Bioconjugate Chem. 1999, 10, 861−866
861
Identification of Fluorescein-5′-Isothiocyanate-Modification Sites in Proteins by Electrospray-Ionization Mass Spectrometry Volker Schnaible and Michael Przybylski* Fakulta¨t fu¨r Chemie, Universita¨t Konstanz, 78457 Konstanz, Germany. Received April 9, 1999; Revised Manuscript Received June 1, 1999
Model peptides and proteins, such as hen eggwhite lysozyme, have been modified with fluorescein5′-isothiocyanate (FITC) to yield the corresponding fluorescein-thiocarbamoyl (FTC) conjugates (N,N′disubstituted thiourea and dithiourethane adducts). The extent of FITC incorporation, i.e., number of modified residues, has been identified by direct molecular weight determination using matrixassisted laser desorption-ionization and electrospray-ionization mass spectrometry (MALDI-MS; ESIMS). A specific fragmentation by cleavage of the FTC moiety from modified residues occurs by nozzleskimmer dissociation in ESI mass spectra at increased declustering potential. This fragmentation pathway is easily obtained and renders ESI-MS an efficient tool for the characterization of FITCmodified proteins, and identification of modification sites in FTC-peptide mixtures.
INTRODUCTION
The modification of proteins by fluorescein-5′-isothiocyanate (FITC)1 is an established method to generate fluorescent proteins, e.g., in antibodies (1), which then can be detected in low quantities according to the number of FITC molecules incorporated into the protein. FITC reacts with amino and thiol groups of proteins to yield fluorescein-5′-thiocarbamoyl (FTC) conjugates, N,N′-disubstituted thiourea and dithiourethane adducts (as shown in the reaction scheme in Figure 1) (2, 3). According to the general pathway of the Edman coupling with aryl-isothiocyanate, only the thiourea adducts are stable at alkaline pH and in the presence of nucleophiles such as β-mercaptoethanol (4). Eukaryotic ATPases (5) and recombinant partial ATPase sequences have been specifically modified by FITC at the ATP-binding region (6, 7). To obtain structural information about FITC modified ATPases, e.g., by fluorescence energy transfer (8), the molecular characterization of the number and the precise location of FITCmodified amino acid residues are crucial. To determine the sites modified by FITC, enzymatic digestion of the modified proteins is usually carried out, and the proteolytic peptide mixture separated by HPLC. FITClabeled peptides are then identified by Edman degradation (9) or mass spectrometry (10, 11). However, pure peptide samples for Edman degradation are frequently difficult to obtain (11). The direct analysis of proteolytic peptides from complex mixtures, e.g., upon enzymatic digestion has been employed by mass spectrometric peptide mapping with both electrospray-ionization and * To whom correspondence should be addressed. Phone: +49 7531882249.Fax: +497531883097.E-mail: Michael.Przybylski@ uni-konstanz.de. 1 Abbreviations: DMF, dimethylformamide; DMSO, dimethyl sulfoxide; DTT, 1,4-dithiothreitol; ESI, electrospray-ionization; FITC, fluorescein-5′-isothiocyanate; FTC, fluorescein-5′-thiocarbamoyl; HCCA, R-cyano-4-hydroxy cinnamic acid; HEL, hen eggwhite lysozyme; MALDI, matrix assisted laser desorptionionization; PTFE, poly(tetrafluoroethylene); TFA, trifluoroacetic acid; TPCK, L-1-tosylamido-2-phenylethyl chloromethyl ketone.
Figure 1. Scheme for the reaction of FITC with thiol and amino groups.
matrix-assisted laser desorption-ionization mass spectrometry (ESI-MS, MALDI-MS) (12-15). As shown in several recent studies, mass spectrometric peptide mapping provides the exact information of the number and distribution of covalent modification sites in the protein (16, 17). Furthermore, it has been shown that peptide backbone fragmentation in ESI-MS can be induced at increased declustering potential (18, 19) (∆CS), normally used to assist the desolvation of the initial charged microdroplets. This fragmentation occurs in the nozzleskimmer region of the electrospray ion source, and is therefore readily observed, e.g., with single quadrupole instruments (18, 20). In the present study, a specific fragmentation of FITC-modified side chains at increased declustering potential has been investigated and used for the identification of FITC-modification sites. This is illustrated by an FITC-modified model peptide (LGYLGYLCONH2), hen eggwhite lysozyme (HEL) as a well characterized model protein, and by peptides of FITC modified HEL following trypsin digestion.
10.1021/bc990039x CCC: $18.00 © 1999 American Chemical Society Published on Web 09/01/1999
862 Bioconjugate Chem., Vol. 10, No. 5, 1999 EXPERIMENTAL PROCEDURES
Materials. Solvents and reagents were of highest available purity from Merck (Darmstadt, Germany). The following reagents and proteins were obtained from Sigma (St. Louis, MO): DTT, FITC Isomer I, HCCA, hen eggwhite lysozyme (HEL), and trypsin (TPCK treated). The model peptide LGYLGYL-CONH2 was synthesized by solid-phase peptide synthesis (SPPS) using the Fmoc strategy on a semiautomatic peptide synthesizer EPS 221 from Abimed (Langenfeld, Germany). The synthesis product was purified by HPLC. Homogeneity of the peptide was ascertained by MALDI and ESI-MS. FITC Modification of HEL. (a) A total of 100 µg of HEL was dissolved in 100 µL of 50 mM NaH2PO4 buffer, pH 8. A total of 39 µg of FITC was added using a freshly prepared stock solution (c ) 1.95 µg/µL) in DMSO. The molar ratio of FITC:HEL is 14 under these conditions. The reaction was carried out for 17 h at 25 °C in the dark and quenched by addition of 400 µL of acetone at -20 °C followed by centrifugation (5 min at 13000g) after storage at -20 °C for 1 h. The supernatant containing free excess FITC was discarded and the pellet washed twice with 500 µL of acetone at -20 °C. The pelleted protein was then redissolved in 200 µL of acetonitrile/ aqueous 0.1% TFA (v/v ) 2:1) and purified by HPLC. (b) A total of 3 mg of HEL were dissolved in 1.08 mL of 50 mM NaH2PO4-buffer, pH 8. A total of 480 µg of FITC were added using a freshly prepared stock solution in (c ) 3.9 µg/µL) 120 µL of DMF. The molar ratio FITC: HEL is 6 under these conditions. The reaction was carried out at 25 °C. A total of 200 µL samples were taken from the reaction mixture after 0, 30, 60, 120, and 300 min and 55 h reaction time. The reaction was quenched by the addition of 800µL of -20 °C cold acetone to the 200 µL samples. The protein was pelleted after 1 h storage at -20 °C by centrifugation (5 min at 13000g). The supernatant was discarded and the pellet washed twice with 500 µL of -20 °C cold acetone. The pelleted protein then was subjected to trypsin digestion. FITC Modification of Synthetic Peptide LGYLGYL-CONH2. FITC modification of the synthetic peptide, LGYLGYL-CONH2, was carried out with 90 µg of the peptide dissolved in 280 µL of 100 mM sodium borate buffer, pH 9. A total of 20 µL of a freshly prepared 20 mM solution of FITC in DMF was added. The reaction was terminated after 12 h. The reaction mixture was purified by HPLC, since the removal of unbound reagent by acetone precipitation was not possible. The HPLC fraction containing FITC-derivatized peptide was analyzed by ESI-MS. Trypsin Digestion of FITC-Modified HEL. A 500 µg sample of FITC-modified HEL, prepared by a reaction time of 55 h, was redissolved in 50 mM NH4HCO3 buffer, pH 8, containing 30% methanol (21) to yield a final protein concentration of 1 µg/µL. The high methanol content is crucial to achieve digestion of HEL, which is highly resistant to proteolysis in the native state. A solution of freshly prepared trypsin in 50 mM NH4HCO3 was added to yield an enzyme-to-substrate ratio of 1:30. The digestion was carried out for 18 h at 37 °C. A 10fold excess of DTT per cysteine residue was added, the reduction was carried out for 1 h, and then stopped by addition of 10 µL of 10% TFA. A 1 µL aliquot of the reaction mixture was taken for mixture analysis by MALDI-MS. The digestion mixture was centrifuged for 5 min at 13000g in order to remove partially digested
Schnaible and Przybylski
protein which precipitates upon DTT reduction. The supernatant was subjected to HPLC separation and subsequently analyzed by MALDI-MS. A complete coverage of the HEL-sequence was achieved, although some of the protein is lost due to the precipitation. HPLC Separation. All HPLC separations were performed with a Waters/Millipore solvent delivery system (high-pressure pumps model M 510 and M 45) using a binary gradient of 0.1% TFA in water and 0.07% TFA in acetonitrile. Separation was carried out on a (250 × 8 mm) Macherey & Nagel C8-Nucleosil RP-HPLC column using linear gradients of 45 min (1% acetonitrile/min from 40 to 60%) for the FITC-modified HEL and of 80 min (1% acetonitrile/min from 5 to 55%) for the tryptic digestion mixtures. Peak detection was performed with a Waters 490 E multiwavelength detector system at a wavelength of 220 nm. FITC-modified peptides were also detected with a Shimadzu RF-551 fluorescence detector with the excitation wavelength set to 447 nm and the emission wavelength set to 514 nm. All fractions were manually collected and characterized by MALDI-MS. Mass Spectrometry. MALDI-MS analyses were carried out with a Bruker Biflex linear time-of-flight spectrometer (Bruker-Franzen, Bremen, Germany) equipped with a UV nitrogen laser (λ ) 337 nm) and a dual microchannel plate detector. The acceleration voltage was set to 25 kV. Lyophilized HPLC fractions were redissolved in acetonitrile/aqueous 0.1% TFA (v/v ) 2:1) and 1 µL of the solution was mixed with 1 µL of a saturated HCCA solution in acetonitrile/aqueous 0.1% TFA (v/v ) 2:1). The digestion mixture of HEL was prepared by mixing 1 µL of the DTT-containing digestion mixture solution with 1 µL of saturated HCCA solution. The dried sample spot was washed with 3 µL of 0.1% TFA, redissolved in acetonitrile/0.1% TFA (v/v ) 2:1) and spotted again on the target. Spectra were recorded after evaporation of the solvent and processed by means of the X-MASS data system. Peptide spectra were calibrated internally with insulin and HCCA reference ions. Protein spectra were calibrated externally with the singly and doubly charged molecular ions of HEL. Nano-ESI-MS (22, 23) was carried out with a Vestec A201 single-quadrupole mass spectrometer (Vestec, Houston, TX) fitted with a 10 kV conversion dynode and a 2000 m/z mass range. A home-built nano-ESI source was used (24). Borosilicate glass capillaries of the type GC120F10 from Clark Electromedical Instruments (Pangbourne, U.K.) were used for preparation of the microcapillaries. Capillaries were pulled with a capillary puller model P-97 from Sutter Instruments (Novato, CA) in a two-step cycle. The capillaries were then coated with a thin layer of a gold/palladium alloy using a Polaron SC7610 sputter coater (VG Microtech, Uckfield, U.K.), for providing the high voltage connection. Protein and peptide samples were dissolved in 2% aqueous acetic acid:methanol mixtures (v/v ) 9:1 unless otherwise stated). An aliquot of 1-4 µL of the sample solution was loaded by dipping the capillary tip into the sample solution. In case of plugging, the capillary was reopened by briefly touching a glass plate. The loaded capillary was fixed via a PTFE screw in a metal mounting and then pushed forward into the ion source region. The voltage at the capillary tip was set to 1.4-1.5 kV. To obtain a stable spray the capillary was manually adjusted to the nozzle of the ion source. The declustering potential between nozzle and skimmer was adjusted in a range of 10-120 V for recording ESIMS spectra of intact and fragmented peptides and proteins.
ESI-Mass Spectrometry of FITC−Protein Conjugates
Bioconjugate Chem., Vol. 10, No. 5, 1999 863
Figure 3. ESI-mass spectra of the FITC modified model peptide LGYLGYL-CONH2 recorded at three different declustering potentials. (a) 10 V; (b) 40 V; (c) 80 V.
Figure 2. ESI spectra of HPLC purified HEL containing predominantly two FITC groups. Spectra were recorded with different declustering potentials. (a) 10 V; (b) 60 V; (c) 80 V; (d) 120 V. Numbers at the peaks indicate the number of FITC molecules incorporated. RESULTS
HEL was modified for 17 h at 25 °C at pH 8 using a molar ratio of FITC:HEL of 14. After the removal of unbound FITC by acetone precipitation, MALDI-MS was used to ascertain the number of FITC molecules incorporated into the protein. The MALDI-MS spectrum indicated an average incorporation of two FITC groups into the protein. It also revealed that the extent of FITC incorporation is heterogeneous. To isolate homogeneous protein derivatives, FITC-treated HEL was subjected to HPLC separation on a C8-RP-HPLC column. HEL species with one to four FITC groups were isolated, although the isolation of a single, homogeneous HEL derivative was not achieved. ESI-mass spectra of a HPLC fraction of modified HEL containing predominantly two FTC groups were recorded by application of different declustering potentials as shown in Figure 2. With a declustering potential of 10 V (Figure 2a), the main protein species is 2-fold FITC-modified HEL (molecular mass 15 083 Da). An increase of the declustering potential of the ESI-ion source to 60 V revealed significant fragmentation at the FTC residues of HEL as shown in Figure 2b. The base peak at m/z 1677 is still corresponding to the [M + 9H]9+ molecular ion of a HEL derivative containing two FTC groups, but in addition intense ions of mono-FTC-HEL (molecular mass 14 694 Da) and free HEL (molecular
mass 14 305 Da) are detected. At a declustering potential of 80 V (Figure 2c), the [M + 9H]9+ molecular ion of unmodified HEL (i.e., removal of all FTC groups) at m/z 1591 is the base peak of the spectrum. A further increase of the declustering potential up to 120 V (Figure 2d) resulted in a spectrum with broad signals showing complete removal of all FTC residues. To test whether the nozzle skimmer fragmentation of FTC residues occurs also with FITC-modified peptides, a synthetic model peptide (LGYLGYL-CONH2), (molecular mass 797 Da) was investigated. The N-terminus is the only amino group in this peptide. Since the removal of excess FITC was not possible by acetone precipitation, the reaction mixture was purified by HPLC. The HPLC fraction containing the FITC-modified peptide was subjected to ESI-MS analysis. Figure 3 shows ESI spectra of the modified peptide (molecular mass 1186 Da). Under standard conditions (declustering potential set to 10 V, Figure 3a), the singly and doubly charged molecular ions of the peptide carrying one FTC group are detected. A doubly charged species representing the sodium adduct of the modified peptide was additionally found. With the declustering potential set to 40 V (Figure 3b) sequence fragments of the modified peptide belonging to the b series (25), still containing the FTC group and the [M + H]+ ion of the free FTC moiety were observed. With the declustering potential set to 80 V (Figure 3c), predominantly, the [M + H]+ ion of the free FTC moiety was found at m/z 390. This result with the model peptide confirmed the predominant fragmentation of the FTC-N bond by release of the FTC moiety. Tryptic digest mixtures of FITC-modified HEL were analyzed by MALDI-MS. Figure 4 shows the MALDI-MS spectrum of a reduced digestion mixture of HEL after
864 Bioconjugate Chem., Vol. 10, No. 5, 1999
Schnaible and Przybylski
Figure 4. MALDI mass spectrum of a reduced tryptic digest mixture of HEL treated 55 h with 1 mM FITC. Partial tryptic sequences and the number of incorporated FITC labels are assigned.
Figure 5. RP-HPLC chromatogram of a reduced tryptic digest mixture of HEL treated 55 h with 1 mM FITC. Partial peptides of HEL are assigned. Fractions 19 and 23 were subjected to ESIMS (see Figure 6 and Figure 7). Table 1. Localization of FTC-Residues in HPLC Separated HEL Derivatives molecular ion with number of major modification sites highest intensity FTC-residues identified by tryptic (MALDI-MS) incorporated MALDI-MS peptide mapping 14 693 15 110 15 503 15 890
1 2 3 4
R-NH2, K-33, K-97 R-NH2, K-33 K-97 R-NH2, K-1, K-33, K-97, K-116 R-NH2, K-1, K-33, K-97, K-116
modification for 55 h at pH 8 at a molar ratio FITC:HEL of 6. The peptide fragments and the number of FITC groups incorporated are assigned to the molecular ions. These results (summarized in Table 1 for HEL derivatives with different degrees of modification) revealed that R-NH2, K-1, K-33, K-97, and K-116 amino groups were predominantly modified by FITC. The digestion mixture was subsequently separated by RP-HPLC on a C8 column; the corresponding chromatogram is shown in Figure 5. The isolated peptides were analyzed by MALDIMS, which showed that K-13 was detected as an additional modification site. No modification was found at K-96, which is consistent with previous results of the amino-acylation of HEL (26, 27). This lysine residue has a particularly low accesibility to chemical modification due the hydrogen bonding to the carbonyl oxygen of H-15. The HPLC-separated tryptic peptides of FITC-modified HEL were also analyzed by ESI-MS. The effect of different declustering potentials on FTC peptides is illustrated in Figure 6 and Figure 7. Spectra of the homogeneous peptide (97-112) modified with FITC at K-97 (molecular mass 2193 Da) are shown in Figure 6.
Figure 6. ESI mass spectrum of HPLC fraction 23 (see Figure 5) containing the peptide (97-112) carrying a FITC group at K-97. The sample was dissolved in 2% acetic acid/methanol (v/v ) 8/2). Spectra were recorded with declustering potentials of (a) 20 V, (b) 40 V, and (c) 80 V.
The spectrum recorded with a declustering potential of 20 V (Figure 6a) is dominated by signals of the intact FTC peptide. Upon increasing the declustering potential to 40 V (Figure 6b), the signal intensity of the unmodified peptide (97-112) is comparable to the intensity of the FTC peptide, and the free FTC moiety is detected at m/z 390. Upon further increase of the declustering potential up to 80 V (Figure 6c), the molecular ions of the FTC peptide had disappeared and only an intense [M + H]+ signal of the FTC moiety together with a weak [M + H]+ signal of the unmodified peptide were observed. Further studies were carried out to identify FITCmodified peptides from more complex mixtures. Figure 7a shows a spectrum from an HPLC fraction containing several peptides at a declustering potential of 20 V. The abundant signal at m/z 1168 (indicated by an asterisk) is due to a trimer of FITC, formed during lyophilization and storage of the HPLC fractions (28). (In MALDI-MS spectra recorded directly after HPLC separation, no signal at m/z 1168 was detected.) The triply charged molecular ion of the FTC peptide (1-5) (molecular mass 995 Da; B) at m/z 333 and the doubly and triply charged molecular ions of the FTC peptide (113-125) (molecular mass 1936 Da; A) at m/z 969 and 647 were identified. The unmodified peptide (74-97) (molecular mass 2466 Da; C) was found as doubly and triply charged ions. The [M + H]+ ions of the FTC moiety (m/z 390) strongly increased by increasing the declustering potential to 50 V (Figure 7b), and a strong signal at m/z 775 corresponding to the doubly charged molecular ion of unmodified peptide (113-125) (molecular mass 1547 Da) was now
ESI-Mass Spectrometry of FITC−Protein Conjugates
Bioconjugate Chem., Vol. 10, No. 5, 1999 865
occurring with peptide and protein derivatives, and for R- and -FTC-amino groups. The site of each FITC modification can be readily assigned by this specific fragmentation. The direct identification of FITC-labeling sites is possible even when other molecular ions of different intensities are present, as shown by the ESI spectra of a peptide mixture (see Figure 7). Thus, the identification of the number of FTC groups by MALDI-MS and the facile characterization of the specific modification sites by peptide mapping using ESI-MS with increased declustering potentials applied should render this approach an efficient tool for the molecular characterization of fluorescence labeled peptides and proteins, even from complex mixtures. ACKNOWLEDGMENT
We thank Klaus Ha¨gele for expert assistance in ESImass spectrometric procedures. This work has been supported by the Deutsche Forschungsgemeinschaft, Bonn, Germany (“Struktur und Funktionssteuerung an zellula¨ren Oberfla¨chen”), the EU network “Peptide and Protein Structure Elucidation by Mass Spectrometry”, and the Fonds der chemischen Industrie, Germany. LITERATURE CITED
Figure 7. ESI mass spectrum of HPLC fraction 19 (see Figure 5) containing a complex mixture of peptides. The sample was dissolved in 2% acetic acid/methanol (v/v ) 8/2). Spectra were recorded with declustering potentials of (a) 20 V, (b) 50 V, and (c) 80 V. A corresponds to the peptide (113-125) (molecular mass 1547 Da). B corresponds to the peptide (1-5) (molecular mass 606 Da). C corresponds to the peptide (74-97) (molecular mass 2466 Da).
observed. The intensity of these ions was further increased at a declustering potential of 80 V (Figure 7c). The identification of the FTC peptide (1-5) was somewhat more difficult since the ion intensities of these signals were very low in all spectra. DISCUSSION
The results of this study show that the FITC modification provides a broad, inhomogeneous distribution of FTC groups at lysine residues at the conditions employed, as shown for the FITC modification of HEL. For example, the modification of the highly reactive K-97 (26) is not quantitative as shown by the intense ions for the peptide (98-112) (molecular mass 1676 Da) in the digestion mixture (see Figure 4); the quantitative modification of K-97 would have abolished the tryptic cleavage at this site. Furthermore, signals of the peptide (1-5) as singly (molecular mass 995 Da) and doubly modified species (molecular mass 1384 Da) were detectable with high intensity, indicating partial modification of the amino groups of K-1. These results are consistent with the MALDI-MS data of the intact, modified proteins where 2-5-fold modified species were found (see Table 1). The ESI-MS spectra of FITC-modified peptides and proteins show that covalently bound FITC is specifically removed by simple increase of the declustering potential in the ESI ion source. This fragmentation is equally
(1) Coons, A. H., Creech, H. J., Jones, R. N., and E, B. (1942) The demonstration of pneumococcal antigen in tissues by the use of fluorescent antibody. J. Immunol. 45, 159-70. (2) Swoboda, G., and Hasselbach, W. (1985) Reaction of fluorescein isothiocyanate with thiol and amino groups of sarcoplasmic ATPase. Z. Naturforsch. 40c, 863-75. (3) Hucho, F., and Tsetlin, V. I. (1998) Modifizierung als Mittel zur Einfu¨hrung von Reportergruppen. In Bioanalytik (F. Lottspeich and H. Zorbas, Eds.) pp 113-8, Spektrum Akademischer Verlag GmbH, Heidelberg, Berlin. (4) Breier, A., Ziegelhoffer, A., Famulsky, K., Michalak, M., and Slezak, J. (1996) Is cysteine residue important in FITCsensitive ATP-binding site of P-type ATPases? A commentary to the state of the art. Mol. Cell Biochem. 160, 89-93. (5) Kirley, T. L., Wang, T., Wallick, E. T., and Lane, L. K. (1985) Homology of ATP binding sites from Ca2+ and (Na,K)ATPases: comparison of the amino acid sequences of fluorescein isothiocyanate labeled peptides. Biochem. Biophys. Res. Commun. 130, 732-8. (6) Gatto, C., Wang, A. X., and Kaplan, J. H. (1998) The M4M5 cytoplasmic loop of the Na,K-ATPase, overexpressed in Escherichia coli, binds nucleoside triphosphates with the same selectivity as the intact native protein. J. Biol. Chem. 273, 10578-85. (7) Moutin, M. J., Rapin, C., Miras, R., Vincon, M., Dupont, Y., and McIntosh, D. B. (1998) Autonomous folding of the recombinant large cytoplasmic loop of sarcoplasmic reticulum Ca2+-ATPase probed by affinity labeling and trypsin digestion. Eur. J. Biochem. 251, 682-90. (8) Corbalan-Garcia, S., Teruel, J. A., and Gomez-Fernandez, J. C. (1993) Intramolecular distances within the Ca(2+)ATPase from sarcoplasmic reticulum as estimated through fluorescence energy transfer between probes. Eur. J. Biochem. 217, 737-44. (9) Ohta, T., Morohashi, M., Kawamura, M., and Yoshida, M. (1985) The amino acid sequence of the fluorescein-labeled peptides of electric ray and brine shrimp (Na,K)-ATPase. Biochem. Biophys. Res. Commun. 130, 221-8. (10) Hentz, N. G., Richardson, J. M., Sportsman, J. R., Daijo, J., and Sittampalam, G. S. (1997) Synthesis and characterization of insulin-fluorescein derivatives for bioanalytical applications. Anal. Chem. 69, 4994-5000. (11) Pavela-Vrancic, M., Pfeifer, E., Schroder, W., von Dohren, H., and Kleinkauf, H. (1994) Identification of the ATP binding site in tyrocidine synthetase 1 by selective modification with fluorescein 5′-isothiocyanate. J. Biol. Chem. 269, 14962-6.
866 Bioconjugate Chem., Vol. 10, No. 5, 1999 (12) Vorm, O., Roeppstorff, P., and Mann, M. (1994) Improved resolution and very high sensitivity in MALDI TOF of matrix surfaces made by fast evaporation. Anal. Chem. 66, 3281-7. (13) Spengler, B., and Kaufmann, R. (1992) Gentle probe for tough molecules: matrix assisted laser desorption mass spectrometry. Analysis 20, 91-101. (14) Denzinger, T., Przybylski, M., Savoca, R., and Sonderegger, P. (1997) Mass spectrometric characterisation of primary structure, sequence heterogeneity, and intramolecular disulfide loops of the cell adhesion protein axonin-1 from chicken. Eur. Mass Spectrom. 3, 379-89. (15) Macht, M., Fiedler, W., Ku¨rzinger, K., and Przybylski, M. (1996) Mass Spectrometric Mapping of Protein Epitope Structures of Myocardial Infarct Markers Myoglobin and Troponin T. Biochemistry 35, 15633-9. (16) Glocker, M. O., Nock, S., Sprinzl, M., and Przybylski, M. (1998) Characterization of Surface Topology and Binding Area in Complexes of the Elongation Factor Proteins EF-Ts and EF-Tu*GDP form Thermus thermophilus: A Study by Protein Chemical Modification and Mass Spectrometry. Chem. Eur. J. 4, 707-15. (17) Fiedler, W., Borchers, C., Macht, M., Deininger, S. O., and Przybylski, M. (1998) Molecular Characterization of a Conformational Epitope of Hen Egg White Lysozyme by Differential Chemical Modification of Immune Complexes and Mass Spectrometric Peptide Mapping. Bioconjugate Chem. 9, 236-41. (18) Allen, M. H., and Vestal, M. L. (1992) Design and performance of a novel electrospray interface. J. Am. Soc. Mass Spectrom. 3, 18-26. (19) Huddleston, M. J., Annan, R. S., Bean, M. F., and Carr, S. A. (1993) Selective detection of phosphopeptides in complex mixtures by electrospray liquid chromatography/mass spectrometry. J. Am. Soc. Mass Spectrom. 4, 710-7. (20) Lehmann, W. D. (1996) Vergleich zwischen API-CID und Kollisionszellen-CID Massenspektrometrie in der Biochemie,
Schnaible and Przybylski pp 282-6, Spektrum Akademischer Verlag, Heidelberg, Berlin, Oxford. (21) Glocker, M. O., Kalkum, M., Yamamoto, R., and Schreurs, J. (1996) Selective Biochemical Modification of Functional Residues in Recombinant Human Macrophage Colony-Stimulationg Factor β (rhM-CSF β): Identification by Mass Spectrometry. Biochemistry 35, 14625-33. (22) Wilm, M. S., and Mann, M. (1994) Electrospray and TaylorCone theory, Dole’s beam of macromolecules at last? Int. J. Mass Spec. Ion Proc. 136, 167-80. (23) Wilm, M. S., and Mann, M. (1996) Analytical properties of the nanoelectrospray ion source. Anal. Chem. 68, 1-8. (24) Fligge, T. A., Bruns, K., and Przybylski, M. (1998) Analytical development of electrospray and nanoelectrospray mass spectrometry in combination with liquid chromatography for the characterization of proteins. J. Chromatogr. B 706, 91100. (25) Roepstorff, P., and Fohlmann, J. (1984) Proposal for a Common Nomenclature for Sequence Ions in Mass Spectra of Peptides. Biomed. Mass Spectrom. 11, 601. (26) Suckau, D., Mak, M., and Przybylski, M. (1992) Protein surface topology probing by selective chemical modification and mass spectrometric peptide mapping. Proc. Natl. Acad. Sci. U.S.A. 89, 5630-4. (27) Glocker, M. O., Borchers, C., Fiedler, W., Suckau, D., and Przybylski, M. (1994) Molecular characterization of surface topology in protein tertiary structures by amino-acylation and mass spectrometric peptide mapping. Bioconjugate Chem. 5, 583-90. (28) Richter, R., and Ulrich, H. (1977) Oligomerization and polymerization of isocyanates. In The chemistry of cyanates and their thio derivatives (S. Patai, Ed.) Vol. 2, pp 667-80, John Wiley & Sons, Chichester.
BC990039X
