Determination of Recombinant Monoclonal Antibodies and Noncovalent Antigen TNF Trimer Using Q-TOF Mass Spectrometry

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1 Determination of Recombinant Monoclonal Antibodies and Noncovalent Antigen TNF Trimer Using Q-TOF Mass Spectrometry L.C. Santora, I.S. Krull, and K. Grant This article discusses how quadrupole time-of-flight mass spectrometry was used to measure the intact molecular weights of large proteins and some noncovalent protein complexes. L.C. Santora* is a senior scientist at Abbott Bioresearch Center, 100 Research Drive, Worcester, MA 01605; (508) , fax (508) I.S. Krull is an associate professor of chemistry at Northeastern University (Boston, MA) and is an editorial advisory board member of LCGC, Spectroscopy s sister magazine. K. Grant is a group leader at Abbott Bioresearch Center. *To whom all correspondence should be addressed. I n the 1980s, the molecular weight (MW) of a protein could be determined by electrophoretic, lightscattering, chromatographic, or ultracentrifugation methods; however, the MW was not very accurate. Some of these methods depended on characteristics other than MW, such as conformation, Stoke s radius, and hydrophobicity. Recently, advances in mass spectrometry (MS) such as electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) allow for more accurately determining biomolecule MW (1 6). MS enables the precise measurement of the MW of peptides and proteins, as well as sequencing the peptides and proteins, especially with tandem MS techniques (7 12). ESI-MS produces multiple charged ions that extend the effective mass range of the mass analyzer. Some intact antibodies as large as 200,000 Da were measured using a quadrupole (Q) mass spectrometer having a mass-to-charge ratio (m/z) range of 2400 (13). However, resolution of the quadrupole instrument was too low to resolve the antibody heterogeneities. An advantage of time-of-flight (TOF) instrumentation is that the m/z range is limited only by detector efficiency. Resolution of TOF is limited by the effects of spatial and velocity dispersions and energy focusing. The Micromass (Beverly, MA) Q-TOF mass spectrometer applies an orthogonal acceleration ion source with an electric mirror to accomplish a long flight path, high transmission, and high resolution. A fully human tumor necrosis factor alpha (TNF ) IgG 1 monoclonal antibody D2E7 is presently in clinical trials as a therapy for rheumatoid arthritis. D2E7 is heterogeneous and has heavychain C-terminal Lys variants and some acidic isoforms (14). In addition, D2E7 is N-glycosylated at heavy-chain Asn (position 301) with fucosylated biantennary oligosaccharides containing zero, one, or two galactoses. Therefore, multiple masses should be measured for D2E7, due to the heterogeneities in anti- Figure 1. Deglycosylated antibody D2E7 analysis using Q-TOF. Operational conditions are described in the Experimental section. (a) The ESI/Q-TOF mass spectrum, which shows the antibody ion envelope in the m/z range. (b) The deconvolution result using MassLynx software (Micromass). Peaks A, B, and C are D2E7 heavy chain C-terminal Lys variants. Peak D is an unknown compound. 50 Spectroscopy 17(5) May

2 bodies. Two MS approaches can handle higher MW heterogeneous antibodies: MALDI and ESI. MALDI produces primarily singly charged molecular ions. However, the heterogeneous glycoproteins broaden the peak, which reduces the intensity of a glycoprotein molecular ion by spreading its abundance over a broader range. To resolve the different glycosylated and C-termini variants of D2E7, the MWs of glycosylated and deglycosylated D2E7 were measured by ESI/Q-TOF. The antibody charged variants, which were generated by incomplete posttranslational cleavage of the C-terminal lysine, could be determined not only by cation ionexchange chromatography or capillary isoelectric focusing (14), but also by Q- TOF MS. By using a Mass-Lynx deconvolution tool (Micromass, Beverly, MA), the antibody N-linked zero-galactose, onegalactose, and two-galactose oligosaccharide variants and deglycosylated antibody heavy chain C-terminal zero-lys, one-lys, and two-lys variants were resolved. Figure 2. Glycosylated antibody D2E7 analysis using Q-TOF. Operational conditions are described in the Experimental section. (a) The ESI/Q-TOF mass spectrum shows the antibody ion envelope in the m/z range. (b) The deconvolution result obtained using MassLynx software (Micromass). Peaks A, B, and C are D2E7 N-linked oligosaccharide isoforms. Peaks D and E are unknowns. An x-ray crystallographic study found that the biologically active TNF exists as a trimer, which is a noncovalent complex (15 17). Studying noncovalent interactions of proteins by MS is a developing field of research (19 26). The study of noncovalent complexes of antibody and antigen TNF using cation-exchange, size-exclusion chromatography (SEC), and a BIAcore instrument also provided strong evidence that antigen TNF is the trimer, in the neutral solution phase (18). The analysis of noncovalent TNF trimer by Q-TOF demonstrated that TNFa trimer was a more stable form than the dimer and monomer. Circle 47 May (5) Spectroscopy 51

3 Figure 3. Comparison of the m/z peak widths between deglycosylated and glycosylated D2E7. (a) Enlarged one m/z ion peak ( 50) for deglycosylated D2E7. The peak width at half height is about 2 Da and there are two other minor peaks besides the main peak. (b) One m/z ion peak ( 50) for glycosylated D2E7. The peak width at half height is about 3 Da, and there are some minor peaks beside the main peak. Experimental Materials and sample handling.recombinant human IgG antibodies (Abbott Bioresearch Center, Worcester, MA) were diluted with high performance liquid chromatography (HPLC) grade water to a concentration of 1.0 mg/ml and dialyzed into 50 mm ammonium acetate (ph 5.5) buffer using a Spectra/Por membrane with a molecular weight cutoff of (Spectrum Laboratories, Rancho Dominguez, CA). Then, all samples were further diluted to 0.5 mg/ml in 25% methanol and 0.5% formic acid for MS. N- Glycosidase F (PNGase F) enzyme (Boehringer Mannheim, Indianapolis, IN) was used for antibody deglycosylation. All other chemicals were from Sigma (Sigma Chemical, St. Louis, MO). Enzymatic reactions for antibody deglycosylation were in a 5 mm n- octyl- -d-glucopyranoside, 5 mm phosphate, and 10 mm sodium chloride (ph 7.0) solution. Samples were incubated at 37 C overnight. A ratio of 2.0 mg monoclonal antibody (mab) to 4 U (activity units) of PNGase F was used. All samples were analyzed immediately after the enzymatic reactions. Antibodies were reduced by adding excess amounts of dithiothreitol. The light chain and heavy chain mixture of reduced and glycosylated D2E7 was infused into the Q-TOF source directly without any separation. Mass spectrometry. A Q-TOF mass spectrometer (Micromass), with the standard Z-spray source fitted electrospray probe, was used for ESI experiments. The needle voltage was 3500 V and the cone voltage was 55 V. The 52 Spectroscopy 17(5) May 2002 Circle 48 Circle 49

4 Table I. Theoretical MWs of D2E7 Deglycosylated D2E7 with zero Lys Deglycosylated D2E7 with one Lys Deglycosylated D2E7 with two Lys Glycosylated D2E7 with zero Lys and zero galactose Glycosylated D2E7 with zero Lys and one galactose Glycosylated D2E7 with zero Lys and two galactose Glycosylated D2E7 with one Lys and zero galactose Glycosylated D2E7 with one Lys and one galactose Glycosylated D2E7 with one Lys and two galactose Glycosylated D2E7 with two Lys and zero galactose Glycosylated D2E7 with two Lys and one galactose Glycosylated D2E7 with two Lys and two galactose TNF monomer TNF dimer TNF trimer source block and desolvation temperatures were 85 C and 120 C, respectively. The nominal flow rates of desolvation gas and nebulizer gas were 250 L/h and 4 L/h, respectively. All samples were continuously infused through the 145,192 Da 145,320 Da 145,448 Da 148,080 Da 148,242 Da 148,404 Da 148,208 Da 148,370 Da 148,532 Da 148,336 Da 148,498 Da 148,660 Da 17,352.7 Da 34,705.4 Da 52,058.1 Da electrospray probe at 5 or 10 ml/min from a Harvard syringe pump (Harvard Apparatus, Holliston, MA). Infusion flow rates were used in the experiments to achieve experimental simplicity and low fluctuations in ionization. For nanospray, all samples were loaded into borosilicate nanoflow probe tips, coated with gold (Micromass). The needle voltage was 1200 V and the cone voltage was 45 V. The source block temperature was 60 C. The scan duration and the interscan delay for all experiments were 0.90 and 0.10 s, respectively. All data were acquired in about 1 min by using the tune page. Data analysis. All spectra shown were obtained by averaging the signals from multiple scans. Because the protein charge-state peak width actually reflects the measurement precision and accuracy, the standard deviation of the molecular weights was calculated either from three repeated measurements or by the measurement of the m/z peak width, whichever number was larger. MassLynx software was used for sample deconvolution. Figure 4 (left). Reduced antibody D2E7 analysis using Q-TOF. (a) The ESI/Q-TOF mass spectrum shows different antibody ion envelopes in the m/z range of (b) The deconvolution result obtained using MassLynx software. Peak A is D2E7 light chain and peak B is N-linked oligosaccharide heavy chain. Figure 5 (above). Analysis of in-purification process samples by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The operational conditions were according to the Bio-Rad (Hercules, CA) manual. Lane 1 contains MW standards, lane 2 contains an eluate sample from a conventional cation-exchange column, and lane 3 contains a regeneration sample from the same column. Figure 6 (above). Analysis of an eluate sample purified from a conventional cation-exchange column by Q-TOF. (a) The ESI/Q-TOF mass spectrum, which shows different antibody ion envelopes in the m/z range of (b,c) Deconvolution results using MassLynx software, in which peak A is a fulllength antibody and peak B is an antibody fragment of one heavy chain connected with one light chain. Peak C is an antibody fragment of double light chain and peak D is a free light chain. May (5) Spectroscopy 53

5 Figure 7. Analysis of a regeneration sample from a conventional cation-exchange column by Q-TOF. (a) The ESI/Q-TOF mass spectrum shows different antibody ion envelopes in the m/z range of (b,c) Deconvolution results obtained using MassLynx software, in which Peak E is an antibody fragment of double light chain and peak F is a free light chain. Peaks G and H are antibodyrelated species. Figure 8. Analysis of TNF trimer by Q-TOF. The sample was in 20 mm ammonium acetate (ph 5.5) solution. (a) The ESI/Q-TOF mass spectrum shows TNF protein ion envelopes in the m/z range of (b) The deconvolution result obtained using MassLynx software. The 52,068 Da peak is TNF trimer and the 34,710 Da peak is the dimer. The 17,364 Da peak is the monomer. 54 Spectroscopy 17(5) May 2002 Results and Discussion Analysis of deglycosylated and glycosylated monoclonal antibodies. The theoretical MWs of D2E7, based on amino acid sequence and posttranslational modifications, are given in Table I. There are basically three isoforms for deglycosylated D2E7 due to heavy-chain C-terminal Lys variants. Because the MW of Lys is 128 Da, the mass differences among these isoforms should be 128 Da. For glycosylated D2E7, the isoforms are Table II. Comparison of Antibody MWs Obtained by MALDI and Q-TOF D2E7 Deglycosylated D2E7 Glycosylated MALDI-TOF Data 147, Da ESI/Q-TOF Data (A) 145,204 7 Da 148, Da ESI/Q-TOF Data (B) 145,328 7 Da 148, Da ESI/Q-TOF Data (C) 145,456 7 Da 148, Da D2E7 isoform fractions 145,205 Da (zero Lys) using nanospray/q-tof 145,334 Da (one Lys) 145,493 Da (two Lys) mainly due to the presence or absence of galactose residues on the oligosaccharides. Because the MW of galactose is 162 Da, the mass differences among these isoforms are 162 Da. Because antibodies precipitate under reversedphase HPLC conditions, the reversedphase HPLC system cannot be used for the separation. All of the antibody samples demonstrated a very similar protein envelope spectral behavior, producing charge distribution profiles in the high m/z range ( ) of the instrument. Charge states ranging from 65 to 35 were observed for these monoclonal antibodies. Figures 1b and 2b show the MW values after using MassLynx deconvolution. The deglycosylated D2E7 main peak resulted in a MW of 145,204 7 Da. The predicted mass for deglycosylated D2E7 is 145,192 Da. Besides the main peak A, three minor components were detected in the deglycosylated D2E7, which yielded MWs of 145,328 7 Da (component B), 145,456 7 Da (component C), and 145,052 7 Da (component D), respectively (Figure 1b). The mass differences of 124 Da and 128 Da among these components were due to the heavy chain C-terminal zero-lys, one-lys, and two-lys isoforms (14). The mass of amino acid Lys is 128 Da. MW determined by the Q-TOF was in an accuracy range of 100 ppm (or 0.01%) of the theoretical MW. It is not clear whether the MW difference of 152 Da between components D and A was due to an artifact or to amino acids cleaved from D2E7 during the ionization process. To confirm the D2E7 heavy chain C-termini Lys variants, the D2E7 heavy chain C-terminal zero-lys, one-lys, and two-lys isoforms were separated and fractionated by using the weak-cation-exchange column (WCX-10 column) (14). These fractions were analyzed using the nanospray/q-tof system (Table II). The MWs of D2E7 fractions 1, 2, and 3 were 145,205 Da, 145,334 Da, and 145,493 Da, respectively. The mass difference of 129 Da between fractions 1 and 2 corresponded to the mass of the amino acid Lys. Because fraction 3 had a very low concentration, the accuracy of mass determination by Q-TOF for

6 fraction 3 was not as good as for fractions 1 and 2. As noted, masses of the common sugars found in glycoproteins are Da for fucose, Da for galactose or mannose, Da for N-acetylgalactosamine or N-acetylglucosamine, and Da for N-acetylneuraminic acid. Because glycosylated D2E7 only has N- linked oligosaccharides, without any sialic acids on the residues, the mass differences of 162 Da should be observed among these isoforms. When glycosylated D2E7 was analyzed by Q- TOF, a main peak with the MW of 148, Da was observed. The theoretical mass for glycosylated D2E7 with zero-lys and zero-galactose residues is 148,080 Da. Besides the main peak A, four other components were detected for glycosylated D2E7, which yielded MWs of 148,256 Da (component B), 148,412 Da (component C), 147,872 Da (component D), and 147,652 Da (component E), respectively (Figure 2b). The mass difference of 156 Da between A and B was due to one galactose difference of glycosylated D2E7 with zero Lys. The mass difference of 312 Da between A and C was due to two galactoses on glycosylated D2E7 with zero Lys. It was unclear whether the MW differences of 228 Da between components D and A, and 220 Da between D and E, were due to an artifact or to amino acids cleaved from D2E7 during the ionization process. The HPLC results showed that the relative percentages of zero-lys, one- Lys, and two-lys D2E7 isoforms were about 74% to 21% to 5% (14). The D2E7 oligosaccharide analysis showed that the relative percentages of zerogalactose, one-galactose, and twogalactose D2E7 isoforms were 80% to 17% to 3% (data not shown here). Therefore, the amounts of one-lys and two-lys D2E7 with one-galactose and two-galactose D2E7 isoforms were very low and the sensitivity, resolution, and accuracy of Q-TOF were significantly decreased for determination of those D2E7 species with one-lys and onegalactose or two-lys with two-galactose components. Figure 3 compares the m/z peak spectra for both deglycosylated and glycosy- Figure 9. Analysis of TNF monomer by Q- TOF. The sample was in 0.5% formic acid, ph 3.0 solution. (a) The ESI/Q- TOF mass spectrum shows TNF protein ion envelopes in the m/z range of (b) The deconvolution result obtained using MassLynx software. Figure 10. Analysis of TNF trimer by Q-TOF. The sample was in the 15% methanol, 20 mm ammonium acetate (ph 5.5) solution. (a) The ESI/Q-TOF mass spectrum shows TNF protein ion envelopes in the m/z range of (b) The deconvolution result obtained using MassLynx software. The 52,055 Da, 34,699 Da, and 17,357 Da peaks are the TNF trimer, dimer, and monomer, respectively. lated D2E7. Figure 3a shows that the m/z peak width at half height was about 2 Da for deglycosylated D2E7. There were at least two peaks. Figure 3b shows that the m/z peak width at half height was about 3 Da for glycosylated D2E7 and there were at least four peaks. These results indicated that there was more than one component in these samples. In general, by using deconvolution software, the main peaks of D2E7 heterogeneities, which were in agreement with the mass predicted (Table I), could be resolved by Q-TOF. Compared with MALDI-TOF data (Table II), ESI/Q-TOF provided much higher resolution and accuracy. Analysis of a Mixture of Antibody Fragments When the reduced glycosylated D2E7 samples were infused into Q-TOF source directly, without any separation, two protein envelopes were observed in the m/z spectra. The analysis of light chain and heavy chain mixtures of reduced D2E7 was done by ESI/Q-TOF. This resulted in the molecular mass of 23,420 3 Da (mass predicted: 23,412 Da) for the light chain and 50, Da (mass predicted: 50,651 Da) for the heavy chain, as shown in Figure 4. First, these results confirmed that the light chain had no posttranslational modifications. Second, the MW measured for the heavy chain was the sum of the peptide chain and oligosaccharides attached at Asn 301. A similar strategy was used for monitoring antibodies in the purification process. When a conventional cationexchange column was used to purify antibodies, the in-process samples were monitored by a Q-TOF MS. Figure 5 shows the analysis of antibodies using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The first lane shows the MW markers and the second lane shows the intact antibody and antibody fragments purified May (5) Spectroscopy 55

7 from a strong-cation-exchange column (eluate). This sample of the elution contained full-length antibodies A and A1 (approximately 170 kda and 150 kda), minor bands X (approximately 117 kda), B (approximately 74 kda), C (approximately 46 kda), and D (approximately 23 kda). The third lane shows impurities, which were collected during column regeneration (regeneration). There were four main bands stained in the third lane: bands E (approximately 46 kda), F (approximately 23 kda), G (approximately 14 kda), and H (approximately 10 kda). All these bands observed from SDS-PAGE were antibody-related species. These eluate and regeneration samples were analyzed by Q-TOF. Molecular masses detected by Q-TOF, for the eluate sample, are given in Figure 6. The m/z spectra gave many different protein envelopes. Deconvolution of these protein envelopes resulted in five peaks. The species with a MW of 146,900 Da (peak A) was a full-length antibody, and the protein with the MW of 73,466 Da (peak B) was one heavy chain connected with one light chain fragment (Figure 6b). The protein with a MW of 143,464 Da (peak X) was a deglycosylated Ab (Figure 6c). The 46,302 Da (peak C) species was a double light chain and the 23,240 Da peak (peak D) was a free light chain (Figure 6c). These data were consistent with the presence of antibody and antibody fragments analyzed by SDS-PAGE. Figure 7 gives the results for a regeneration sample from the cation-exchange column analyzed by Q-TOF. The 46,264 Da peak (peak E) was a double light chain and the 23,210 Da peak (peak F) was a free light chain (Figure 7b). The 15,424 Da (G) and 11,568 Da (H) peaks were shown to be some antibody-related species (Figure 7c), which were in agreement with the analysis by SDS-PAGE (Figure 5). Analysis of Antigen TNF Trimer By using ESI-MS to study noncovalent protein complexes, several important features of MS can be exploited over other traditional methods. Speed and accuracy are the most obvious advantages of the MS-based method compared with electrophoretic, lightscattering, chromatographic, or ultracentrifugation methods. Recently demonstrated advantages of low-flow ESI or nanoelectrospray offer additional sensitivity and enhancements. However, the solution conditions needed to maintain an intact, noncovalently bound complex do not match those typically used for routine ESI operations. Solution ph of 2 4 for positive ionization and ph 8 10 for negative ion ESI are typical for maximum sensitivity. The addition of an organic cosolvent such as methanol or acetonitrile also enhances sensitivity and ionsignal stability. Unfortunately, these conditions generally disrupt the stability of noncovalent complexes. It is very challenging to find a balance between sufficient desolvation of the gas-phase complexes and no dissociation of the complexes, especially, for high MW complexes. The MW of TNF monomer (17,352.7 Da) is given in Table I. When TNF is in phosphatebuffered saline or 20 mm ammonium acetate (ph 5.5) buffer, it exists as a trimer, which was determined by SEC/ultraviolet (UV)/light-scattering/ refractive index detection (20). The ph of the buffer had to be higher than 5.5 so that TNF trimer complex was in solution before ionization. The needle voltage and other apparatus parameters were held constant for all TNF measurements to ensure maximum comparability. Figure 8 shows a mass spectrum of the TNF trimer in 20 mm ammonium acetate (ph 5.5) buffer. High charge states of m/z peaks 3253 and 3470 are preferably produced in the mass spectrum. By using MassLynx deconvolution software, the TNF trimer complex was observed as a dominant peak, and only small portions of TNF monomer and dimer peaks were observed, as indicated in Figure 8b. The peak with a MW of 52,068 Da is the TNF trimer, the 34,710 Da peak is the dimer, and the 17,364 Da peak is the monomer. To show that the observed TNF trimer complex is from TNF, the solution was acidified to ph 3.0 and then analyzed again. As already known from the literature, under these conditions, most complexes are completely dissociated in solution. Consequently, at ph 3.0, TNF monomer was clearly observed in Figure 9b, after MW deconvolution. In the mass spectrum, high charge states of m/z peaks were shifted down to lower m/z peaks (1240, 1920, and 2160). The overall signal intensity and the signal-to-noise ratio was much better than in Figure 8. This was largely attributed to the fact that the MS detector was more sensitive in the lower mass region, and that on dissociation a trimer complex would give rise to a three-fold higher concentration of TNF. When 15% of methanol, as an organic cosolvent, was added into the TNF trimer buffer, at least half of the TNF trimer complex was dissociated during the ionization process (Figure 10). However, there was only the TNF trimer and no TNF monomer in solution phase, as was determined by SEC/UV under the same conditions. This situation indicated that the TNF trimer was dissociated into the monomer during the ESI ionization process. The MW deconvolution demonstrated that there were mixed compounds of TNF monomer, dimer, and trimer in the gas phase (Figure 10b). These results showed that, generally, an acid or buffer ph is a stronger dissociation factor than an organic cosolvent for noncovalent protein complexes. It clearly indicated that TNF exists as a trimer even under less acidic solution conditions. Conclusions The MWs of monoclonal antibodies were determined by using an ESI/Q- TOF MS. Compared with MALDI-TOF, the Q-TOF MS was capable of resolving the microheterogeneities of both deglycosylated and glycosylated antibodies, in the MW range of 140, ,000 Da. The MWs of the microheterogeneity of antibodies can be determined by using MassLynx deconvolution software. Differences among deglycosylated antibodies were due to heavy chain, C- terminal Lys variants. Differences among glycosylated antibodies were mainly due to a combination of the 56 Spectroscopy 17(5) May

8 number of galactoses on the N-linked oligosaccharides and on the heavy chain C-terminal Lys variants. Compared with these MW peaks, the peak widths of glycosylated antibodies were wider than that of deglycosylated antibodies. In other words, peak widths can also be used to assess the degree of microheterogeneity of antibodies. The D2E7 MW, determined by Q-TOF, had an accuracy of 200 ppm (or 0.02%) to the theoretical MW. Q-TOF resolved the Lys isoforms for deglycosylated D2E7, and zero lysine with zero-galactose, one-galactose, and two-galactose variants for glycosylated D2E7. However, it could not resolve either one-lys with zero-galactose, one-galactose, and twogalactose or two-lys with zerogalactose, one-galactose, and twogalactose isoforms, because of low percentages of these isoforms in the samples. The noncovalent complex, the TNF trimer, was analyzed by using Q-TOF. The acid-induced dissociation of the TNF trimer confirmed the noncovalent character of the active trimer form of TNF, under less acidic solution phase conditions. Acknowledgments The authors wish to thank B. Turner for stimulating discussion and insights. The authors appreciate P. Moesta for supporting this research. We especially thank B. Perilli-Palmer for her SDS- PAGE imaging and antibody purification work. Finally, the authors thank M. Tomlinson and B. Janssen for instrument time on the Q-TOF. References 1. J.B. Fenn, M. Mann, C.K. Meng, and C.M. Whitehouse, Science 246, 64 (1989). 2. M. Karas and F. Hillenkamp, Anal. Chem. 60(14), 2299 (1988). 3. R.D. Smith, J.A. Loo, C.G. Edmonds, C.J. Barinaga, and H.R. Udseth, Anal. Chem. 62(5), 882 (1990). 4. R. Feng and Y. Konishi, Anal. Chem. 64(18), 2090 (1992). 5. M. Tito, K. Tars, K. Valegard, J. Hajdu, and C.V. Robinson, J. Am. Chem. Soc. 122(14), 3550 (2000). 6. A. Wattenberg, F. Sobott, and B. Brutschy, Rapid Commun. Mass Spectrom. 14(10), 859 (2000). 7. A.L. Burlingame, D.S. Millington, D.L. Norwood, and D.H. Russel, Anal. Chem. 62, 268R (1990). 8. J.C. Cotter, Anal. Chem. 64(5), 1027 (1992). 9. S.A. Carr, M.E. Hemling, M.F. Bean, and G.D. Roberts, Anal. Chem. 63(24), 2802 (1991). 10. G.J. Feistner, K.F. Faull, D.F. Barofsky, and P. Roepstorff, J. Mass Spectrom. 30(6), 519 (1995). 11. K. Eckart, Mass Spectrom. Rev. 13(1), 23 (1994). 12. P. Roepstorff, Trends Anal. Chem. (Pers. Ed.) 12, 413 (1993). 13. R. Feng and Y. Konishi, Anal. Chem. 64(18), 2090 (1992). 14. L.C. Santora, I.S. Krull, and K. Grant, Anal. Biochem. 275(1), 98 (1999). 15. M.J. Eck, B. Beutler, G. Kuo, J.P. Merryweather, and S.R. Sprang, J. Biol. Chem. 263(26), 12,816 (1988). 16. M.J. Eck and S.R. Sprang, J. Biol. Chem. 267(4), 2119 (1992). 17. S. Saijo, N. Watanabe, and Y. Kobayashi, J. Biochem. 118(1), (1995). 18. L.C. Santora, Z. Kaymacklan, P. Sakorafas, I.S. Krull, and K. Grant, Anal. Biochem. 299(2), 119 (2001). 19. J.H. Willem, V. Berkel, H.H. Robert, H. VanDen, V. Cees, and J.R.H. Albert, Protein Science 9(3), 435 (2000). 20. A.A. Rostom and C.V. Robinson, J. Am. Chem. Soc. 121(19), 4718 (1999). 21. K.F. Blom, B.S. Larsen, and C.N. McEwen, J. Combinatorial Chem. 1(1), 82 (1999). 22. A.V. Tolmachev, H.R. Udseth, and R.D. Smith, Anal. Chem. 72(5), 970 (2000). 23. M.E. Belov, M.V. Gorshkov, H.R. Udseth, G.A. Anderson, A.V. Tolmachev, D.C. Prior, R. Harkewicz, and R.D. Smith, J. Am. Soc. Mass Spectrom. 11(1), 19 (2000). 24. N. Xu, L. Pasa-Tolic, R.D. Smith, S. Ni, and B.D. Thrall, Anal. Biochem. 272(1), 26 (1999). 25. J.E. Bruce, V.F. Smith, C. Liu, L.L. Randall, and R.D. Smith, Protein Science 7(5), 1180 (1998). 26. L.P. Tolic, J.E. Bruce, Q.P. Lei, G.A. Anderson, and R.D. Smith, Anal. Chem. 70(2), 405 (1998). Circle 50 May (5) Spectroscopy 57