Purification and High-Resolution Top-Down Mass Spectrometric

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Purification and High-Resolution Top-Down Mass Spectrometric Characterization of Human Salivary α-Amylase Ying Peng,† Xin Chen,† Takuya Sato,‡ Scott A. Rankin,§ Ryohei F. Tsuji,∥ and Ying Ge*,†,⊥ †

Human Proteomics Program, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin 53706, United States ‡ Kikkoman USA R&D Laboratory, Inc., Madison, Wisconsin 53719, United States § Department of Food Science, University of Wisconsin-Madison, Madison, Wisconsin 53706, United States ∥ Kikkoman Foods, Inc., Walworth, Wisconsin 53184, United States ⊥ Department of Cell and Regenerative Biology, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin 53706, United States S Supporting Information *

ABSTRACT: Human salivary α-amylase (HSAMY) is a major component of salivary secretions, possessing multiple important biological functions. Here we have established three methods to purify HSAMY in human saliva for comprehensive characterization of HSAMY by high-resolution top-down mass spectrometry (MS). Among the three purification methods, the affinity method based on the enzyme-substrate specific interaction between amylase and glycogen is preferred, providing the highest purity HSAMY with high reproducibility. Subsequently, we employed Fourier transform ion cyclotron resonance MS to analyze the purified HSAMY. The predominant form of α-amylase purified from saliva of various races and genders is nonglycosylated with the same molecular weight of 55 881.2, which is 1885.8 lower than the calculated value based on the DNA-predicted sequence. High-resolution MS revealed the truncation of the first 15 N-terminal amino acids (−1858.96) and the subsequent formation of pyroglutamic acid at the new N-terminus Gln (−17.03). More importantly, five disulfide bonds in HSAMY were identified (−10.08) and effectively localized by tandem MS in conjunction with complete and partial reduction by tris (2-carboxyethyl) phosphine. Overall, this study demonstrates that top-down MS combined with affinity purification and partial reduction is a powerful method for rapid purification and complete characterization of large proteins with complex and overlapping disulfide bond patterns.

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aliva is an important body fluid essential for health.1,2 It is secreted from multiple salivary glands such as parotid, submandibular, and sublingual glands. Saliva has a range of fundamental functions including but not limited to oral digestion, food swallowing and tasting, lubrication, antibacterial and antiviral protection, as well as maintenance of the tooth integrity.1,2 Thus, human saliva is an ideal source in the search of diagnostic markers especially for oral disease given its easy availability and a noninvasive collection process.2−4 α-Amylase (EC 3.2.1.1) is a major protein in saliva, which catalyzes hydrolysis of 1,4-α-glucosidic linkages in starch and other polysaccharides.5 It also plays an important role in the colonization and metabolism of bacteria leading to dental plaque formation.6,7 Salivary and pancreatic α-amylase are produced by two closely related genetic loci, AMY1 and AMY 2, respectively.5 It is believed that human salivary α-amylase (HSAMY) contains a major nonglycosylated component (∼56 kDa) and a minor glycosylated component at a higher molecular mass (∼59 kDa).5,8,9 Interestingly, only nonglycosylated amylase was obtained when recombinant salivary amylase was expressed in a baculovirus system.10 Nevertheless, © 2012 American Chemical Society

the secreted nonglycosylated amylase exhibited antigenicity and bacterial binding activity comparable to the native amylase. With regards to the potential use of HSAMY as a predictive marker, it is essential to characterize its sequence and posttranslational modifications (PTMs).10 Previous studies used a bottom-up approach involving in-gel digestion and matrixassisted laser desorption/ionization time-of-flight (MALDITOF) MS analysis of HSAMY.11,12 However, an accurate molecular weight (MW) of HSAMY has not been obtained, and the protein sequence and PTMs have not been thoroughly characterized. Top-down MS shows clear advantages in analysis of protein with complex post-translational modifications (PTM) and sequence variants.13−27 Compared to the traditional bottomup MS, top-down MS shows several advantages for characterization of protein modifications. It analyzes intact proteins without proteolytic digestion, providing universal protein Received: January 10, 2012 Accepted: March 5, 2012 Published: March 5, 2012 3339

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Figure 1. Flow diagram of HSAMY purification and characterization procedures. Method 1 was based on size-sieving effect, method 2 used reversedphase chromatography, and method 3 was based on enzyme-substrate specific interaction between amylase and glycogen. Method 3 is the preferred method for purification of HSAMY with the highest purity and reproducibility.

modification information without a priori knowledge. Individual proteins with modifications can be isolated in the gas phase and fragmented by tandem MS such as collision-activated dissociation (CAD)28 and electron capture dissociation (ECD), 29 allowing a highly reliable mapping of the modification sites with full sequence coverage.23,24,30 To date, top-down MS has been employed in deciphering molecular complexity of intact proteins,17 mapping complex, even unexpected, post-translational modifications (PTM)21,30 and quantification of modified protein levels.17,31 Recently, topdown MS has been employed to characterize human salivary cystatins and peptide P-C.32,33 In this study, we have employed three methods to purify HSAMY based on the size-sieving effect (method 1), reversedphase chromatography (method 2), and the enzyme-substrate specific interaction between glycogen and amylase (method 3) (Figure 1). Method 3 is preferred providing the highest purity HSAMY with the highest reproducibility. Top-down Fourier transform ion cyclotron resonance (FTICR) MS was employed to accurately determine the MW and comprehensively characterize HSAMY. We have unambiguously identified the N-terminal sequence truncation and the subsequent formation of pyroglutamic acid at the new N-terminal Gln. Importantly, we have precisely localized five disulfide bonds (S−S) in this 56 kDa nonglycosylated HSAMY by top-down MS/MS in conjunction with complete and partial reduction by tris (2carboxyethyl) phosphine (TCEP). To our knowledge, this is the first comprehensive characterization of large proteins (>55 kDa) with complex and overlapping S−S patterns by top-down MS/MS without the need for proteolytic digestion.

Corp. (Indianapolis, IN). All solutions were prepared in Milli-Q water (Millipore Corp., Billerica, MA). Human Saliva Collection. All saliva samples were collected according to a protocol approved by Institutional Review Board at University of Wisconsin-Madison. Adult saliva donors of various ethnic and racial origins were recruited from students at University of Wisconsin-Madison. Donors were in good health and exhibited normal salivary function and refrained from drinking and eating for at least 2 h prior to donation. Unstimulated whole saliva was collected into sterilized plastic centrifuge tubes that were placed on ice and centrifuged (10 000g, 10 min, 4 °C). A volume of 1 mL of supernatant was then treated with 20 μL of protease and phosphatase inhibitors dissolved in 50 mL of water and immediately frozen at −80 °C until use. HSAMY Purification. Three different methods were used to purify HSAMY (Figure 1). Method 1 is based on size sieving using Amicon 50 K molecular weight cutoff (MWCO) filter (Millipore Corp., Billerica, MA). In detail, 200 μL of the saliva samples in 50 K Amicon filter were centrifuged (10 000g, 4 °C) and washed with 0.1% formic acid (FA) aqueous solution to remove salt. Smaller size proteins (50 kDa) remained on the filter for further MS analysis. Method 2 is based on reversed-phase chromatography34 and fraction collection. In detail, 500 μL of the saliva samples was dried by centrifugal evaporation and redissolved in 200 μL of 8 M urea and centrifuged (10 000g, 10 min, 4 °C). A volume of 120 μL was injected into a reversedphase HPLC column (PLRP/S 10 μm, 1000 Å, 0.5 mm × 100 mm, Varian Inc., Palo Alto, CA). Amylase fractions were collected in microcentrifuge tubes at 1 min intervals and stored at −80 °C for high-resolution MS analysis. Method 3 is based on enzyme-substrate specific interaction between amylase and glycogen.35 In this method, all samples and operations were carried out at 4 °C. In detail, 200 μL of ethanol was added into 300 μL of saliva drop-by-drop. The mixture was centrifuged at 10 000g for 10 min. A volume of 450 μL of supernatant was

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MATERIALS AND METHODS Chemicals and Reagents. All reagents were purchased from Sigma Chemical Co. (St. Louis, MO) unless noted otherwise. Complete protease and phosphatase inhibitor cocktail tablets were purchased from Roche Diagnostics 3340

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then mixed with the following reagents in the order (25 μL of 0.2 M phosphate buffer, pH 8.0, 50 μL of glycogen (20 mg/ mL), 50 μL of ethanol). The mixture was shaken for 5 min and then centrifuged at 5 000g for 6 min. The resulting precipitate was washed twice with 0.01 M phosphate buffer (pH 8.0) containing 40% ethanol and then suspended in 0.5 mL of 0.2 M phosphate buffer (pH 8.0) containing 5 mM CaCl2 and incubated at room temperature for 2 h. Finally, the solution was centrifuged at 10 000g for 3 min to remove precipitate before desalting by the Amicon 50 K MWCO filter. Reduction of HSAMY. A volume of 50 μL of purified HSAMY was mixed with 50 μL of 50 mM TCEP hydrochloride in 0.1 M citric buffer at pH 3.0 to completely reduce S−S in amylase. Complete reduction was performed at 65 °C for 30 min. Partial reduction was performed by mixing 50 μL of purified HSAMY with 50 μL of 10 mM TCEP solution in 0.1 M citric buffer (pH 3.0), followed by shaking 10 min at room temperature. After reduction, a 50 K Amicon filter was used to desalt the reduced HSAMY with 0.1% FA aqueous solution. Top-Down Mass Spectrometry. Desalted HSAMY was analyzed using a 7 T linear ion trap/FTICR (LTQ FT Ultra) hybrid MS (Thermo Scientific Inc., Bremen, Germany) equipped with an automated chip-based nano ESI source (Triversa NanoMate; Advion BioSciences, Ithaca, NY) as described previously.24 For CAD and ECD fragmentation, protein molecular ions of individual charge states were first isolated and then dissociated using 10−35% normalized collision energy for CAD or 2−3% electron energy for ECD with a 70 ms duration with no delay. Typically, 1 000−3 000 transients were averaged to obtain high-quality CAD and ECD spectra. FTICR spectra were processed with Xtract Software (FT programs 2.0.1.0.6.1.4, Xcalibur 2.0.5, Thermo Scientific Inc., Bremen, Germany) with S/N threshold of 1.5 and a fit factor of 40% and manually validated. The resulting mass lists were further assigned using in house developed “Ion Assignment” software (version 1.0) based on the protein sequence of HSAMY obtained from the Swiss-Prot protein knowledgebase (Unit-ProtKB/Swiss-Prot). Allowance was made for possible PTMs, such as the removal of signal peptides and blocking the N-terminus, using a 1 Da tolerance for precursor and fragment ions, respectively. The assigned ions were manually validated to ensure the quality of assignments. All reported masses are the most abundant masses.

HSAMY (Figure S1A in the Supporting Information). Additionally, the reproducibility of method 1 is not as good as the other two methods (data not shown). Method 2 exhibited better reproducibility compared to method 1. Nonetheless, there are still other proteins that coelute with HSAMY even with the most optimized LC conditions (Figure S1B in the Supporting Information). Method 3 takes advantage of the specific binding of amylase to glycogen35 for purification (Figure S1C in the Supporting Information). The glycogen−amylase complex is insoluble in phosphate buffer and thus can be specifically precipitated from the solution. The complex can later be dissociated after the incubation in the phosphate buffer with CaCl2, which breaks down glycogen into small pieces. Removal of digested glycogen yields essentially pure HSAMY. Obviously, HSAMY purified with this method showed much higher purity (Figure S1C in the Supporting Information) than those prepared with the other two methods (Figure S1A,B in the Supporting Information). Method 3 also has very high reproducibility as demonstrated in Figure S2 in the Supporting Information. Since method 3 exhibited the highest effectiveness and reproducibility, it was used to prepare purified HSAMY from different sources for comprehensive top-down MS/MS analysis. High-Resolution MW Measurement of HSAMY. Highresolution MS spectra of purified HSAMY from various races and genders (Figure 2) exhibited the highly comparable profiles. The observed MW of 55 881.21 for the major peak does not match with the reported HSAMY in the UniprotKB/ SwissProt protein database. The calculated MW based on the HSAMY sequence in protein database is 57 767.00, which is larger than the experimental MW by 1885.79. It is reasonable to hypothesize that sequence truncation has occurred in this

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RESULTS AND DISCUSSION Rapid Purification of HSAMY. Since there are hundreds of proteins in saliva,36 it is important to isolate and purify HSAMY from other salivary proteins for top-down MS analysis. We have employed and compared three methods for the purification of α-amylase from human saliva based on size sieving (method 1), reversed-phase chromatography (method 2), and enzymesubstrate specific interaction between amylase and glycogen (method 3) (Figure 1). HSAMYs purified with the three methods were subsequently detected by high-resolution FTICR MS. The broadband FTICR MS analysis of the proteins purified from human saliva revealed HSAMY as the major protein, suggesting the effectiveness of these purification methods (Figure S1 in the Supporting Information). Among the three methods, method 1 is the simplest, only requiring a 50 K MWCO filter. Purified HSAMY can be obtained within 60 min. However, in most occasions, other small proteins (55 kDa) without the need of proteolytic digestion, providing effective and precise localization of the complex S−S positions.

(9) Kandra, L.; Gyemant, G. Carbohydr. Res. 2000, 329, 579−585. (10) Ragunath, C.; Sundar, K.; Ramasubbu, N. Protein Express. Purif. 2002, 24, 202−211. (11) Hirtz, C.; Chevalier, F.; Centeno, D.; Rofidal, V.; Egea, J. C.; Rossignol, M.; Sommerer, N.; de Periee, D. D. Proteomics 2005, 5, 4597−4607. (12) Yao, Y.; Berg, E. A.; Costello, C. E.; Troxler, R. F.; Oppenheim, F. G. J. Biol. Chem. 2003, 278, 5300−5308. (13) Kelleher, N. L.; Lin, H. Y.; Valaskovic, G. A.; Aaserud, D. J.; Fridriksson, E. K.; McLafferty, F. W. J. Am. Chem. Soc. 1999, 121, 806−812. (14) Ge, Y.; Lawhorn, B. G.; ElNaggar, M.; Strauss, E.; Park, J. H.; Begley, T. P.; McLafferty, F. W. J. Am. Chem. Soc. 2002, 124, 672−678. (15) Ge, Y.; ElNaggar, M.; Sze, S. K.; Bin Oh, H.; Begley, T. P.; McLafferty, F. W.; Boshoff, H.; Barry, C. E. J. Am. Soc. Mass Spectrom. 2003, 14, 253−261. (16) Han, X. M.; Jin, M.; Breuker, K.; McLafferty, F. W. Science 2006, 314, 109−112. (17) Zabrouskov, V.; Ge, Y.; Schwartz, J.; Walker, J. W. Mol. Cell. Proteomics 2008, 7, 1838−1849. (18) Xie, Y. M.; Zhang, J.; Yin, S.; Loo, J. A. J. Am. Chem. Soc. 2006, 128, 14432−14433. (19) Ayaz-Guner, S.; Zhang, J.; Li, L.; Walker, J. W.; Ge, Y. Biochemistry 2009, 48, 8161−8170. (20) Ge, Y.; Rybakova, I. N.; Xu, Q.; Moss, R. L. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 12658−12663. (21) Solis, R. S.; Ge, Y.; Walker, J. W. J. Muscle Res. Cell Motil. 2008, 29, 203−212. (22) Jebanathirajah, J. A.; Pittman, J. L.; Thomson, B. A.; Budnik, B. A.; Kaur, P.; Rape, M.; Kirschner, M.; Costello, C. E.; O’Connor, P. B. J. Am. Soc. Mass Spectrom. 2005, 16, 1985−1999. (23) Zhang, J.; Guy, M. J.; Norman, H. S.; Chen, Y. C.; Xu, Q. G.; Dong, X. T.; Guner, H.; Wang, S. J.; Kohmoto, T.; Young, K. H.; Moss, R. L.; Ge, Y. J. Proteome Res. 2011, 10, 4054−4065. (24) Zhang, J.; Dong, X. T.; Hacker, T. A.; Ge, Y. J. Am. Soc. Mass Spectrom. 2010, 21, 940−948. (25) McLafferty, F. W.; Breuker, K.; Jin, M.; Han, X. M.; Infusini, G.; Jiang, H.; Kong, X. L.; Begley, T. P. FEBS J. 2007, 274, 6256−6268. (26) Tipton, J. D.; Tran, J. C.; Catherman, A. D.; Ahlf, D. R.; Durbin, K. R.; Kelleher, N. L. J. Biol. Chem. 2011, 286, 25451−25458. (27) Zhang, H.; Ge, Y. Circ. Cardiovasc. Genet. 2011, 4, 711. (28) Senko, M. W.; Speir, J. P.; McLafferty, F. W. Anal. Chem. 1994, 66, 2801−2808. (29) Zubarev, R. A.; Horn, D. M.; Fridriksson, E. K.; Kelleher, N. L.; Kruger, N. A.; Lewis, M. A.; Carpenter, B. K.; McLafferty, F. W. Anal. Chem. 2000, 72, 563−573. (30) Kuhn, P.; Xu, Q. G.; Cline, E.; Zhang, D.; Ge, Y.; Xu, W. Protein Sci. 2009, 18, 1272−1280. (31) Pesavento, J. J.; Mizzen, C. A.; Kelleher, N. L. Anal. Chem. 2006, 78, 4271−4280. (32) Whitelegge, J. P.; Zabrouskov, V.; Halgand, F.; Souda, P.; Bassiliana, S.; Yan, W.; Wolinsky, L.; Loo, J. A.; Wong, D. T. W.; Faull, K. F. Int. J. Mass Spectrom. 2007, 268, 190−197. (33) Halgand, F.; Zabrouskov, V.; Bassilian, S.; Souda, P.; Wong, D. T.; Loo, J. A.; Faull, K. F.; Whitelegge, J. P. J. Am. Soc. Mass Spectrom. 2010, 21, 868−877. (34) Ryan, C. M.; Souda, P.; Halgand, F.; Wong, D. T.; Loo, J. A.; Faull, K. F.; Whitelegge, J. P. J. Am. Soc. Mass Spectrom. 2010, 21, 908− 917. (35) Loyter, A. S., M. Biochim. Biophys. Acta 1962, 65, 200−206. (36) Ghafouri, B.; Tagesson, C.; Lindahl, M. Proteomics 2003, 3, 1003−1015. (37) Nakamura, Y.; Ogawa, M.; Nishide, T.; Emi, M.; Kosaki, G.; Himeno, S.; Matsubara, K. Gene 1984, 28, 263−270. (38) Sato, T.; Tsunasawa, S.; Nakamura, Y.; Emi, M.; Sakiyama, F.; Matsubara, K. Gene 1986, 50, 247−257. (39) Cline, D. J.; Redding, S. E.; Brohawn, S. G.; Psathas, J. N.; Schneider, J. P.; Thorpe, C. Biochemistry 2004, 43, 15195−15203.

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CONCLUSIONS We have utilized three methods to purify and separate HSAMY from other proteins in saliva, which was subsequently characterized using high-resolution FTICR MS and MS/MS. Method 3 based on amylase and glycogen interaction is the preferred method with the highest purity and reproducibility. HSAMY purified from various races and genders showed the same mass profiles, indicating that race and gender have no significant effect on amylase primary sequence. We have identified the truncation of the N-terminal 15 amino acids and formation of pyroglutamic acid at the new N-terminus. Importantly, we have precisely mapped 5 S−S bond positions in HSAMY by top-down MS in combination with partial reduction by TCEP. Taken together, our study demonstrates that top-down MS combined with affinity purification (based on enzyme-substrate interaction) and partial reduction is a powerful new method for rapid purification and complete characterization of proteins with complex, overlapping S−S positions.

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ASSOCIATED CONTENT

* Supporting Information S

Supplemental figures (Figures S1−S5) as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Address: Ying Ge, Ph.D., 1300 University Ave., SMI 130, Madison, WI, 53706. E-mail: [email protected]. Phone: 608-2639212. Fax: 608-265-5512. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Y.P. and X.C. contributed equally to this work. This project was funded by Kikkoman USA R&D Laboratory, Inc. We are grateful to Dr. Han Zhang, Matt Lawrence, Yi-chen Chen, Lisa Xu, and Huseyin Guner for the helpful discussion and technical assistance. We also would like to thank Wisconsin Partnership Funds for the establishment of the University of Wisconsin Human Proteomics Program Mass Spectrometry Facility.

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REFERENCES

(1) Edgar, W. M. Brit. Dent. J. 1992, 172, 305−312. (2) Spielmann, N.; Wong, D. T. Oral Dis. 2011, 17, 345−354. (3) Pfaffe, T.; Cooper-White, J.; Beyerlein, P.; Kostner, K.; Punyadeera, C. Clin. Chem. 2011, 57, 675−687. (4) Chen, Y. C.; Li, T. Y.; Tsai, M. F. Rapid Commun. Mass Spectrom. 2002, 16, 364−369. (5) Nishide, T.; Nakamura, Y.; Emi, M.; Yamamoto, T.; Ogawa, M.; Mori, T.; Matsubara, K. Gene 1986, 41, 299−304. (6) Ramasubbu, N.; Paloth, V.; Luo, Y.; Brayer, G. D.; Levine, M. J. Acta Crystallogr. D 1996, 52, 435−446. (7) Alhashimi, I.; Levine, M. J. Arch. Oral Biol. 1989, 34, 289−295. (8) Karn, R. C. Adv. Comp. Physiol. Biochem. 1978, 7, 1−103. 3345

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(40) Loo, J. A.; Edmonds, C. G.; Udseth, H. R.; Smith, R. D. Anal. Chem. 1990, 62, 693−698. (41) Lin, J.; Lin, Y. S.; Kuo, S. T.; Jiang, C. M.; Wu, M. C. J. Sci. Food Agric. 2009, 89, 574−578. (42) Ferey-Roux, G.; Perrier, J.; Forest, E.; Marchis-Mouren, G.; Puigserver, A.; Santimone, M. Biochim. Biophys. Acta 1998, 1388, 10− 20. (43) Wu, J.; Watson, J. T. Methods Mol. Biol. 2002, 194, 1−22. (44) Thevis, M.; Loo, R. R. O.; Loo, J. A. J. Am. Soc. Mass Spectrom. 2003, 14, 635−647. (45) Gorman, J. J.; Wallis, T. P.; Pitt, J. J. Mass Spectrom. Rev. 2002, 21, 183−216. (46) Narayan, M.; Welker, E.; Zhai, H. L.; Han, X. M.; Xu, G. Q.; McLafferty, F. W.; Scheraga, H. A. Nat. Biotechnol. 2008, 26, 427−429. (47) Tie, J. K.; Mutucumarana, V. P.; Straight, D. L.; Carrick, K. L.; Pope, R. M.; Stafford, D. W. J. Biol. Chem. 2003, 278, 45468−45475. (48) Krokhin, O. V.; Cheng, K.; Sousa, S. L.; Ens, W.; Standing, K. G.; Wilkins, J. A. Biochemistry 2003, 42, 12950−12959. (49) Pitt, J. J.; Da Silva, E.; Gorman, J. J. J. Biol. Chem. 2000, 275, 6469−6478. (50) Wallis, T. P.; Huang, C. Y.; Nimkar, S. B.; Young, P. R.; Gorman, J. J. J. Biol. Chem. 2004, 279, 20729−20741. (51) Wu, J.; Watson, J. T. Protein Sci. 1997, 6, 391−398. (52) Zhang, H.-M.; McLoughlin, S. M.; Frausto, S. D.; Tang, H.; Emmett, M. R.; Marshall, A. G. Anal. Chem. 2010, 82, 1450−1454. (53) Yen, T. Y.; Yan, H.; Macher, B. A. J. Mass Spectrom. 2002, 37, 15−30.

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