Heat-induced Rearrangement of Disulfide Bond of Lactoglobulin

Publication Date (Web): August 24, 2018 ... While both mass spectrometry-based bottom-up and top-down proteomic are widely used in identification of ...
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Heat-induced Rearrangement of Disulfide Bond of Lactoglobulin Characterized by Multiply Charged MALDI-TOF/TOF Mass Spectrometry Lingpeng Zhan, Yu Liu, Xiaobo Xie, Caiqiao Xiong, and Zongxiu Nie Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b02563 • Publication Date (Web): 24 Aug 2018 Downloaded from http://pubs.acs.org on August 27, 2018

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Analytical Chemistry

Heat-induced Rearrangement of Disulfide Bond of Lactoglobulin Characterized by Multiply Charged MALDI-TOF/TOF Mass Spectrometry Lingpeng Zhan†‡¶, Yu Liu‡¶, Xiaobo Xie†‡, Caiqiao Xiong†, Zongxiu Nie*†§ †Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China ‡University of Chinese Academy of Sciences, Beijing 100049, China §National Center for Mass Spectrometry in Beijing, Beijing 100190, China ABSTRACT: Disulfide bonds are an important post-translational modification of proteins and play a significant role in stabilizing protein structure. While both mass spectrometry-based bottom-up and top-down proteomic are widely used in identification of disulfide linkages, top-down approach can avoid potential information loss of disulfide linkage occurring in the bottom-up analysis. In the present work, we applied matrix-assisted laser desorption/ionization tandem Time-of-Flight (MALDI-TOF/TOF) mass spectrometry to investigate the heat-induced disulfide rearrangement of β-lactoglobulin (β-LG). Since β-LG (18kDa) is too large for common TOF/TOF analysis, we use 2-nitrophloroglucinol (2-NPG) as matrix to generate multiply charged proteins by MALDI. Fragmentation of doubly charged protein ions yields characteristic triplet peaks of disulfide bond. We found that the characteristic fragments derived from heterolytic cleavage of disulfide bond decreased sharply when the incubation temperature of β-LG solution reached the critical point 75ºC. These results indicate that the disulfide linkage between C160 and C66 has been broken during the heating process, and probably new disulfide formed. In conclusion, our work highlights the analytical value of multiply charged MALDI-TOF/TOF method in identification of larger proteins (>12kDa) and disulfide-containing proteins.

The disulfide linkages between two cysteine residues play crucial roles in stabilizing protein structure and maintaining its functionality1. The accurate identification and assignment of the disulfide connectivity are essential in characterizing protein structure. With its advantages of low sample consumption, high speed and high sample throughput, mass spectrometry (MS) has been demonstrated as an important tool in identification of disulfide bond. MS-based bottom-up and top-down proteomics are two main approaches for identification of protein and its post-translational modifications (PTMs), including disulfide bond. The most widely used approach to assign disulfide linkage is to compare the reduced and unreduced peptide maps after digestion using the conventional bottom-up methods. However, this process may introduce artificial modifications of peptides, such as disulfide scrambling2, deamidation or unspecific cleavage. In contrast, top-down proteomics, which fragment the intact protein ions directly to avoid the time-consuming sample handling steps, loss of PTMs and scrambling of disulfide in bottom-up analysis, have been developed to discriminate proteoforms in recent years3-9. However, cleaving disulfide bonds at the protein level is still an issue. Only a few studies reported that collision-induced dissociation (CID), the most widely used MS/MS approach, could break down protein backbone and disulfide bond concurrently.7 The electron-based tandem MS methods, i.e. electron capture dissociation (ECD) and electron transfer dissociation (ETD), were reported to preferentially cleave disulfide bonds.10 However, full preservation of disulfide bonds and non-preferential disulfide cleavage in several multiple disul-

fide-containing proteins have been observed upon ECD, suggesting the unpredictable nature of disulfide cleavages via ECD and ETD.6 Meanwhile, complete and partial reduction coupled with top-down analyses have been proposed to localize the disulfide bonds.11 MALDI-based tandem mass spectrometry has also been demonstrated as a powerful tool in characterizing disulfidecontaining peptide.12-16 The MALDI-in source decay (ISD) approach have been used to screen disulfide-linked peptide, based on detecting and fragmenting peptides released by ISD of disulfide bond.14 Furthermore, MALDI-TOF/TOF analysis of disulfide-bonded peptides was found to produce distinctive triplet peaks,12,17 which correspond to the homolytic and heterolytic cleavages of disulfide bonds. Moreover, the intrachain disulfide bonds and backbone bonds of a ~8kDa protein β-Stx2 were cleaved concurrently by MALDI-TOF/TOF, producing abundant triplet peaks separated by ~m/z 33.18 Nevertheless, the main drawback of MALDI top-down approach is that it works in a relatively small range of molecular mass.19 Though this approach has been applied to rapid identification of protein biomarker20 and microorganisms21 for many years, the application was only limited to small proteins (mass12kDa) by fragmenting multiply charged ions. Moreover, the heatinduced disulfide rearrangement of β-lactoglobulin (β-LG) has been investigated in detail. Lactoglobulin is a 18kDa protein with 162 residues, including five cysteine residues and two disulfide bonds. The relative high mass of β-LG makes it beyond the reach of common MALDI-TOF/TOF analysis. Here, we attempted to fragment multiply charged protein ions by using 2-nitrophloroglucinol (2-NPG) as matrix.22 Fragmentation of doubly charged β-LG ions yielded abundant product ions, where the concurrent cleavages of disulfide bond and protein backbone were found. Interestingly, the asymmetric cleavage products of disulfide bonds (yn-SH and yn+SH) decreased suddenly when the incubation temperature of protein solution reached a critical point, 75ºC. Moreover, it is worth noting that the charge state distributions (CSDs) and ion mobility of β-LG from native mass spectrometry were similar at various incubation temperatures. Experimental Chemicals. Equine myoglobin and bovine β-lactoglobulin B were purchased from Sigma Aldrich (St. Louis, MO, U.S.A.). 2-Nitrophloroglucinol was purchased from Alfa Aesar. Myoglobin was prepared in water at 20µM, lactoglobulin was solubilized in water at 200µg/mL. The matrix 2-NPG was prepared at 20mg/mL (ACN:H2O:TFA=49.9:49.9:0.2). Water was prepared using a Milli-Q water purification system from Millipore (Milford, MA, U.S.A.). Acetonitrile (HPLC grade) were purchased from Thermo Fisher. The pH of the protein solution was measured at 7.18. MALDI-TOF/TOF. The mass spectra were acquired with reflectron geometry MALDI-TOF/TOF (Ultraflextreme, Bruker Daltonics, Germany) equipped with a 355nm and 2kHz solid state Nd:YAG Smart Beam laser. The MS spectra were summed up by 2000 shots in the linear mode at a laser repetition rate of 1kHz, while the MS/MS spectra were accumulated of 20000 shots. The mass selection window was set at about 1% of the precursor, for example, 90Da for 9000Da. The CID gas is off. The instrument was calibrated with myoglobin in both MS and MS/MS mode. Heat treatment. The protein β-LG B solutions were incubated in temperature-controlled water bath at definitive temperatures for 15min, and then analyzed by MALDI-TOF/TOF. Data analysis. The tandem mass spectra of proteins were interpreted manually. Theoretical average mass of protein fragments were calculated from the website http://prospector.ucsf.edu/prospector/mshome.htm. To calculate the fraction of asymmetric cleavages of disulfide bond (Fig.4), raw data was firstly exported from flexAnalysis (Bruker Daltonics, Germany) and then imported in Excel. Peak area was summed with intensity of every data point from yn-3Da to yn+3Da. Results and Discussion Fragmentation of β-LG B ions by TOF/TOF. First, we investigated the performances of multiply charged MALDITOF/TOF method with myoglobin, a ~17kDa protein. The fragmentation spectra of apo-myo 2+ and 3+ were shown in the Fig.S1 and Fig.S2, respectively. Abundant fragment ion

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peaks and specifically, a sequence “tag” (fragments derived from cleavage of a series of adjacent amino acid residues), have been obtained in the tandem mass spectra of doubly (Fig.S1) and triply (Fig.S2) charged myoglobin ions, suggesting the validity of this approach. Then we applied this approach to analyze β-lactoglobulin. The MALDI mass spectrum acquired with 2-NPG as matrix has generated highly charged ions22, from 1+ to 8+ with 2+ being the most abundant, as shown in Fig.1a. Previous studies have shown that α-cyano-4hydroxycinnamic

Figure 1. Fragmentation of β-LG B 2+ by MALDITOF/TOF. (a) MALDI-TOF mass spectrum of β-LG B; (b) LIFT TOF-TOF spectrum of β-LG B 2+. acid (CHCA) is the best matrix in producing multiply charged ions.24,28 However, when using CHCA as the matrix, few multiply charged ions were observed in the reflectron mode.22 With 2-NPG as matrix, abundant multiply charged protein ions were stable enough to pass through the flight in the reflectron mode. Fragmentation of doubly charged β-LG B ions (m/z 9139) yielded plentiful product ions, as shown in Fig.1b. Although top-down analysis of doubly charged β-LG B ions by MALDI-TOF/TOF has been achieved via ultra-thin-layer sample preparation method with CHCA as matrix,24 the sample preparation was time consuming and the fragments of βLG deserved more detailed investigation. The identified fragments of our results are listed in Table S1. Since the precursor ions are doubly charged, the fragments may contain doubly charged ions. The charge state of fragment ions can be easily identified from the accompanying neutral group (NH3/H2O)

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Analytical Chemistry loss peak,21,24 such as doubly charged fragment y93(D-L) and triply charged fragment b141(D-I) from the dissociation of triply charged myoglobin ions, as shown in Fig.S3. Actually, doubly charged fragments were barely found in the tandem mass spectra of doubly charged β-LG B ions. The fragmentation of peptide/protein by MALDI-TOF/TOF always occurs at sites adjacent to the acidic amino acid, such as aspartic acid (D) and glutamic acid (E), and proline (P). Indeed, the product ions obtained here followed these rules, since the most intensive peaks all attributed to the fragments adjacent to Asp residues, such as y25(D-K), y32(D-E), y33(D-D). Moreover, fragmentation of triply charged β-LG B yielded several peaks as well(Fig.S4). Particularly, characteristic “triplet” peaks separated by ~m/z 33 were found in the tandem MS spectra.18 As shown in Fig.2, those triplet peaks were joint results of intra-chain disulfide bond cleavage and polypeptide backbone cleavage, corresponding to yn, yn-SH, yn+SH,7 respectively. No triplet patterns were found in the product ions cleaved outside the disulfide loop, such as b44(E-E), b45(E-L), b47(K-P), b51(E-G), b53(D-L) (Fig.1b and Table S1). If only protein backbone enclosed in the disulfide loop is cleaved, no fragments will be obtained since the assumed product ions are still linked by the disulfide bond. The preferential cleavage sites in the disulfide bond loop also happened adjacent to residues D, E and P, such as y25(D-K), y32(D-E) and y33(D-D). We also performed nanoESI-CID analysis of β-LG B with

tion behaviors of β-LG B after incubation at different temperatures. Interestingly, we found that the relative intensity of fragments coming from disulfide-cleavage changed along with the incubation temperatures. Figure 3 displays four MS/MS spectra corresponding to each of the incubation temperatures, i.e. room temperature, 70ºC, 75 ºC and 80 ºC, respectively. The color-shaded peaks represent asymmetric cleavage products of disulfide, i.e. y25-SH (red), y32-SH (yellow) and y33-SH (green). It was obvious that when the temperature reached 75ºC, the fragments derived from asymmetric disulfide cleavage (yn-SH or yn+SH) decreased dramatically. All of the studied fragments supported this phenomenon. Additionally, topdown analyses of triply charged β-LG B ions were similar, as shown in Fig.S6. We attribute this to the reduced content of native proteoform which contain disulfide linked C160 and another cysteine. Thus, a proportion of β-LG B ions contain free C160 residue. The connections of disulfide bond in native β-lacglobulin B monomer were determined as C66-C160 and C106-C119. It can be seen that the characteristic disulfide cleavage peaks were derived from the disulfide loop C66C160 (Fig.2b). Thus, the decrease of those peaks implies that the disulfide linkage between C66 and C160 broke down in the heating process, consistent with the previous study.33,34 Moreover, no new fragments cleaved between C66 and C121 were found after heat treatment, indicating the probable formation of new disulfide bond between C66 and other cysteine. These results are consistent with the study by employing bottom-up method33 and cysteine block approach34. There are five cysteine residues and two disulfides in β-LG B, which means one cysteine residue was in the reduced form. It

Figure 2. Concurrent cleavage of disulfide and peptide bond of β-LG B. a) enlarged MALDI-LIFT-TOF/TOF spectrum of β-LG B 2+, the triplet peaks of disulfide cleavage were revealed; b) schematic view of the concurrent cleavage of disulfide bond and backbone bond. The color-shaded peaks in panel a were correlated with the fragments in panel b. high resolution QTOF mass spectrometer. As shown in Fig.S5, due to the existence of disulfide loop, the fragmentation efficiency of β-LG B 9+ was low. A series of b ions were identified from the spectra (Table S2), which derived from cleavage of the polypeptide backbone outside the disulfide loop. However, no concurrent cleavages of disulfide bond and protein backbone were found in our tandem mass spectra. Heat-induced disulfide rearrangement of β-LG B. Temperature is an important factor affecting protein structure and functionality.29 The influence of heating on the disulfidecontaining protein, such as soy protein30, milk protein31 and antibody32, has been broadly investigated via bottom-up proteomics. The disulfides in these proteins were reported to reshuffle upon heat stress. Thus we investigated the fragmenta-

Figure 3. MALDI-TOF/TOF spectra of β-LG B 2+ underwent incubation at different temperatures. The color shades represent the fragments of asymmetric cleavage of disulfide. The protein β-LG B was incubated at different temperature: a) room temperature, b) 70 ºC, c) 75 ºC, d) 80 ºC for 15 minutes.

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has been reported that disulfide scrambling can happen in the presence of free cysteine residues,32,35 which can act as a nuclei for initiating scrambling reaction. The tandem mass spectra of β-LG B after incubation at a series of temperatures, i.e. from 50ºC to 90ºC, were acquired. The relative amount of asymmetric cleavage fragment of the disulfide (C66-C160) was calculated to reveal the heatinduced disulfide rearrangement at various temperatures, as shown in Fig.4a. The data was analyzed as follows, P[y n -SH]+P[y n +SH] (1) F= 2P[y n ]

P means the peak area of the particular fragments (y25(D-K)), F means the ratio of asymmetric cleavage fragments to symmetric cleavages. The ratios were all about 0.6 through the temperatures from 50ºC to 70ºC. When the incubation temperature reached critical point 75 ºC (critical temperature Tc), the ratio was suddenly decreased to about 0.4. Take y25(D-K) as an example, the homolytic cleavage product y25(D-K) would increase since the content of non-native lac B monomer increase after heat treatment (Fig.4b). It’s worth noting that the fragmentation patterns of doubly charged ions are not disturbed by the acquisition positions, even though the spots are inhomogeneous, as shown in Fig.S7. Since pH is a critical factor affecting the formation of nonnative disulfide bonds, we studied the heat-stressed protein in buffer as well. The results indicate that the fragmentation pattern of β-LG B 2+ ions after heating were similar with that in water, as shown in Fig.S8 and Fig.S9. Moreover, the fragmentation behavior of disulfide bonds were also not affected by the laser influence (Fig.S10) or the matrix concentration and acetonitrile fraction (Fig.S11). While previous studies have demonstrated that the heatdenatured β-LG B form large aggregates, no dimers or larger aggregates were found in the MALDI-TOF and nanoESIQTOF mass spectra (Fig.S12). This may attribute to lower concentration of β-LG B used in our experiment. An interesting observation was that the charge state distributions (CSD) of native mass spectra of β-LG B after incubation at various temperatures were similar (Fig.S12). Only a slight shift to higher charge state of β-LG B after heat-treatment was found, even though the disulfide linkage was changed. The CSDs have been widely used to inform on the conformation of protein.36 This result indicated that the conformation of β-LG B changed little after heat treatment. The consequence of disulfide scrambling could also be investigated further with hydrogen-deuterium exchange (HDX)37 and ion mobility mass spectrometry (IM-MS)38. However, only a slight change in the protein conformation was found in ion mobility mass spectra, as shown in Fig.S13. The circular dichroism spectra showed that the trough shifted to lower wavelengths after heat treatment39, indicating that the secondary structure was disturbed (Fig.S14). In summary, the consequence of disulfide bond reshuffling was not easy to probe, since heat would alter the secondary structure of proteins.

Figure 4. The thermal induced disulfide rearrangement. a) Relative amount of asymmetric cleavages of disulfide bond at different temperatures. Representative LIFT TOF/TOF spectra at 70ºC, 75 ºC, 80 ºC and 90 ºC were displayed with different colors. The data were averaged with three measurements; b) schematic view of heat-induced disulfide rearrangement, native monomer was transformed into non-native monomer after heat treatment. Conclusion Top-down analysis of lactoglobulin (18kDa) has been successfully achieved by MALDI-TOF/TOF, via generating and fragmenting multiply charged protein ions with matrix 2-NPG. A number of fragments were obtained by tandem mass spectra of doubly charged β-LG B, especially the cleavage at sites adjacent to aspartic acid and glutamic acid. Importantly, the concurrent cleavages of disulfide bond and protein backbone bonds lead to characteristic triplet peaks, corresponding to ynSH, yn, yn+SH, respectively. It was found that the relative abundance of diagnostic ions, which arise from asymmetric disulfide cleavage between Cys66 and Cys160, sharply decreased at the critical temperature 75ºC. These results indicate that the Cys66-Cys160 disulfide bond was broken during heating. These findings have highlighted the ability of MALDITOF/TOF in characterizing large and disulfide-containing protein. In considering the unique characters of MALDITOF/TOF, such as unique dissociation pathway, simple mass spectra and low sample consumption, this approach has the potential for rapid top-down analysis of large protein (>12kDa) and characterization of protein post-translational modification. This approach can also be extended to intact protein biomarkers for virus or protein toxin identification.21 We envision that the application of this method would be expanded to unknown proteins’ identification when the mass accuracy and

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Analytical Chemistry resolution of TOF/TOF were improved and the instrument and calibration method were optimized. 40 Moreover, the capability of MALDI source of in situ analysis would promote the profiling of large protein directly from the tissue26 and complex medium18. Since disulfide bonds exist extensively in biosimilar13, venom41 and food protein30, the presence of characteristic fragment ion triplets of disulfide bonds would facilitate disulfide detection in these proteins.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Supplementary figures and tables(PDF)

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected].

Author Contributions

¶L.Zhan and Y.Liu contributed equally to this work. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by grants from the National Natural Sciences Foundation of China (Grant Nos. 21625504, 21827807, 21475739, 21675160 and 21621062), and Chinese Academy of Sciences. The authors would also like acknowledge the help of TIMS measurement from Dr. Yu Xia and Dr. Xiaoyun Yang from Tsinghua Univesrtiy.

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