biological compounds, as compared to simple salt clusters, are also investigated. This is done through a systematic examination of the fragmentation patterns of a few model peptides as a function of experimental conditions, combined with simulations of ion motion. In addition, valuable data are obtained from ion tomography experiments (Hemberger et al., 1992), in which the actual positions of the ions in the trap are measured by laser photodissociation. All these results, in combination, suggest that additional control over ion motion is needed to optimize performance of the ion trap for peptides and other biomolecules. One way to achieve this is to pulse the ions from the center of the trap into larger radii, and then to implement the dissociation process. In addition, results of using higher order (higher than quadrupole) electric fields for ion manipulation will be given. This work is supported by the National Science Foundation, CUE 87-21768. The contributions of S. A.

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Lammert, K. L. Morand, and R. K. Julian, Jr. are gratefully acknowledged.

References

Cooks, R. G., Glish, G. L., McLuckey, S. A., and Kaiser, R. E. (1991). Chem. and Eng. News 69, 26. Hemberger, P. H., Nogar, N. S., Williams, J. D., Cooks, R. G., and Syka, J. E. P. (1992). Chem. Phys. Lett. (in press). Johnson, J. V., Yost, R., Kelley, P., and Bradford, D. (1990). Anal. Chem. 62, 2162. Julian, R. K., Reiser. H.-P., and Cooks, R. G. (1992). Int. J. Mass Spectrom. and Ion Proc. (in press). Kaiser, R. E., Cooks, R. G., Stafford, G. C., Syka, J. E. P., and Hemberger, P. E. (1991). Int. J. Mass Spectrom. Ion Proc. 106, 79. Louris, J. N., Brodbelt-Lustig, J. S., Cooks, R. G., Glish, G. L., Van Berkel, G. J., and McLuekey, S. A. (1990). Int. J. Mass Spectrom. Ion Proc. 96, 117. Paul, W., and Steinwedel, H. (1953). Z. Natusforsch 8a, 448. Stafford, G., Kelley, P., Syka, J., Reynolds W., and Todd, J. (1984). Int. J. Mass Spectrom. and Ion Proc. 60, 85.

Donald F. Hunt, 1 Jeffrey Shabanowitz, 1 Hanspeter Michel, 1 Andrea L. Cox, I Tracey Dickinson, ~ Theresa Davis, ~ Wanda Bodnar, ~ Robert A. Henderson, 2 Noelle Sevilir, 2 Victor H. Engdhard, 2 Kazuyasu Sakagachi, 3 Ettore Appella, 3 Howard M. Grey, 4 and Alessandro Sette. 4 Sequence Analysis of Peptides Presented to the Immune System by Class I and Class II MHC Molecules. (i Department of Chemistry, University of Virginia, Charlottesville, Virginia 22901, U.S.A.; 2 Department of Microbiology and Beirne Center for Immunological Research, University of Virginia School of Medicine, Charlottesville, Virginia 22901, U.S.A. ; 3 Laboratory of Cell Biology, National Cancer Institute, NIH, Bethesda, Maryland 20892, U.S.A.; 4 Cytel Corporation, 3525 John Hopkins Court, San Diego, California 92121, U.S.A.)

Recognition of peptide fragments by T-lymphocytes is a central event in the immune response to foreign proteins. This recognition occurs in association with either class I or class II molecules of the major histocompatibility complex (MHC). Current evidence indicates that the assembly of the complexes of the two classes of MHC molecules with peptides takes place in two different intracellular compartments. Viral and self peptides that are derived from the degradation of cytosolic proteins and transported into the endoplasmic reticulum are found in complexes with MHC class I molecules. Cytotoxic T-cells recognize these complexes. MHC class II molecules bind peptides derived mainly from exogenous proteins that enter the cell by phagocytosis, endocytosis, or internalization of part of the