Capture of Peptides with N-Terminal Serine and Threonine: A

Dec 12, 2002 - The sequence-specific peptide selection not only reduces the complexity of digest mixtures, but also provides additional information fo...
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Bioconjugate Chem. 2003, 14, 205−211

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Capture of Peptides with N-Terminal Serine and Threonine: A Sequence-Specific Chemical Method for Peptide Mixture Simplification Dirk Chelius* and Thomas A. Shaler Thermo Finnigan, 355 River Oaks Parkway, San Jose, California. Received September 13, 2002; Revised Manuscript Received October 24, 2002

The objective of this study was to evaluate a sequence-specific chemistry for the ability to specifically capture peptides that contain N-terminal serine or threonine residues from mixtures. The first step is the oxidation of the 1,2-amino alcohol structure -CH(NH2)CH(OH)- of peptides containing N-terminal serine or threonine with periodate. The newly formed aldehyde reacts with a labeling reagent containing a hydrazide, RCONHNH2, to form a hydrazone-peptide conjugate, RCONHNdCHpeptide. Biotin-labeled conjugates can then be isolated by affinity purification with streptavidin. The method described in this report can be useful in simplifying the complex mixtures of peptides that are generated in typical proteomic analysis, where proteins are digested with trypsin and analyzed using liquid chromatography mass spectrometry data. The sequence-specific peptide selection not only reduces the complexity of digest mixtures, but also provides additional information for peptide identification. The targeted peptides are those that have either serine or threonine adjacent to a protease cleavage site. The sequence information should greatly aid in both database matching for protein identification and for de novo sequence determination.

INTRODUCTION

Large-scale analysis of a proteome typically includes separation of complex biological mixtures into individual proteins followed by the identification of the fractionated proteins. Typically, two-dimensional gel electrophoresis followed by mass spectrometry analysis is used (1-3). Alternative methods are based on two-dimensional chromatography coupled with mass spectrometry (4). The identification of proteins in both approaches includes tryptic digestion of the protein and identification of the tryptic peptides either directly by mass or by fingerprinting of these peptides during tandem mass spectrometry. Large numbers of peptides are typically generated during such experiments and the separation and identification remains a challenge. The objective of this study was to develop a method to reduce the large number of peptides that are generated during large-scale protein identification techniques. To evaluate if peptides containing N-terminal serine or threonine are distributed in sufficient amounts in all proteins, a computer program was written to generate and identify all peptides that fit the criteria from the C. elegans database. The sequence-specific chemistry to isolate peptides containing N-terminal serine or threonine residues was optimized for our purpose, including oxidation with periodate, linkage to biocytin hydrazide, and binding and release from strepdavidine-coated beads. The oxidation by periodate of N-terminal 1,2-amino alcohols serine (R ) CH2OH) or threonine [R ) CH(OH)CH3] residues NH2-CHR-CO- of a polypeptide to an aldehyde func* Corresponding author. Address: ThermoFinnigan, Proteomics Division, 355 River Oaks Parkway, San Jose, CA 95134. Phone: (408) 965-6326. Fax: (408) 965-6138. E-mail: dchelius@ thermofinnigan.com.

tion OdCH-CO- has been known for many years (5). This mild oxidation has been exploited for the synthesis of bioconjugates through formation of hydrazone, thiazolidine, or oxime (-ONdCH-CO-) bonds (6-11). Reaction of the aldehyde with a hydrazide was reported to be very effective and could be achieved quantitatively (6). The conjugation reaction was used to generate biocytinylated peptides that could be isolated on streptavidinecoated beads. The biocytin is similar to biotin and has a very strong affinity to streptavidine. Elution of the captured peptides could be achieved by incubation in acid conditions. The results presented in this study described a method to specifically capture peptides containing N-terminal serine or threonine residues. The method can potentially lead to improvements in large-scale protein identification strategies. Additionally the potential for an alternative method for protein quantitation similar to the isotopecoded affinity tags(ICAT) method is demonstrated (12). EXPERIMENTAL PROCEDURES

Theoretical Modeling. Bioinformatic modeling of theoretical digests and the simplifications resulting for different types of peptide selection for the C. Elegans database was performed with software written in-house. The C. Elegans protein database was obtained from the National Center for Biotechnology Information (NCBI). Chemistry. Ten microgram pure peptides (Sigma, St. Louis, MO) or peptide mixtures (Table 1) were incubated with 10 µL sodium periodate (40 µmol) in 20 µL PBS (pH 7.2) at room temperature for 10 min in the dark. Peptides were exchanged into 100 mM sodium acetate (pH 4.5), and 100 µg biocytin hydrazide (EZ-Link, Pierce, Rockford, IL) was added. The mixture was incubated for 1 h at room temperature and then incubated with streptavidin-coated magnetic beads (CPG Inc., Palo Alto, CA). After 1 h at room temperature, the beads were washed five times

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Table 1. Amino Acid Sequence and Molecular Weight (MW) of Peptides Used in This Studya # 1 2 3 4

peptide TFQAYPLREA SGQSWRPQGRF SIPSKDALLK MNYLAFPRM

m/z (2+);

(1+)

598.9 1195.6 436.5 (3+); 653.3 (2+) 1304.7 (1+) 536.7 (2+) 572.0 (2+); 1141.6 (1+)

observed MW

calculated MW

1195.2 ( 0.6 1304.9 ( 0.8 1072.4 1141.3 ( 0.7

1195.3 1304.4 1071.3 1141.4

a Peptides were analyzed by LC/MS as described in the Experimental Procedure section. Although ions were observed in a variety of multiple charge states, observed masses are calculated as molecular masses of neutrals. The masses are identified as average.

Figure 1. Bioinformatic modeling of theoretical digests and the simplifications resulting for different types of peptide selection for the C. Elegans database (NCBI) was performed with software written in-house. A: Distribution of the numbers of peptides per protein generated by tryptic digestion from C. Elegans database. B: Peptides per protein containing cysteine residues from tryptic digestion of the C. Elegans database. C: Peptides per protein containing N-terminal serine or threonine residues from tryptic digestion of the C. Elegans database.

with the sodium acetate buffer to remove nonbiotinylated peptides. The aldehyde form of the peptides could be eluted from the beads by incubation of the beads at 60 °C for 1 h in 10% formic acid. Each step in the reaction scheme (Figure 2) was monitored by LC/MS/MS on an LCQ DECA ion trap mass spectrometer (Thermo Finnigan, San Jose, CA). Sample Analysis. The peptide samples were analyzed using a fully automated nanoflow LC/MS/MS system (13). Aliquots of the sample digest (250 fmol each peptide) were placed in wells of a 96-well plate (Nalge Nunc International, Rochester, NY). The plate was sealed with plastic film to minimize evaporation and inserted into the auto-sampler of a Surveyor HPLC system (ThermoFinnigan, San Jose, CA), where it was kept at 4 °C while waiting for analysis. The auto-sampler was equipped with no-waste injection capability, which enables injection volumes as low as 1 µL. The injected peptides were first loaded onto a reversed-phase poly(styrene-divinylbenzene) peptide trap (Michrom Bioresources, Auburn, CA) with a flow rate of 10 µL/min for 3 min. The peptides were eluted from the trap and separated on a reversedphase capillary column (PicoFrit; 5 µm BioBasic C18, 300 Å pore size; 75 µm × 10 cm; tip 15 µm, New Objective, Woburn, MA) with a 30 min linear gradient of 0-60% acetonitrile in 0.1% formic acid/water at a flow rate of

approximately 0.1 µL/min after split. The HPLC was directly coupled to a ThermoFinnigan LCQ Deca ion trap mass spectrometer equipped with a nano-spray ionization source. The spray voltage was 2.0 kV, and the capillary temperature was 200 °C. The ion-trap collisional fragmentation spectra were obtained using collision energies of 35%. Each full-scan mass spectrum was followed by one MS/MS spectra of the most intense peak. Full mass spectra and tandem mass spectra were analyzed manually by comparing the data with the predicted data based on the amino acid sequence of the peptides. RESULTS

Computer Modeling. A computer program was written to generate and identify all peptides from a tryptic digest that contain N-terminal serine or threonine residues from the C. elegans database. The results are summarized in Figure 1. Only 1500 proteins from the C. elegans database do not contain such peptides (Figure 1C). Therefore, our proposed method of peptide reduction would not be able to identify these proteins. However, the C. elegans database contains 63 394 proteins, so that only 2.3% of all proteins would be missed. This result was compared to peptides containing cysteine residues (Figure 1b). Capture of cysteine residue is commonly used for reduction of peptide complexity and for quantitation

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Table 2. Calculated and Observed Molecular Weights (MW) of Peptides after Oxidation with Periodatea #

m/z

observed MW

1 2 3 4

1151.3 (1+); 1168.8 (1+) 646.9 (2+); 530.2 (2+); 522.0 (2+) 588.0 (2+); 1173.6 (1+)

1150.3; 1167.8 1291.8 1058.4; 1042.0 1174.0

calculated MW (hydrate) 1168.3 1291.4 1058.3 1173.4b

calculated MW (aldehyde) 1150.3 1273.4 1040.3 -

a Peptides were oxidized with periodate and the products were analyzed by LC/MS as described in the Experimental Procedures section. Although ions were observed in a variety of multiple charge states, observed masses are calculated as molecular masses of neutrals. The masses are identified as average. b The molecular weight of peptide 4 MNYLAFPRM was calculated under the assumption that both methionines were oxidized which correlates to the addition of 32 Da to the molecular weight.

Figure 3. LC/MS/MS analysis of reaction products after each step during capture of peptide 1 (TFQAYPLREA). See the Experimental Procedures for details.

Figure 2. Schematically representation of the peptide derivatization chemistry performed in this study.

applications such as the ICAT method. The distribution of cysteine containing tryptic peptides is similar to the distribution of tryptic peptides containing N-terminal serine or threonine residues. The result demonstrates that our proposed method would generate sufficient amounts of peptides to identify more than 97% of all proteins of the C. elegans database. Chemistry. The four peptides used in this study, including two peptides with N-terminal serine residue and one peptide with N-terminal threonine residue, were analyzed by LC/MS/MS analysis to ensure the purity and to confirm the molecular weight. The results are summarized in Table 1. The oxidation of N-terminal 1,2 amino alcohols with periodate was optimized using one synthetic peptide containing a N-terminal threonine residue and one peptide containing an N-terminal serine residue. Oxidation of peptide 1 (TFQAYPLREA) resulted in the formation of the aldehyde form and the aldehydehydrate form (to be referred to as hydrate). The calculated molecular weight and the measured molecular weight are shown in Table 2. The singly charged ion with an m/z value of 1151.3 correlates well with the calculated molecular weight of the aldehyde (1150.3). Additionally the hydrate form could be detected with an m/z value of 1168.8 (calculated 1168.3). The corresponding chromato-

Figure 4. LC/MS/MS analysis of reaction products after each step during capture of peptide 3 (SIPSKDALLK). See the Experimental Procedures for details.

grams and mass spectra are shown in Figure 3. The reaction was found to be quantitative. Similar results could be obtained with the oxidation of peptide 3 (SIPSKDALLK). The doubly charged ions with m/z values of 522.0 and 530.2 correspond to peptides with apparent molecular weight of 1042.0 and 1058.4 Da, which agrees well with the calculated molecular weight for the aldehyde (1040.4 Da) and the hydrate-form (1058.3). The results are shown in Table 2 and Figure 4. The alde-

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hyde-hydrate was the major product in both reactions. Incubation of the oxidation products with biocytin hydrazide yields the biocytinylated peptides. For peptide 1 the bioconjugate was calculated to have a molecular weight of 1518.8 (the molecular weight of the linker 386.5 Da plus the molecular weight of the aldehyde 1150.3 Da minus 18 (H2O) ) 1518.8). This correlates very well with the major peak found in the chromatogram, where the doubly charge ion of 760.4 corresponds to a molecular weight of 1518.8 Da (Figure 3). The conjugation reaction of peptide 3 shows two major peaks with m/z values of 471.8 (3+) and 705.2 (2+), corresponding to a molecule with a molecular weight of 1410.4 ( 2.0 Da (Figure 4). This corresponds well with the calculated molecular weight of peptide 3 linked to biocytin hydrazide of 1408.8 Da (same calculation as above). The reaction appears to be quantitative on the basis of the chromatogram. In the final step the biocytinylated peptides were bond to streptavidin-coated beads, washed and eluted with 10% formic acid. LC/MS analysis of the eluted samples showed the aldehyde form of either peptide 1 or peptide 3 as major peaks. Elution of peptide 1 shows one major peak with an m/z value of 1153.2 corresponding to the aldehyde form of peptide 1 (Figure 3). Several smaller peaks could be detected but not identified. Most likely, these peaks correspond to breakdown products of peptide 1. Elution of peptide 3 shows one major peak with an m/z value of 522.2, corresponding to the aldehyde form of peptide 3 (Figure 4). Additionally, several smaller peaks could be also detected but not identified. LC/MS analysis of the products after every step of chemical synthesis confirms that the peptide modifications were performed as planned and complete. The method was further evaluated to test if this approach could be used to specifically capture peptides, which contain N-terminal serine or threonine residues from a mixture of four synthetic peptides, two containing N-terminal serine (peptide 2 SGQSWRPQGRF and peptide 3 SIPSKDALLK), one containing N-terminal threonine (peptide 1 TFQAYPLREA), and one containing neither N-terminal serine nor threonine but N-terminal methionine (peptide 4 MNYLAFPRM). The peptide mixture was first oxidized with periodate as described in the Experimental Procedure section. The sample mixture was analyzed by LC/MS, and the results are shown in Figure 5. The four major peaks in the chromatogram could be identified as the oxidized isoforms of peptides 1, 2, 3, and 4. The aldehyde form and the hydrate form of peptide 1 (calculated MW of 1150.3 and 1168.3 Da) can be identified as a singly charged ions with an m/z value of 1151.4 and 1167.8, respectively. The doubly charge ion with an m/z value of 646.9 corresponds to the hydrate form of peptide 2, and the doubly charge ions with an m/z value of 521.7 and 530.3 corresponds to the aldehyde form and the hydrate form of peptide 3. Additionally peptide 4 was identified in the oxidized form as a doubly charged ion with an m/z value of 588.0 and a singly charge ion with an m/z value of 1173.6. The observed molecular weight of peptide 4 differs by 32 from the calculated molecular weight of peptide 4 (1141.4). The difference can be explained with the oxidation of the two methionines in peptide 4. The oxidized peptide mixture was incubated with biocytin hydrazide as described in the Experimental Procedures section, and the bioconjugates were bound to the streptavidine-coated beads. After several washing steps to remove all unspecific bound material, the peptides were eluted with 10% formic acid as described in the Experimental Procedures section. The eluate was analyzed by LC/MS/MS, and the ion chromatograms and

Chelius and Shaler

Figure 5. LC/MS/MS analysis of the four peptides after oxidation with periodate. See the Experimental Procedures for details. The amino acid sequence of the four peptides is shown in Table 1. The full-scan mass spectrum is the sum of all mass spectra recorded during the elution of the peptides (elution time: 28-35 min).

mass spectra are shown in Figure 6. As predicted peptide 4 could not be identified. However, peptides 1, 2, and 3 could be identified as both, the aldehyde form and the hydrate form. Peptide 1 showed ions with m/z values of 1150.8 (1+) and 577.4 (2+) that correlate well with the predicted molecular weight of the aldehyde form of peptide 1 (see Table 2). Additionally the hydrate form of peptide 1 could be identified as singly charge ion with a m/z value of 1168.6 (calculated molecular weight of hydrate 1168.3 Da) and as a doubly charged ion with a m/z value of 585.6. Peptide 2 could be identified as doubly charge ions at m/z values of 646.9 (hydrate) and 638.7 (aldehyde), and peptide 3 could be identified as doubly charged ions with m/z values of 530.5 (hydrate) and 522.4 (aldehyde) (see Table 2). Additionally a species with an m/z value of 727.6 (singly charged ion) could be detected. Fragmentation of the 727.6 ion resulted in a major peak with an m/z value of 387.4 (data not shown), which correlates with the molecular weight of the linker (386.5) indicating that the unidentified peak at 727.6 might be a linker related adduct. The results demonstrated that the peptide modifications were performed as planned and the specific capture of peptides, containing N-terminal serine or threonine residues was successful. DISCUSSION

Analysis of the theoretical tryptic digest of the C. elegans database showed that 1500 proteins do not generate tryptic peptides that contain N-terminal serine or threonine residues. These proteins could not be identified if the proposed method for protein identifications would be used. However, the NCBI database contains a

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Bioconjugate Chem., Vol. 14, No. 1, 2003 209

Figure 6. A mixture of peptides 1-4 (the amino acid sequences of the peptides are shown in Table 1) was oxidized and biotinylated as described in the Experimental Procedures section. The biotinylated peptides were captured on magnetic beads and released by acid hydrolysis. The eluate was analyzed by LC/MS/MS as described in the Experimental Procedures section. A: Base peak ion chromatogram. B: Full mass spectra at a retention time of 34.2 min (peptide 1 TFQAYPLREA). C: Full mass spectra at a retention time of 31.3 min (peptide 2 SGQSWRPQGRF). D: Full mass spectra at a retention time of 32.0 min (peptide 3 SIPSKDALLK).

total of 63394 protein entries for C. elegans so that only 2.3% of all proteins would be missed. The reduction of total numbers of peptides in a complex mixture should greatly enhance the peptide ionization efficiency during electrospray process and should therefore facilitate the identification of low abundant proteins. The percentage of proteins missed using this technology depends on the database and it is possible that more proteins would be missed for other organisms. The ICAT (isotope-coded affinity tags) method for protein quantitation uses cysteine residues to specifically label and isolate certain peptides (12). The distribution of tryptic peptides containing cysteine residues is very similar to tryptic peptides containing N-terminal serine or threonine residues in the C. elegans database. The distribution of tryptic peptides containing cysteine or N-terminal serine or threonine residues might also be different for other databases. After theoretical proof of the concept, that indeed the capture of peptides containing N-terminal serine or threonine residues could reduce the complexity of a complex mixture of tryptic peptides without loosing a significant amount of protein information, a chemistry was developed to capture those peptides. The oxidation of N-terminal 1,2 amino alcohols serine or threonine with periodate was optimized. Alcohols were converted to aldehydes within 10 min reaction time, and the reaction was found to be quantitative. The aldehyde is in equilibrium with the diol form (in the text revert to as aldehyde-hydrate or hydrate), and under the conditions

used in this experiment, the hydrate form is the predominant species. This fact does not affect the next reaction step with hydrazide as shown in results. The reaction was performed under mild conditions however oxidation of methionine was also observed. This unwanted side reaction should not present any problems for our proposed method, since oxidation of methionine to the thiol form seems to be quantitative, too. The next step was the linkage of the generated aldehydes to the biocytin hydrazide reagent. The reaction was also found to be quantitative demonstrating that our method of specifically labeling peptides containing Nterminal serine or threonine was successful. As expected, peptides that do not contain N-terminal serine or threonine do not react with the biocytin hydrazide. Finally the labeled peptides could be bound to streptavidin-coated beads, allowing the isolation of the labeled peptides. Elution of the peptides was found to be somewhat tricky. The biocytin-streptavidine interaction is very strong. In fact, initial attempts to remove the peptides with guanidine hydrochloride and incubation at high temperature resulted in very bad yields (data not shown). Fortunately the peptides could be released by incubation in 10% formic acid at 60° for 30 min. The chemistry was not studied in detail, however hydrolysis of hydrazone bonds is well reported in the literature (6). The main product for the three peptides tested was the aldehyde form of those peptides. Several other fragments could be eluted from the beads. These fragments could not be identified but are most likely breakdown product

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Figure 7. Tandem mass spectra analysis of peptides eluted from the streptavidin-coated magnetic beads (see Experimental Procedures for details). The full scan mass spectra are shown in Figure 6B-D. A: Tandem mass spectrum derived by collision induced dissociation of the (M + 2H)2+ precursor ion, m/z ) 577.4 of peptide 1 (retention time 34.2). B: Tandem mass spectrum derived by collision induced dissociation of the (M + H)+ precursor ion, m/z ) 1150.8 of peptide 1 (retention time 34.2). C: Tandem mass spectrum derived by collision induced dissociation of the (M + 2H)2+ precursor ion, m/z ) 646.9 of peptide 2 (retention time 31.3). And D: Tandem mass spectrum derived by collision induced dissociation of the (M + 2H)2+ precursor ion, m/z ) 522.4 of peptide 3 (retention time 32.0). Fragment ions in all four spectra represent mainly singly event preferential cleavage of the peptide bonds resulting in the sequence information recorded from both N (b-ions) and C (y-ions) termini of the peptides simultaneously.

of the bioconjugates. Either peptide bonds are hydrolyzed under the acid conditions or other parts of the linker are labile to acid hydrolyzation. Clearly the acid hydrolysis for peptide release is not the optimal solution. Although all three peptides could be identified, the appearance of additional peptide fragments is not desirable and could complicate the peptide identification. However, several linkers are commercially available that contain a cleavable linker attached to a biotin or biocytin label and a hydrazine reactive group. The use of such linkers should greatly enhance the yield of our proposed method and reduce the formation of breakdown products. Additionally one could imagine the construction of linkers, which are different only in certain isotopes that could be used similar to the ICAT method in protein quantitation (12). The idea behind such quantitation method is the labeling of control and experimental sample with tags that are different in mass but chemically identical (H/D or C12/C13). After labeling, the control and experimental samples are combined and differences in the protein profile can be analyzed by comparing the signal intensity of the labeled peptides. The labeling of peptides containing N-terminal serine or threonine residues could complement the ICAT method, which labels peptides containing cysteine residues. The huge advantage of our proposed method becomes obvious when looking at the tandem mass spectra in

Figure 7. Although all peptides could be identified in the full mass spectra, the tandem mass spectra frequently do not contain sufficient fragmentation information for automatic peptide identification. Our method is sequencespecific, and the targeted peptides are those that have either serine or threonine adjacent to a protease cleavage site. This additional sequence information should greatly aid in both database matching for protein identification and for de novo sequence determination. CONCLUSIONS

Peptides that contain serine or threonine at the Nterminus can be specifically modified and captured from mixtures. The initial oxidation step with periodate occurs under mild conditions and is rapid and quantitative, although methionine sulfoxide formation occurs. Treatment of the N-terminal aldehydes with biocytin hydrazide allows the modified peptides to be tagged with affinity selection groups. These bioconjugates can be captured on streptavidin-coated magnetic beads. The release of the peptide aldehydes from the solid support was achieved by acidic hydrolysis of the hydrazone. This release reaction can lead to peptide degradation. However, all peptides containing N-terminal serine or threonine could be isolated from a mixture of peptides with this method. The described approach to simplify complex

Capture of Peptides with N-Terminal Ser and Thr

peptide mixture could have an impact on large-scale protein identification and quantification. ACKNOWLEDGMENT

The authors would like to thank Chris Becker for the encouragement and fruitful discussions. LITERATURE CITED (1) Hanash, S. M. (2000) Biomedical applications of twodimensional electrophoresis using immobilized pH gradients: current status. Electrophoresis 21, 1202-1209. (2) Pandey, A., and Mann, M. (2000) Proteomics to study genes and genomes. Nature 405, 837-846. (3) Washburn, M. P., and Yates, J. R., III. (2000) Analysis of the microbial proteome. Curr. Opin. Microbiol. 3, 292-297. (4) Washburn, M. P., Wolters, D., and Yates, J. R., III. (2001) Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat. Biotechnol. 19, 242-247. (5) Fields, R., and Dixon, H. B. (1968) A spectrophotometric method for the microdetermination of periodate. Biochem. J. 108, 883-887. (6) Geoghegan, K. F., and Stroh, J. G. (1992) Site-directed conjugation of nonpeptide groups to peptides and proteins via periodate oxidation of a 2-amino alcohol. Application to modification at N-terminal serine. Bioconjugate Chem. 3, 138-146. (7) Gaertner, H. F., Rose, K., Cotton, R., Timms, D., Camble, R., and Offord, R. E. (1992) Construction of protein analogues by site-specific condensation of unprotected fragments. Bioconjugate Chem. 3, 262-268.

Bioconjugate Chem., Vol. 14, No. 1, 2003 211 (8) Mikolajczyk, S. D., Meyer, D. L., Starling, J. J., Law, K. L., Rose, K.; Dufour, B., and Offord, R. E. (1994) High yield, sitespecific coupling of N-terminally modified beta-lactamase to a proteolytically derived single-sulfhydryl murine Fab′. Bioconjugate Chem. 5, 636-646. (9) Zhang, L., and Tam, J. P. (1996) Thiazolidine formation as a general and site-specific conjugation method for synthetic peptides and proteins. Anal. Biochem. 233, 87-93. (10) Rose, K., Zeng, W., Regamey, P. O., Chernushevich, I. V., Standing, K. G., and Gaertner, H. F. (1996) Natural peptides as building blocks for the synthesis of large protein-like molecules with hydrazone and oxime linkages. Bioconjugate Chem. 7, 552-556. (11) Nardin, E. H., Calvo-Calle, J. M., Oliveira, G. A., Clavijo, P., Nussenzweig, R., Simon, R., Zeng, W., and Rose, K. (1998) Plasmodium falciparum polyoximes: highly immunogenic synthetic vaccines constructed by chemoselective ligation of repeat B-cell epitopes and a universal T-cell epitope of CS protein. Vaccine 16, 590-600. (12) Gygi, S. P., Rist, B., Gerber, S. A., Turecek, F., Gelb, M. H., and Aebersold, R. (1999) Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat. Biotechnol. 17, 994-99. (13) Chelius, D., Hu¨hmer, A. F. R., Shieh, C. H., Lehmberg, E., Traina, J. A., Slattery, T. K., and Pungor, E., Jr. (2002) Analysis of the adenovirus type 5 proteome by liquid chromatography and tandem mass spectrometry methods. J. Proteome Res. 501-513.

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