- Email: [email protected]
Chemical crosslinking and mass spectrometric identification of interaction sites within soluble aggregate of protein therapeutics
Journal of Pharmaceutical and Biomedical Analysis 73 (2013) 99–102
Contents lists available at SciVerse ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Chemical crosslinking and mass spectrometric identification of interaction sites within soluble aggregate of protein therapeutics Alan Zhao a , Gang Hao b , Jane Gu a,∗ a b
Massoverz Technologies, Albany, NY 12203, USA Weill Medical College of Cornell University, New York, NY 10021, USA
a r t i c l e
i n f o
Article history: Received 27 January 2012 Received in revised form 30 April 2012 Accepted 6 May 2012 Available online 22 May 2012 Keywords: Aggregation Biotechnology Antibody Crosslinking Mass spectrometry
a b s t r a c t Protein aggregation presents a major challenge to bioengineering. In the present study, chemical crosslinking and mass spectrometry are employed to probe the interaction amongst soluble oligomer formed by Fc molecule of monoclonal antibody. Aggregation was induced by thermal stress at 42 ◦ C for 6 h under physiological pH. Contacting residues on adjacent molecules were captured with bifunctional crosslinker BS3, followed by mass spectrometric identification of the crosslinked sites. The approach provides site-specific information regarding the binding interface that would have broad application in the field. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Aggregation of soluble proteins into fibrillar species is a ubiquitous phenomenon occurring in both in vivo and in purified protein solutions [1,2]. Protein aggregation is closely associated with many neurodegenerative diseases [3,4], whereas coagulation of purified biotherapeutics presents a major problem for biotechnology manufacturing [5,6]. The mechanism for association remained unclear despite intensive research. Historically it has been viewed as nonspecific gathering of misfolded molecules. However, accumulating data suggested that specific intermolecular interactions underlie the process [7–9]. According to this view, aggregation proceeds via two sequential steps. In the first step, small soluble oligomer was formed, which served as nucleation for the second step, in which larger insoluble species developed and eventually precipitated [10,11]. Hence, defining the precise interaction sites within the initial oligomerization phase would provide key insight into the process. While a number of analytical methods are available for the characterization of protein aggregate, such as size-exclusion chromatography, light-scattering spectroscopy and differential scanning calorimetry, none of these conventional methods provide sub-molecular resolution that can pinpoint the interaction loci. Hydrogen deuterium exchange coupled to mass spectrometry (HDX-MS) is a powerful tool for gaining detailed
∗ Corresponding author. E-mail address: [email protected] (J. Gu). 0731-7085/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jpba.2012.05.006
structure information and has been applied to amyloid protein [12,13] and to monoclonal antibody recently [14,15]. The technique is generally considered as time-consuming and requires specialized equipment. In the present study, we seek to adopt a simpler approach that entails chemical crosslinking and mass spectrometry. While the technique has been extensively applied to the study of protein–protein interactions, for reviews, see [16,17], few study concerned protein aggregate. The applicable scope of the method was extended to soluble oligomer formed by the Fc region of monoclonal antibody. 2. Experimental Fc molecule was expressed in Chinese Hamster Ovary cells and purified on Protein A and size-exclusion chromatography. 50 g of Fc at a concentration of 10 mg/ml in phosphate buffered saline (PBS, pH 7.4) was stressed by incubating at 42 ◦ C for 6 h with gentle agitation. Following the incubation, the sample was filtered through 0.22 m spin filter to remove insoluble aggregates. Size exclusion chromatography was performed on a 1100 series HPLC (Agilent, Palo Alto, CA). 5 g of Fc was injected to a TSK SuperSW3000 size exclusion column (Tosoh Bioscience, King of Prussia, PA) and the protein was monitored by UV absorbance at 280 nM. The control sample was stored at 4 ◦ C. Lysine specific crosslinker, bis(sulfosuccinimidyl) suberate BS3 (Pierce, Rockland, IL), was added to the sample at final concentration of 2 mM and the crosslinking reaction was carried out at 37 ◦ C for 30 min. The crosslinker was removed by methanol precipitation of the
100
A. Zhao et al. / Journal of Pharmaceutical and Biomedical Analysis 73 (2013) 99–102
Fig. 1. Identification of interaction sites on soluble aggregate of Fc molecule by chemical crosslinking and mass spectrometry. Fc molecule was stressed at 42 ◦ C for 6 h in PBS buffer to induce aggregation. Panel A: size exclusion chromatogram (SEC) of the control and stressed Fc samples. Panel B: total ion chromatogram for the tryptic digest of the crosslinked Fc. The proteins were incubated with the bifunctional amine-specific crosslinker BS3 and the tryptic digest was analyzed by LC–MS/MS to identify crosslinked products. Inset: extracted chromatograms of the intermolecular crosslinked species. A peak eluting around 40 min (denoted with an arrow sign) contained a crossslinked species that was uniquely presented in the aggregated sample. Panel C: CID fragmentation spectrum of the inter-molecular crosslinked peptides containing CH2 region peptide TISK124 AK and CH3 region peptide LTVDK200 SR. Panel D: proposed model for the aggregation of three Fc molecules mediated by the interaction between the CH2 and CH3 region peptides. The arrows point to the identified crosslinking sites.
protein. The precipitated protein was resuspended in 1 M guanidine hydrochloride and 10 mM ammonium bicarbonate, reduced by 10 mM dithiothreitol and alkylated by 30 mM iodoacetamide. Sequencing grade trypsin (Promega, Madison, WI) was added and the digestion was carried out at 37 ◦ C for 4 h. 5 g of the digest was injected to a LC–MS system comprised of an Agilent 1100 HPLC
coupled via electrospray to a Micro Q-TOF mass spectrometer (Waters, Milford, MA). The peptides were separated on a 1.0 mm × 50 mm ZORBAX C18 column (Agilent). A linear gradient of 10–40% solvent B (0.1% formic acid in acetonitrile) was employed to elute the peptides off the column. Solvent A was 0.1% formic acid in water. The cone voltage was 35 V and the capillary voltage
A. Zhao et al. / Journal of Pharmaceutical and Biomedical Analysis 73 (2013) 99–102
was 4500 V. Data dependant MS/MS acquisition was performed in which each MS scan was followed by four data-dependant MS/MS analysis using a charge and m/z dependant collision energy profile. The mass spectrum peak list was searched against a theoretical list of intermolecular crosslinked peptides masses using a program written in Perl. 3. Results and discussion Fc (fragment crystallizable) of monoclonal antibody was selected as a model system to evaluate the feasibility of the method, because of the modest size more amenable to bottom-up LC–MS analysis. The sample was subjected to mild thermal stress to induce the formation of aggregated species. Insoluble aggregates were removed by passing through a 0.22 m filter and the soluble aggregate was profiled on size exclusion chromatography. As shown in Fig. 1A, the stress treatment lead to significant aggregation, which eluted before the native peak as a broad peak, whereas no aggregate species was observed in the untreated sample. While intermolecular disulfide was often involved in aggregation formation, both SDS and guanidine completely disrupted the formation of the aggregate species, revealing the noncovalent nature of the species (data not shown). The size of the aggregate peak was estimated to be 3–5 Fc dimer molecules based on referencing size exclusion protein standards. It was previously shown that oligomerization could serve as the seed for subsequent formation of larger complex [10] and thus the identification of the interaction loci is of critical importance in elucidation of the aggregation pathway. The samples were labeled with the homo-bifunctional aminespecific crosslinker, bis-sulfosuccinimidyl suberate (BS3) to capture lysine pairs located in close proximity [18]. Following removal of the excessive crosslinker by protein precipitation and buffer exchange, the protein was subjected to tryptic digestion followed by LC–MS analysis to identify crosslinked species. The total ion chromatograms (TIC) for the control and stressed fractions were very similar (Fig. 1B), suggesting that no significant conformational changes occurred during the aggregation process. Automatic searching of the precursor peak list against the theoretical intermolecular crosslinked masses returned 11 crosslinked species from the control sample. For the stressed sample, the same set of 11 peptides were found plus an additional triply charged species at m/z of 534.97, consistent with theoretical mass for the crosslinked peptide TISK124 AK and LTVDK200 SR. Extracted ion chromatogram (EIC) confirmed the absence of the ion in the native sample (Fig. 1B inset), indicating the species was formed between two different Fc molecules. Consistently, close inspection of the TIC revealed that the signal intensity around the elution time window for the crosslinked species slightly increased in the aggregated sample (denoted with an arrow in Fig. 1B). The sequence of the crosslinked peptide was verified by MS/MS analysis (Fig. 1C), which yielded multiple y ions from both peptides. The predominant fragmentation product is the isobartic y4 ion from TISK124 AK or y5 ion from LTVDK200 SR, generated upon cleavage of the two N-terminal residues from either peptide. The abundance of these ions might be attributed to additional fragmentation pathway, namely the diketopiperazine-yN-2 pathway [19]. The theoretical and experimental m/z values for the precursor and product ions are listed in Table 1. Inspection of the crystal structure of the Fc domain revealed that Lys124 and Lys200 are located in the CH2 and CH3 domain, respectively, facing opposite direction into the solvent [20]. Based on the observed crosslinking pattern, a model for the aggregation process is proposed in Fig. 1D, illustrating the association between the CH2 and CH3 domains from three Fc molecules. The side chains surrounding the two lysine residues are mainly hydrophilic, suggesting that the interaction was mediated by ionic interactions,
101
Table 1 m/z values in CID spectrum of the crosslinked peptide. Ion
Theoretical
Experimental
precursor+++ y2 y3++ y4++ y5/y4␣++ y5␣++ y6++
534.986 262.151 587.861 645.375 694.909 751.451 745.433
534.978 262.150 587.857 645.376 694.905 751.448 745.429
e.g. hydrogen bonding. This is in contrast to the conventional theory that hydrophobic surface exposure upon denaturation cause aggregation. Notwithstandingly, Lys124 is relatively solvent inac˚ cessible and also engages in a strong salt bridge with Glu216 (2.4 A), which suggested that at least partial structural shift was required to bring these two regions to proximity. Intriguingly, recent study of an aggregated monoclonal antibody by hydrogen deuterium exchange (HDX) mass spectrometry [17] revealed several sites on the Fab region as the main aggregation interface that underwent extensive conformational changes. The discrepancy with our data could be reconciled by the difference in the target molecule (IgG vs. Fc), thermal treatment (65 ◦ C vs. 42 ◦ C) and protein concentration (0.5 mg/ml vs. 10 mg/ml). Conceivably, higher temperature opened up new channels for more stable interaction as observed in the HDX study. Works are undergoing to construct mutant Fc molecules with polar to neutral substitutions surrounding Lys124 and Lys200 in order to verify the proposed model. 4. Conclusion Collectively, the results demonstrate that chemical crosslinking combined with mass spectrometry offer a viable approach for characterization of protein aggregation. More complex system could be studied using similar methodology to detail the mode of intermolecular association, which would greatly improve the current understanding of this important biophysical process. References [1] A.M. Morris, M.A. Watzky, R.G. Finke, Protein aggregation kinetics, mechanism, and curve-fitting: a review of the literature, Biochim. Biophys. Acta 1794 (2009) 375–397. [2] M. Stefani, Protein misfolding and aggregation: new examples in medicine and biology of the dark side of the protein world, Biochim. Biophys. Acta 1739 (2004) 5–25. [3] C.A. Ross, M.A. Poirier, Protein aggregation and neurodegenerative disease, Nat. Med. 10 (Suppl.) (2004) 10–17. [4] P.T. Lansbury, H.A. Lashuel, A century-old debate on protein aggregation and neurodegeneration enters the clinic, Nature 443 (2006) 774–779. [5] M. Vázquez-Rey, D.A. Lang, Aggregates in monoclonal antibody manufacturing processes, Biotechnol. Bioeng. 108 (2011) 1494–1508. [6] W. Wang, S. Nema, D. Teagarden, Protein aggregation—pathways and influencing factors, Int. J. Pharm. 390 (2010) 89–99. [7] R.S. Rajan, M.E. Illing, N.F. Bence, R.R. Kopito, Specificity in intracellular protein aggregation and inclusion body formation, Proc. Natl. Acad. Sci. U.S.A. 98 (2001) 13060–13065. [8] R. Wetzel, For protein misassembly, it’s the I decade, Cell 86 (1996) 699– 702. [9] M.A. Speed, D.I. Wang, J. King, Specific aggregation of partially folded polypeptide chains: the molecular basis of inclusion body composition, Nat. Biotechnol. 14 (1996) 1283–1287. [10] S. Auer, C.M. Dobson, M. Vendruscolo, A. Maritan, Self-templated nucleation in peptide and protein aggregation, Phys. Rev. Lett. 101 (2008) 258101– 258105. [11] T.R. Serio, A.G. Cashikar, A.S. Kowal, G.J. Sawicki, J.J. Moslehi, L. Serpell, M.F. Arnsdorf, S.L. Lindquist, Nucleated conformational conversion and the replication of conformational information by a prion determinant, Science 289 (2000) 1317–1321. [12] Z.P. Yao, P. Tito, C.V. Robinson, Site-specific hydrogen exchange of proteins: insights into the structures of amyloidogenic intermediates, Methods Enzymol. 402 (2005) 389–402.
102
A. Zhao et al. / Journal of Pharmaceutical and Biomedical Analysis 73 (2013) 99–102
[13] A.E. Ashcroft, Mass spectrometry and the amyloid problem—how far can we go in the gas phase? J. Am. Soc. Mass Spectrom. 21 (2010) 1087–1096. [14] A. Zhang, W. Qi, S.K. Singh, E.J. Fernandez, A new approach to explore the impact of freeze–thaw cycling on protein structure: hydrogen/deuterium exchange mass spectrometry (HX-MS), Pharm. Res. 28 (2011) 1179–1193. [15] A. Zhang, S.K. Singh, M.R. Shirts, S. Kumar, E.J. Fernandez, Distinct aggregation mechanisms of monoclonal antibody under thermal and freeze–thaw stresses revealed by hydrogen exchange, Pharm. Res. 29 (2012) 236–250. [16] A. Sinz, Chemical cross-linking and mass spectrometry to map threedimensional protein structures and protein–protein interactions, Mass Spectrom. Rev. 25 (2006) 663–682.
[17] E.V. Petrotchenko, C.H. Borchers, Crosslinking combined with mass spectrometry for structural proteomics, Mass Spectrom. Rev. 29 (2010) 862–876. [18] M.D. Partis, D.G. Griffiths, G.C. Roberts, R.B. Beechey, Cross-linking of protein by co-maleimido alkanoyl N-hydroxysuccinimido esters, J. Protein Chem. 2 (1983) 263–277. [19] B. Paizs, S. Suhai, Fragmentation pathways of protonated peptides, Mass Spectrom. Rev. 24 (2005) 508–548. [20] E.O. Saphire, P.W. Parren, R. Pantophlet, M.B. Zwick, G.M. Morris, P.M. Rudd, R.A. Dwek, R.L. Stanfield, D.R. Burton, I.A. Wilson, Crystal structure of a neutralizing human IGG against HIV-1: a template for vaccine design, Science 293 (2001) 1155–1159.
Contents lists available at SciVerse ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Chemical crosslinking and mass spectrometric identification of interaction sites within soluble aggregate of protein therapeutics Alan Zhao a , Gang Hao b , Jane Gu a,∗ a b
Massoverz Technologies, Albany, NY 12203, USA Weill Medical College of Cornell University, New York, NY 10021, USA
a r t i c l e
i n f o
Article history: Received 27 January 2012 Received in revised form 30 April 2012 Accepted 6 May 2012 Available online 22 May 2012 Keywords: Aggregation Biotechnology Antibody Crosslinking Mass spectrometry
a b s t r a c t Protein aggregation presents a major challenge to bioengineering. In the present study, chemical crosslinking and mass spectrometry are employed to probe the interaction amongst soluble oligomer formed by Fc molecule of monoclonal antibody. Aggregation was induced by thermal stress at 42 ◦ C for 6 h under physiological pH. Contacting residues on adjacent molecules were captured with bifunctional crosslinker BS3, followed by mass spectrometric identification of the crosslinked sites. The approach provides site-specific information regarding the binding interface that would have broad application in the field. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Aggregation of soluble proteins into fibrillar species is a ubiquitous phenomenon occurring in both in vivo and in purified protein solutions [1,2]. Protein aggregation is closely associated with many neurodegenerative diseases [3,4], whereas coagulation of purified biotherapeutics presents a major problem for biotechnology manufacturing [5,6]. The mechanism for association remained unclear despite intensive research. Historically it has been viewed as nonspecific gathering of misfolded molecules. However, accumulating data suggested that specific intermolecular interactions underlie the process [7–9]. According to this view, aggregation proceeds via two sequential steps. In the first step, small soluble oligomer was formed, which served as nucleation for the second step, in which larger insoluble species developed and eventually precipitated [10,11]. Hence, defining the precise interaction sites within the initial oligomerization phase would provide key insight into the process. While a number of analytical methods are available for the characterization of protein aggregate, such as size-exclusion chromatography, light-scattering spectroscopy and differential scanning calorimetry, none of these conventional methods provide sub-molecular resolution that can pinpoint the interaction loci. Hydrogen deuterium exchange coupled to mass spectrometry (HDX-MS) is a powerful tool for gaining detailed
∗ Corresponding author. E-mail address: [email protected] (J. Gu). 0731-7085/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jpba.2012.05.006
structure information and has been applied to amyloid protein [12,13] and to monoclonal antibody recently [14,15]. The technique is generally considered as time-consuming and requires specialized equipment. In the present study, we seek to adopt a simpler approach that entails chemical crosslinking and mass spectrometry. While the technique has been extensively applied to the study of protein–protein interactions, for reviews, see [16,17], few study concerned protein aggregate. The applicable scope of the method was extended to soluble oligomer formed by the Fc region of monoclonal antibody. 2. Experimental Fc molecule was expressed in Chinese Hamster Ovary cells and purified on Protein A and size-exclusion chromatography. 50 g of Fc at a concentration of 10 mg/ml in phosphate buffered saline (PBS, pH 7.4) was stressed by incubating at 42 ◦ C for 6 h with gentle agitation. Following the incubation, the sample was filtered through 0.22 m spin filter to remove insoluble aggregates. Size exclusion chromatography was performed on a 1100 series HPLC (Agilent, Palo Alto, CA). 5 g of Fc was injected to a TSK SuperSW3000 size exclusion column (Tosoh Bioscience, King of Prussia, PA) and the protein was monitored by UV absorbance at 280 nM. The control sample was stored at 4 ◦ C. Lysine specific crosslinker, bis(sulfosuccinimidyl) suberate BS3 (Pierce, Rockland, IL), was added to the sample at final concentration of 2 mM and the crosslinking reaction was carried out at 37 ◦ C for 30 min. The crosslinker was removed by methanol precipitation of the
100
A. Zhao et al. / Journal of Pharmaceutical and Biomedical Analysis 73 (2013) 99–102
Fig. 1. Identification of interaction sites on soluble aggregate of Fc molecule by chemical crosslinking and mass spectrometry. Fc molecule was stressed at 42 ◦ C for 6 h in PBS buffer to induce aggregation. Panel A: size exclusion chromatogram (SEC) of the control and stressed Fc samples. Panel B: total ion chromatogram for the tryptic digest of the crosslinked Fc. The proteins were incubated with the bifunctional amine-specific crosslinker BS3 and the tryptic digest was analyzed by LC–MS/MS to identify crosslinked products. Inset: extracted chromatograms of the intermolecular crosslinked species. A peak eluting around 40 min (denoted with an arrow sign) contained a crossslinked species that was uniquely presented in the aggregated sample. Panel C: CID fragmentation spectrum of the inter-molecular crosslinked peptides containing CH2 region peptide TISK124 AK and CH3 region peptide LTVDK200 SR. Panel D: proposed model for the aggregation of three Fc molecules mediated by the interaction between the CH2 and CH3 region peptides. The arrows point to the identified crosslinking sites.
protein. The precipitated protein was resuspended in 1 M guanidine hydrochloride and 10 mM ammonium bicarbonate, reduced by 10 mM dithiothreitol and alkylated by 30 mM iodoacetamide. Sequencing grade trypsin (Promega, Madison, WI) was added and the digestion was carried out at 37 ◦ C for 4 h. 5 g of the digest was injected to a LC–MS system comprised of an Agilent 1100 HPLC
coupled via electrospray to a Micro Q-TOF mass spectrometer (Waters, Milford, MA). The peptides were separated on a 1.0 mm × 50 mm ZORBAX C18 column (Agilent). A linear gradient of 10–40% solvent B (0.1% formic acid in acetonitrile) was employed to elute the peptides off the column. Solvent A was 0.1% formic acid in water. The cone voltage was 35 V and the capillary voltage
A. Zhao et al. / Journal of Pharmaceutical and Biomedical Analysis 73 (2013) 99–102
was 4500 V. Data dependant MS/MS acquisition was performed in which each MS scan was followed by four data-dependant MS/MS analysis using a charge and m/z dependant collision energy profile. The mass spectrum peak list was searched against a theoretical list of intermolecular crosslinked peptides masses using a program written in Perl. 3. Results and discussion Fc (fragment crystallizable) of monoclonal antibody was selected as a model system to evaluate the feasibility of the method, because of the modest size more amenable to bottom-up LC–MS analysis. The sample was subjected to mild thermal stress to induce the formation of aggregated species. Insoluble aggregates were removed by passing through a 0.22 m filter and the soluble aggregate was profiled on size exclusion chromatography. As shown in Fig. 1A, the stress treatment lead to significant aggregation, which eluted before the native peak as a broad peak, whereas no aggregate species was observed in the untreated sample. While intermolecular disulfide was often involved in aggregation formation, both SDS and guanidine completely disrupted the formation of the aggregate species, revealing the noncovalent nature of the species (data not shown). The size of the aggregate peak was estimated to be 3–5 Fc dimer molecules based on referencing size exclusion protein standards. It was previously shown that oligomerization could serve as the seed for subsequent formation of larger complex [10] and thus the identification of the interaction loci is of critical importance in elucidation of the aggregation pathway. The samples were labeled with the homo-bifunctional aminespecific crosslinker, bis-sulfosuccinimidyl suberate (BS3) to capture lysine pairs located in close proximity [18]. Following removal of the excessive crosslinker by protein precipitation and buffer exchange, the protein was subjected to tryptic digestion followed by LC–MS analysis to identify crosslinked species. The total ion chromatograms (TIC) for the control and stressed fractions were very similar (Fig. 1B), suggesting that no significant conformational changes occurred during the aggregation process. Automatic searching of the precursor peak list against the theoretical intermolecular crosslinked masses returned 11 crosslinked species from the control sample. For the stressed sample, the same set of 11 peptides were found plus an additional triply charged species at m/z of 534.97, consistent with theoretical mass for the crosslinked peptide TISK124 AK and LTVDK200 SR. Extracted ion chromatogram (EIC) confirmed the absence of the ion in the native sample (Fig. 1B inset), indicating the species was formed between two different Fc molecules. Consistently, close inspection of the TIC revealed that the signal intensity around the elution time window for the crosslinked species slightly increased in the aggregated sample (denoted with an arrow in Fig. 1B). The sequence of the crosslinked peptide was verified by MS/MS analysis (Fig. 1C), which yielded multiple y ions from both peptides. The predominant fragmentation product is the isobartic y4 ion from TISK124 AK or y5 ion from LTVDK200 SR, generated upon cleavage of the two N-terminal residues from either peptide. The abundance of these ions might be attributed to additional fragmentation pathway, namely the diketopiperazine-yN-2 pathway [19]. The theoretical and experimental m/z values for the precursor and product ions are listed in Table 1. Inspection of the crystal structure of the Fc domain revealed that Lys124 and Lys200 are located in the CH2 and CH3 domain, respectively, facing opposite direction into the solvent [20]. Based on the observed crosslinking pattern, a model for the aggregation process is proposed in Fig. 1D, illustrating the association between the CH2 and CH3 domains from three Fc molecules. The side chains surrounding the two lysine residues are mainly hydrophilic, suggesting that the interaction was mediated by ionic interactions,
101
Table 1 m/z values in CID spectrum of the crosslinked peptide. Ion
Theoretical
Experimental
precursor+++ y2 y3++ y4++ y5/y4␣++ y5␣++ y6++
534.986 262.151 587.861 645.375 694.909 751.451 745.433
534.978 262.150 587.857 645.376 694.905 751.448 745.429
e.g. hydrogen bonding. This is in contrast to the conventional theory that hydrophobic surface exposure upon denaturation cause aggregation. Notwithstandingly, Lys124 is relatively solvent inac˚ cessible and also engages in a strong salt bridge with Glu216 (2.4 A), which suggested that at least partial structural shift was required to bring these two regions to proximity. Intriguingly, recent study of an aggregated monoclonal antibody by hydrogen deuterium exchange (HDX) mass spectrometry [17] revealed several sites on the Fab region as the main aggregation interface that underwent extensive conformational changes. The discrepancy with our data could be reconciled by the difference in the target molecule (IgG vs. Fc), thermal treatment (65 ◦ C vs. 42 ◦ C) and protein concentration (0.5 mg/ml vs. 10 mg/ml). Conceivably, higher temperature opened up new channels for more stable interaction as observed in the HDX study. Works are undergoing to construct mutant Fc molecules with polar to neutral substitutions surrounding Lys124 and Lys200 in order to verify the proposed model. 4. Conclusion Collectively, the results demonstrate that chemical crosslinking combined with mass spectrometry offer a viable approach for characterization of protein aggregation. More complex system could be studied using similar methodology to detail the mode of intermolecular association, which would greatly improve the current understanding of this important biophysical process. References [1] A.M. Morris, M.A. Watzky, R.G. Finke, Protein aggregation kinetics, mechanism, and curve-fitting: a review of the literature, Biochim. Biophys. Acta 1794 (2009) 375–397. [2] M. Stefani, Protein misfolding and aggregation: new examples in medicine and biology of the dark side of the protein world, Biochim. Biophys. Acta 1739 (2004) 5–25. [3] C.A. Ross, M.A. Poirier, Protein aggregation and neurodegenerative disease, Nat. Med. 10 (Suppl.) (2004) 10–17. [4] P.T. Lansbury, H.A. Lashuel, A century-old debate on protein aggregation and neurodegeneration enters the clinic, Nature 443 (2006) 774–779. [5] M. Vázquez-Rey, D.A. Lang, Aggregates in monoclonal antibody manufacturing processes, Biotechnol. Bioeng. 108 (2011) 1494–1508. [6] W. Wang, S. Nema, D. Teagarden, Protein aggregation—pathways and influencing factors, Int. J. Pharm. 390 (2010) 89–99. [7] R.S. Rajan, M.E. Illing, N.F. Bence, R.R. Kopito, Specificity in intracellular protein aggregation and inclusion body formation, Proc. Natl. Acad. Sci. U.S.A. 98 (2001) 13060–13065. [8] R. Wetzel, For protein misassembly, it’s the I decade, Cell 86 (1996) 699– 702. [9] M.A. Speed, D.I. Wang, J. King, Specific aggregation of partially folded polypeptide chains: the molecular basis of inclusion body composition, Nat. Biotechnol. 14 (1996) 1283–1287. [10] S. Auer, C.M. Dobson, M. Vendruscolo, A. Maritan, Self-templated nucleation in peptide and protein aggregation, Phys. Rev. Lett. 101 (2008) 258101– 258105. [11] T.R. Serio, A.G. Cashikar, A.S. Kowal, G.J. Sawicki, J.J. Moslehi, L. Serpell, M.F. Arnsdorf, S.L. Lindquist, Nucleated conformational conversion and the replication of conformational information by a prion determinant, Science 289 (2000) 1317–1321. [12] Z.P. Yao, P. Tito, C.V. Robinson, Site-specific hydrogen exchange of proteins: insights into the structures of amyloidogenic intermediates, Methods Enzymol. 402 (2005) 389–402.
102
A. Zhao et al. / Journal of Pharmaceutical and Biomedical Analysis 73 (2013) 99–102
[13] A.E. Ashcroft, Mass spectrometry and the amyloid problem—how far can we go in the gas phase? J. Am. Soc. Mass Spectrom. 21 (2010) 1087–1096. [14] A. Zhang, W. Qi, S.K. Singh, E.J. Fernandez, A new approach to explore the impact of freeze–thaw cycling on protein structure: hydrogen/deuterium exchange mass spectrometry (HX-MS), Pharm. Res. 28 (2011) 1179–1193. [15] A. Zhang, S.K. Singh, M.R. Shirts, S. Kumar, E.J. Fernandez, Distinct aggregation mechanisms of monoclonal antibody under thermal and freeze–thaw stresses revealed by hydrogen exchange, Pharm. Res. 29 (2012) 236–250. [16] A. Sinz, Chemical cross-linking and mass spectrometry to map threedimensional protein structures and protein–protein interactions, Mass Spectrom. Rev. 25 (2006) 663–682.
[17] E.V. Petrotchenko, C.H. Borchers, Crosslinking combined with mass spectrometry for structural proteomics, Mass Spectrom. Rev. 29 (2010) 862–876. [18] M.D. Partis, D.G. Griffiths, G.C. Roberts, R.B. Beechey, Cross-linking of protein by co-maleimido alkanoyl N-hydroxysuccinimido esters, J. Protein Chem. 2 (1983) 263–277. [19] B. Paizs, S. Suhai, Fragmentation pathways of protonated peptides, Mass Spectrom. Rev. 24 (2005) 508–548. [20] E.O. Saphire, P.W. Parren, R. Pantophlet, M.B. Zwick, G.M. Morris, P.M. Rudd, R.A. Dwek, R.L. Stanfield, D.R. Burton, I.A. Wilson, Crystal structure of a neutralizing human IGG against HIV-1: a template for vaccine design, Science 293 (2001) 1155–1159.