Global Disulfide Bond Profiling for Crude Snake Venom Using

Aug 1, 2014 - ABSTRACT: Snake venom consists of toxin proteins with multiple disulfide linkages to generate unique structures and biological functions...
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Global Disulfide Bond Profiling for Crude Snake Venom Using Dimethyl Labeling Coupled with Mass Spectrometry and RADAR Algorithm Sheng Yu Huang,† Sung Fang Chen,‡ Chun Hao Chen,‡ Hsuan Wei Huang,§,∥ Wen Guey Wu,∥ and Wang Chou Sung*,§ †

Mithra Biotechnology Inc., 7F, No. 104, Sec. 1, Xintai 5th Road, Xizhi Dist., New Taipei City 221, Taiwan National Taiwan Normal University, Department of Chemistry, No. 88, Sec. 4, Tingchow Road, Taipei 116, Taiwan § National Health Research Institutes, National Institute of Infectious Diseases and Vaccinology, No. 35 Keyan Road, Zhunan, Miaoli County 350, Taiwan ∥ National Tsing Hua University, Institute of Bioinformatics and Structural Biology, No. 101, Sec. 2, Kuang Fu Road, Hsinchu 330, Taiwan ‡

S Supporting Information *

ABSTRACT: Snake venom consists of toxin proteins with multiple disulfide linkages to generate unique structures and biological functions. Determination of these cysteine connections usually requires the purification of each protein followed by structural analysis. In this study, dimethyl labeling coupled with LC-MS/MS and RADAR algorithm was developed to identify the disulfide bonds in crude snake venom. Without any protein separation, the disulfide linkages of several cytotoxins and PLA2 could be solved, including more than 20 disulfide bonds. The results show that this method is capable of analyzing protein mixture. In addition, the approach was also used to compare native cytotoxin 3 (CTX III) and its scrambled isomer, another category of protein mixture, for unknown disulfide bonds. Two disulfide-linked peptides were observed in the native CTX III, and 10 in its scrambled form, X-CTX III. This is the first study that reports a platform for the global cysteine connection analysis on a protein mixture. The proposed method is simple and automatic, offering an efficient tool for structural and functional studies of venom proteins.

D

into the protein family. Because of the lack of automatic data analysis and assignment, only a few studies have emphasized solving the exact cysteine connections. Different platforms such as Edman sequencing, NMR,1,7 and X-ray have been well developed and successfully applied to characterize the molecular structure of venom toxins. However, the demands on quantity and extremely high purity of analytes have limited their applications on single protein characterization. Mass spectrometry has emerged as a valuable tool for disulfide bond analysis. Conventionally, a purified protein is first digested into peptides under nonreduced conditions, and the peptide mixture containing disulfide-linked peptides is analyzed by LC-MS/MS with and without reduction.8 When comparing their peptide maps, the disulfide-linked peptides that are present in one but absent in the other can be screened out. The MS/MS spectra are further assigned to determine the

isulfide bonds are major structural factors that affect the functionality of natural biomolecules. Snake venom, a cysteine-rich protein mixture, is a well-studied example, and more than 70% of it contains two or more disulfide bonds.1 Multiple disulfide linkages not only provide a rigid and stable structure for these extracellular toxins2 but also construct a unique folding to target various receptors with high binding affinity,3,4 which causes severe syndromes on the victim by blocking the signal transduction of the central nervous system or affecting the functions of the cardiovascular, muscular, and vascular systems. Ironically, such lethal bioeffect also highlights the potential of venom proteins as drug leads in human diseases5 and as a tool to understand the physiological processes at the cellular level. Previous studies also indicated that any substitution of disulfide linkages would drastically change the native structure and cause the loss of function and stability.6 Therefore, the determination of the exact connection between cysteine residues is important for functional studies on venom proteins. Most of the venom studies related to disulfide bonds had a focus on determining the number or ratio of oxidized and reduced cysteine, which is useful for preliminary classification of toxins © 2014 American Chemical Society

Received: May 23, 2014 Accepted: August 1, 2014 Published: August 1, 2014 8742

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3 isomer (X-CTX III) was prepared through reduction and refolding to evaluate the approach of characterizing unknown disulfide bonds. Disulfide linkages in the native CTX III and X-CTX III were profiled and compared using the automatic method. Because many disulfide linkage possibilities can be produced through disulfide scrambling, dimethyl labeling coupled with RADAR search represents a useful approach for condition optimization of generating the most effective X-isomer and quality assessment for reproducible and robust X-isomer production.

cysteine connection. Stepwise alkylation approaches have been utilized to analyze disulfide bonds.9,10 Because different disulfide bonds may have a different reduction rate, cysteine connection can be obtained by the use of different alkylation reagents in each reduction step. This method is especially useful for distinguishing disulfide bonds in close proximity. Mass spectrometric methods based on gas phase reduction, including the use of in-source dissociation (ISD) on MALDI,11,12 negative mode on ESI,13,14 and electron transfer dissociation (ETD),15,16 were also applied to analyze disulfide-linked peptides. A reductive matrix such as 1,5 DAN (1,5 diaminonaphthalene) was used for online reduction of disulfide-linked peptide detection with MALDI-MS.17 Several computational algorithms, such as MassMatrix,18 DBond,19 MS2DB+,20 and DisConnect,21 were developed to identify disulfide linkages by considering different fragment ions from MS/MS spectra. SearchXLinks was designed to analyze the ISD data of the disulfide-linked peptides from MALDI analysis.22 The methods mentioned above have been reviewed extensively.23,24 However, they were mainly designed for purified protein or peptide analyses only. Because manual inspection of MS/MS spectra is still required for the majority of approaches, it is difficult to deal with complicated samples such as protein mixtures, which generate too many cysteinyl peptides, hence too many disulfide linkage possibilities have to be considered. Protein separation or purification is usually required for disulfide bond determination using the approaches described previously. In this study, we used dimethyl labeling25 coupled with mass spectrometry and RADAR (Rapid Assignment of Disulfide linkage via a1 ion Recognition) search26,27 to determine the disulfide linkages in crude cobra snake venom, a protein mixture which contains cobrotoxins (neurotoxins), phospholipase A2 (PLA2), cytotoxins (cardiotoxins), metalloprotease enzymes, and other metallic ions.28−30 Dimethyllabeled peptides are known to exhibit enhanced a1 ions on collision-induced dissociation (CID) spectra, which indicate the identities of the N-terminal amino acids.31 Multiple a1 ions can be used to screen out the dimethyl-labeled, disulfide-linked peptides, and the involved disulfide bonds can be determined by the corresponding MS/MS spectra. The process was automated by the development of the customized software named RADAR. In brief, RADAR generates the predicted m/z of all the cysteinyl peptides and provides the targeted N-terminal amino acids according to the imported protein sequences and cleavage sites. Afterward, it screens out the peptide entries which contain the corresponding a1 ions from the peak list. Finally, the disulfide-linked peptides are determined by correlating the observed a1 ions with the cysteinyl peptide list and comparing the theoretical and experimental molecular weights. In the search results, b/y ions are further assigned by RADAR to confirm the identities of each peptide. The simple and automatic approach integrating dimethyl labeling, LC-MS/MS, and RADAR search is advantageous for handling complicated samples without the need of delicate purification. In addition to the crude cobra venom, the method is further applied to scrambled protein isomers, another kind of mixture containing protein isoforms with different disulfide structures. Protein isomerization through disulfide shuffling has been reported to improve the antigenicity compared to that of the native proteins32 and generate antibodies with cross-reactivity, which is potentially useful for developing universal and therapeutic antivenom. In this study, the scrambled cytotoxin



EXPERIMENTAL SECTION Materials. All solvents are chromatography grade or better. Acetonitrile (ACN) was purchased from J.T. Baker (Center Valley, PA). Sequencing-grade modified trypsin was obtained from Promega (Madison, WI). Thermolysin, sodium cyanoborohydride, formaldehyde-D2, N-ethylmaleimide (NEM), sodium acetate, tris(hydroxymethyl) aminomethane hydrochloride (tris-HCl), dithiolthreitol (DTT), guanidine hydrochloride, and formic acid (FA) were purchased from Sigma-Aldrich (St. Louis, MO). Amicon filter (MW cut-off: 3kDa) was from Merck Millipore (Darmstadt, Germany). The cobra snake venom (Naja naja atra) was purchased from a snake farm in Taiwan, and the purified CTX III was isolated from the crude venom following the procedure as described in the reference article.33 Preparation of CTX III Isomer. CTX III (1 mM) was dissolved in 100 mM Tris-HCl buffer (pH 8.4) containing 5.0 M guanidine hydrochloride (GdnCl) and 30 mM DTT. The sample solution was incubated at 56 °C for 3 h to ensure that all disulfide bonds were reduced. The solution containing unfolded CTX III was immediately diluted with 50-fold ice chilled 100 mM Tris-HCl (pH 8.4) to create the scrambled CTX III. The scrambled protein was recovered with 3 kDa Amicon filter, and then analyzed by HPLC-UV (Agilent 1100) equipped with reverse phase C18 column (Jupiter 5um, 250 × 4.6 mm, Phenomenex). The recovered sample from HPLC was lyophilized and stored in refrigerator for further analysis. Enzymatic Digestion and Dimethyl Labeling. For the crude venom analysis, the protein mixture was diluted with 100 mM sodium acetate pH 6, and then NEM was added at 8.7 mM to block free cysteines at room temperature for 30 min. Enzymatic digestion was performed in sodium acetate pH 6 at 37 °C overnight with a trypsin/protein ratio of 1:50 (w/w) or thermolysin/protein ratio of 1:25 (w/w). All the samples were diluted twice with 100 mM sodium acetate pH 5. To perform dimethyl labeling, 4 μL of 4% (w/v) formaldehyde-D2 was added to 160 μL of protein digest, followed by the addition of 4 μL of 600 mM sodium cyanoborohydride at room temperature for 30 min. Labeled peptide mixtures were ready for LC-MS/MS analysis. The same procedure was applied to the digestion and dimethyl labeling for the native CTX III and its isomer with the difference that only trypsin was used. It is noted that formaldehyde-D2 was originally used instead of formaldehyde to avoid the a1 overlap between Lys and Arg in the previous study. When a high-resolution mass spectrometer is used, both reagents will work without a problem. Special caution should be taken when handling formaldehyde and sodium cyanoborohydride including the use of gloves and a fume hood. LC-MS/MS Analysis. The samples were analyzed with Q-Exactive mass spectrometer (Thermo Scientific, Bremen, Germany) coupled with Ultimate 3000 RSLC system (Dionex, Sunnyvale, CA). The LC separation was performed using the 8743

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C18 column (Acclaim PepMap RSLC, 75 μm ×150 mm, 2 μm, Dionex) with linear gradient from 1% to 35% of mobile phase B (mobile phase A: 5% ACN/0.1% FA; mobile phase B: 95% ACN/0.1% FA) for 35 min, 35% to 80% of mobile phase B for 32 min and 80% mobile phase B for 10 min in a total of 90 min separation time. Full MS scan was performed with the range of m/z 300−2000, and the 10 most intense ions were selected for MS/MS acquisition, which was fixed to start from m/z 50 to include all the a1 ions. For trypsin-digested native CTX III and its isomer, ESI-Q-TOF mass spectrometer Synapt HDMS connected with nanoACQUITY UPLC system (Waters, MA, U.S.A.) was used. The LC separation was performed using a reverse phase C18 column (75 μm × 100 mm, 1.7 μm, Waters, MA, U.S.A.) with a trap column (180 μm × 20 mm, 5 μm, Waters, MA, U.S.A.). Commercially available DI water containing 0.1% FA and acetonitrile with 0.1% FA were purchased from J.T. Baker (Phillipsburg, NJ, U.S.A.) and used as mobile phase A and B, respectively. The gradient conditions were as follows: 1% B to 50% B in 30 min, 50% B to 65% B in 30−40 min, and 65% B for 5 min in a total of 60 min separation time. Full scan was set at the m/z range of 400−1600, and four channels were used for simultaneous MS/MS detection. Data Analysis. Raw data from Q-Exactive was processed into peak list using Proteome Discoverer 1.3 for RADAR search. A new version of the custom-made software RADAR 3.0 (now available for free trial at the Web site http:// www.mass-solutions.com.tw/) was used to screen a1 ions and search for the corresponding molecular weight for disulfide bond assignment. The search was performed against protein sequences in cobra venom as listed in Table 1 with the parameters including: cleavage at the C-terminal of lysine and arginine except for KP and RP when trypsin was employed, cleavage at the N-terminal of isoleucine, leucine, methionine, phenylalanine, and valine (with additional alanine, tyrosine, tryptophan, and threonine as noted in the text) for thermolysin digestion due to its broad specificity. Up to two missed cleavages were allowed. D labeled, intensity ratio cutoff 10%, a1 tolerance ±0.002 Da, mass tolerance ±10 ppm, and maximum chain number 4 were selected for RADAR search. Raw data from Q-TOF for CTX III analysis were converted into peak lists using MassLynx 4.0 for the RADAR search. The search was performed using the sequence #2 as listed in Table 1 with the parameters including the following: Cleavage at the C-terminal of lysine and arginine except for KP and RP. Up to two missed cleavages were allowed. D labeled, intensity ratio cutoff 10%, a1 tolerance ±0.01 Da, mass tolerance ±0.2 Da and maximum chain number 4 were selected.

cobrotoxin(neurotoxin), cytotoxins(cardiotoxins), phospholipase A2 (PLA2), and cysteine-rich venom proteins were identified as the major proteins in the crude venom of the Taiwan cobra, which is consistent with previous studies.34 Among the 15 proteins, neurotoxin and cytotoxins belong to the three finger protein family (TFPx). The proteins in the family share the same morphology but contain different amino acid compositions and disulfide bridges which are the key elements in determining the toxicity of crude venom. Another major component in the Taiwan cobra venom is the enzymatic protein such as PLA2 and metalloproteases, which can hydrolyze sn2-lipid and membrane proteins, respectively.35,36 Except for the two metalloproteases that were identified with low sequence coverage, the complete sequences of the remaining 13 proteins, as listed in Table 1, were used as the database for RADAR search to determine their disulfide connections between cysteines. The sequences as well as their known disulfide bond information were obtained from the Uniprot database. Disulfide Bond Analysis of Crude Snake Venom. To profile the disulfide linkages of the proteins in the crude venom, the conventional digestion procedures were modified as previously detailed.26,27 In brief, N-ethylmaleimide alkylation was first applied to block the free cysteines in venom proteins. Enzymatic digestion was then performed in neutral to acidic conditions in order to avoid disulfide rearrangement. In this study, trypsin and thermolysin were used in parallel to increase the number of the detectable disulfide-linked peptides. After digestion, the peptide mixture was labeled with formaldehyde-D2 and analyzed by LC-MS/MS. Dimethyl-labeled, disulfidelinked peptides exhibit enhanced a1 ions which clearly provide the identities of N-terminal amino acids. The information was then used by RADAR to identify the disulfide linkages. As illustrated in Figure 1, four different a1 ions, 106.12, 62.09, 168.13 and 106.06, can be observed in the MS2 spectrum for the disulfide-linked peptides with m/z 561.4848 (5+), which indicates that the disulfide linked peptide contains four N-terminal amino acids including threonine (T), glycine (G), tyrosine (Y), and cysteine (C). In addition to the a1 ions, the b/y ion series along with the precursor molecular weight, 2802.3849 Da, allow RADAR to identify its four peptide components as *TCPAG*K, *GCIDVCP*K, *YVCCNTDR, and *CN. When one missed cleavage is allowed, the 13 proteins can generate more than one hundred different cysteinyl peptides from either trypsin or thermolysin digestion, which leads to numerous possibilities of disulfide-linkages. To minimize the labor work of manual inspection, a novel edition of algorithm, RADAR 3.0, was designed to analyze disulfide linkages in protein mixtures. The 13 protein sequences can be imported into the software within one text file. In addition to a more efficient running algorithm, a new option called “exclude cross chain disulfide bond” was implanted into the current edition. When the option is enabled, RADAR will only search for disulfide bonds in each protein entry without taking the interprotein disulfide bonds into consideration, otherwise all cysteinyl peptides from different proteins will be treated independently and all possible combinations will be considered, which was the original mode used for analyzing monoclonal antibodies that contain disulfide-linked heavy chains and light chains.27 The disulfide search in protein mixtures can be facilitated by not considering cross chain disulfide bonds as used in this study.



RESULTS AND DISCUSSION Protein Identification for Snake Venom. Previous studies have suggested that venom composition can vary in different geographical regions even for the same snake species such as cobra.34 In order to construct a specific sequence database for the following disulfide bond analysis, the major protein components in the obtained cobra snake venom have to be identified first. Figure S-1 in the Supporting Information shows the HPLC-UV chromatogram of the cobra venom used. Each peak was collected, digested by trypsin, and analyzed with LC-MS/MS. Database search was then performed against a customized cobra protein sequence database derived from UniProt (http://www.uniprot.org/) by searching the keyword Naja naja atra. In total, 15 proteins including different types of 8744

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8745

P60301

Q9W716

P80245

P60770

Q91124

Q98959

Q98962

O93422

P60306

Q9W6W6

P00598

Q7T1K6

2

3

4

5

6

7

8

9

10

11

12

13

cysteine-rich venom protein natrin-1

acidic phospholipase A2 1

cytotoxin 10

long neurotoxin homologue cytotoxin SP13b

cardiotoxin 3d

cytotoxin 3a

cytotoxin 8

cobrotoxin

cytotoxin 6

cytotoxin 5V

cytotoxin 3

cytotoxin 1

protein name*

NLYQFKNMIQCTVPSRSWWDFADYGCYCGRGGSGTPVDDLDRCCQVHDNCYNEAEKISGCWPYFKTYSYECSQGTLTCKGGNNACAAAVCDCDRLAAICFAGAPYNNNNYNIDLKARCQ NVDFNSESTRRKKKQKEIVDLHNSLRRRVSPTASNMLKMEWYPEAASNAERWANTCSLNHSPDNLRVLEGIQCGESIYMSSNARTWTEIIHLWHDEYKNFVYGVGANPPGSVTGHYTQIVWYQTYRAGCAVSYCPSSAWSYFYVCQYCPSGNFQGKTATPYKLGPPCGDCPSACDNGLCTNPCTIYNKLTNCDSLLKQSSCQDDWIKSNCASCFCRNKII

LKCNQHIPPFYKTCAAGKNLCYKIFMVAAPKVPVKRGCIDVCPKSSDLVKYVCCNTDRCN

LTCLICPEKYCNKVHTCLNGEKICFKKYDQRKLLGKRYIRGCADTCPVRKPREIVECCSTDKCNH LKCNKLKPLAYKTCPAGKNLCYKMFMMSNKTVPVKRCIDVCPKNSLLVKYVCCNTDRCN

LKCNKLIPIASKTCPAGKNLCYKMFMVATPKVPVKRGCIDVCPKNSLLVKYVCCNTDRCN

LKCNKLVPLFYKTCPAGKNLCYKMFMVATPKVPVKRGCIDVCPKNSLLVKYVCCNTDRCN

LECHNQQSSQTPTTTGCSGGETNCYKKRWRDHRGYRTERGCGCPSVKNGIEINCCTTDRCNN LKCNKLIPIASKTCPAGKNLCYKMFMVATPKVPVKRGCIDVCPKSSLLVKYVCNTDRCN

LKCNQLIPPFYKTCAAGKNLCYKMFMVAAPKVPVKRGCIDVCPKSSLLVKYVCCNTDRCN

LKCHNTQLPFIYKTCPEGKNLCFKATLRKFPLKFPVKRGCADNCPKNSALLKYVCCSTDKCN

LKCNKLVPLFYKTCPAGKNLCYKMFMVATPKVPVKRGCIDVCPKSSLLVKYVCCNTDRCN

LKCNKLIPIASKTCPAGKNLCYKMFMMSDLTIPVKRGCIDVCPKNSLLVKYVCCNTDRCN

sequence C3−C21, C14−C38, C42−C53, C54−C59 C3−C21, C14−C38, C42−C53, C54−C59 C3−C22, C15−C40, C44−C55, C56−C61 C3−C21, C14−C38, C42−C53, C54−C59 (C3−C24, C17−C41, C43−C54, C55−C60) C3−C21, C14−C38, C42−C53, C54−C59 C3−C21, C14−C38, C42−C53, C54−C59 C3−C21, C14−C38, C42−C53, C54−C59 C3−C24, C6−C11, (C17−C42, C46−C57, C58−C63) C3−C21, C14−C38, C42−C53, C54−C59 (C3−C21), C14−C38, C42−C53, C54−C59 C11−C71, C26−C118, C28−C44, C43−C99, C50−C92, C60−C85, C78−C90 C56−C134, C73−C148, (C129− C145, C167−C174, C170−C179, C183−C216, C192−C210, C201−C214)

expected disulfide linkages (identified ones in bold; not observed ones in parentheses)

The information including protein accession number, protein names, and expected disulfide bonds are obtained from UniProt (http://www.uniprot.org/).

P60304

1

index

protein accession no.

Table 1. Protein List Used for RADAR Search and the Number of Identified Disulfide Linkages in Cobra Snake Venom

2/8

7/7

3/4

4/4

2/5

4/4

4/4

4/4

0/4

4/4

4/4

4/4

4/4

observed/expected disulfide bonds*

Analytical Chemistry Article

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Figure 1. MS2 spectrum of the dimethyl-labeled and disulfide-linked peptides with m/z 561.4848 (5+) showing the peptide consisting of four chains, *TCPAG*K, *GCIDVCP*K, *YVCCNTDR and *CN. The enhanced four a1 ions, 106.12, 62.09, 168.13, and 106.06, clearly indicate that the disulfide linked peptide contains four N-terminal amino acids including T, G, Y, C. Signals above m/z 106.12 are enhanced by 2 times for a clear annotation.

Table 2 contains the RADAR search results of disulfide bond matches in the crude snake venom with trypsin digestion. The disulfide bond locations as well as the a1 ions are shown. Repeated observations/identifications of the matched m/z are all listed in the observed m/z column. There are 13 unique disulfide-linked peptides identified in the search. Some of them belong to more than one protein because proteins of the same family may share conserved sequences as indicated in the “protein index” column. Cytotoxins belong to the short-chain three finger toxin (3FTx) families, which can block the signal transduction and cause severe syndromes of heart and muscle stretching.37,38 Most of them share the same disulfide linkages except for cytotoxins 5 V and 6, which is consistent with previous studies.38 The disulfide bonds of cytotoxins 1 and 3 were assigned by the observation of disulfide-linked peptide sets (1), (2), (3) and (4). The disulfide bonds of cytotoxin 5 V were assigned by the peptide sets (5), (6), (7), and (8), and those of cytotoxin 6 were assigned by the peptide sets (9), (10), (11) and (12). The number of the observed disulfide bonds as well as that of the expected disulfide bonds is shown in the last column of Table 1. The four disulfide bonds from these four proteins were observed and identified in one LC-MS/MS analysis. The MS/MS spectrum of the peptide set (3) is illustrated in Figure 1, whereas the others are shown in the Supporting Information. Long neurotoxin homologue (Kappa-Cobrotoxin) shares a similar three finger structure with cytotoxins (cardiotoxins) but contains five disulfide bonds in their protein skeleton. Two out of five disulfide bonds were identified, and their linkage sites are significantly different from those of the cytotoxins. More disulfide bonds were detected using the second enzyme, thermolysin, as listed in Table 3. Thermolysin is a metallopeptidase which cleaves peptide bonds at the Nterminus of hydrophobic amino acids including alanine, isoleucine, leucine, methionine, phenylalanine, threonine, tryptophan, tyrosine, and valine. In total, 20 unique disulfide-

linked peptides were identified. Most disulfide bonds observed in the trypsin experiment were identified in the thermolysin experiment as well with different peptide lengths due to the complementary enzyme specificity, which further confirms these disulfide linkages. For instance, the peptide sets (1)−(4) in the trypsin data correspond to the peptide sets (1)−(5) in the thermolysin data. The additional peptide set (9) in the thermolysin data can be used to distinguish the disulfide bonds, C42−C53 and C54−C59, in cytotoxin 3 (IDVCPKSS, VCCNTDRCN) from those in cytotoxin 1 (IDVCPKNS, VCCNTDRCN) due to one amino acid difference; those disulfide bonds are indistinguishable in the trypsin experiment. Thermolysin was reported to have a broad specificity. Currently RADAR only allows up to two missed cleavages. In order to locate those peptides with more missed cleavages, five preferred sites were first used for the RADAR search, and then the search was repeated again with additional cleavage sites. Several disulfide bonds which had been absent in the trypsin experiments were observed in the thermolysin digestion for PLA2 and cysteine-rich venom protein natrin-1. In addition to cytotoxins, PLA2 is another major component in cobra venom, which as an enzyme hydrolyzes the sn2 ester bond of membrane lipids and further causes the cell crush. All seven disulfide bonds in PLA2 were identified from the peptide set (12) to (18) in Table 3. The last two disulfide-linked peptide sets, (18) and (19), came from the protein, cysteine-rich venom protein natrin-1, which belongs to the cysteine-rich secretory protein family (CRISP) that can block ion transduction and inhibit the muscle contraction.39 Two out of eight disulfide bonds were identified in the experiment. The reasons that only partial disulfide bonds can be identified in some proteins can be attributed to their relatively low abundance, as observed from the UV signals shown in Figure S-1 (Index 9 and 13 in Table 1). Besides, under nonreduced condition, enzymatic digestion can be either 8746

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Table 2. Identified Disulfide-Linked Peptides from the Trypsin Digestion of Cobra Venom Using RADAR Search query

disulfide-linked peptides

Cys location

N-term A.A.a

(1)

*CN*K *NLCY*K *LKCN*K *NLCY*K *TCPAG*K *GCIDVCP*K *YVCCNTDR *CN *TCPAG*K *GCIDVCP*K *YVCCNTDRCN

C3 C21 C3 C21 C14 C38C42 C53C54 C59 C14 C38C42 C53C54C59

C N L N T G Y C T G Y

*CHNTQLPFIY*K *NLCF*K *LKCHNTQLPFIY*K *NLCF*K *TCPEG*K *GCADNCP*K *YVCCSTD*K *CN *TCPEG*K *GCADNCP*K *YVCCSTD*KCN *CNQLIPPFY*K *NLCY*K *LKCNQLIPPFY*K *NLCY*K *LTCLICPE*K *ICF*K *YCN*K *TCAAG*K *GCIDVCP*K *YVCCNTDR *CN *TCAAG*K *GCIDVCP*K *YVCCNTDRCN

C3 C22 C3 C22 C15 C40C44 C55C56 C61 C15 C40C44 C55C56C61 C3 C21 C3 C21 C3C6 C24 C11 C14 C38C42 C53C54 C59 C14 C38C42 C53C54C59

C N L N T G Y C T G Y C N L N L I Y T G Y C T G Y

(2) (3)

(4)

(5) (6) (7)

(8)

(9) (10) (11)

(12)

(13)

a

experimental mass (Da)

calculated mass (Da)

protein indexb

(2+) (3+) (3+) (4+) (3+) (4+) (5+) (7+) (3+) (4+) (5+) (6+) (3+)

1128.6701 1128.6707 1401.9068 1401.9055 2802.3887 2802.3863 2802.3849 2802.3777 2752.3139 2752.3155 2752.3184 2752.3175 2112.1925

1128.6727

1, 2, 6, 7, 8, 10

1401.9081

1, 2, 6, 7, 8, 10

2802.3849

1, 2, 6, 7, 8, 10

2752.3179

1, 2, 6, 7, 8, 10

2112.1956

3

597.3650 (4+)

2385.4287

2385.4310

3

703.5960 (4+)

2810.3527

2810.3573

3

691.0798 553.0661 461.0559 663.3859

(4+) (5+) (6+) (3+)

2760.2879 2760.2914 2760.2885 1987.1342

2760.2903

3

1987.1366

4

566.1003 (4+)

2260.3699

2260.3721

4

561.5862 (4+)

2242.3135

2242.3145

9

556.2803 (5+) 463.7358 (6+)

2776.3624 2776.3679

2776.3692

4, 11

682.5823 (4+) 546.2680 (5+) 455.3911 (6+)

2726.2979 2726.3009 2726.2997

2726.3022

4, 11

observed m/z 565.3433 377.2314 468.3101 351.4842 935.1374 701.6044 561.4848 401.3475 918.4458 689.0867 551.4715 459.7275 705.0720

N-terminal amino acids derived from the a1 ions on each MS2 spectrum. bProtein index according to Table 1.

inefficient for a larger protein such as CRISP or incapable of cleaving proteins into MS detectable peptides. For instance, no disulfide bond was detected for cobrotoxin (Index 5 in Table 1) because neither trypsin nor thermolysin is able to produce detectable disulfide-linked peptides. Alternative enzymes will be required to solve its cysteine connection. The results demonstrate the capability of the LC-MS/MS combined with dimethyl labeling and the RADAR search for analyzing disulfide bonds in protein mixtures without delicate protein separation. Although some of the observed disulfidelinked peptides may be attributed to more than one protein due to sequence homology, the fact that no data show a different cysteine connection clearly suggests that these disulfide bonds are formed as expected. Disulfide Bond Analysis of CTX III Isomer. The approach was further applied to another kind of protein mixture, protein isomers which contain scrambled disulfide structures. It is generally known that such isomers exist as intermediates when a fully reduced protein folds back to its native form. The analysis of the disulfide bonds of these intermediates provides significant information for investigating

Figure 2. HPLC chromatogram of reduced (unfolded), scrambled (X-CTX III), and native CTX III.

disulfide refolding pathway. Moreover, a recent study has suggested that such protein isomer exhibits the potential to improve the immunogenicity of native protein and generate the antibody with cross reactivity, because it can force more epitopes to be exposed to the immune system.32 The 8747

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Table 3. Identified Disulfide-Linked Peptides from the Thermolysin Digestion of Cobra Venom Using RADAR Search disulfide-linked peptides

Cys location

(1)

*L*KCN*K *LCY*K *LKCN*K *LCY*KM

C3 C21 C3 C21

L L L L

*IAS*KTCPAG*KN *V*KRGC *IDVCP*KNS *VCCNTDRCN

C14 C38 C42 C53C54C59

I V I V

(10)

*IASKTCPAG*KN *V*KRGCIDVCP*KNS *VCCNTDRCN *L*KCHNTQLP *LC *L*KCHNTQLPF *LC *L*KCNQ *LCYK*M *IDVCP*KSS *VCCNTDRCN *ICPE*KYCN*K

I V V L L L L L L I V I

(11)

*LICPE*KYCN*K

(12)

*ISGCWP *ACA *ISGCWPY *AC *ISGCWPY *ACA *YCGRGGSGTP *LDRCCQ *IC *VHDNC *VCDCDR *LTC*KGGNN *YGC *L*KARCQ *IQC *YECSQGT *IQCGES *YCPSGN *ANTCS *VSYCPSS

C14 C38C42 C53C54C59 C3 C22 C3 C22 C3 C21 C42 C53C54C59 C6 C11 C6 C11 C60 C85 C60 C85 C60 C85 C28 C43C44 C99 C50 C90C92 C78 C26 C118 C11 C71 C73 C148 C56 C134

(2)

(3) (4)

(5)

(6) (7) (8) (9)

(13) (14) (15)

(16)

(17) (18) (19) (20) a

N-term A.A.a

query

L I A I A I A Y L I V V L L Y I Y I Y A V

observed m/z

experimental mass (Da)

calculated mass (Da)

protein indexb

644.9402 430.2957 710.4600 473.9759 355.7335 453.0415

(2+) (3+) (2+) (3+) (4+) (4+)

1287.8648 1287.8636 1418.9044 1418.9042 1418.9027 1808.1347

1287.8652

1, 2, 6, 7, 8, 10

1418.9057

1, 2, 6, 7, 8, 10

1808.1369

1, 6, 8

997.4705 665.3168 499.2395 938.7556 751.2061 626.1732 461.2749

(2+) (3+) (4+) (4+) (5+) (6+) (3+)

1992.9254 1992.9269 1992.9267 3750.9911 3750.9919 3750.9923 1380.8012

1992.9280

1, 7, 8, 10

3750.9979

1, 8

1380.8011

3

764.9412 510.2972 694.4142 463.2782 656.3134

(2+) (3+) (2+) (3+) (3+)

1527.8668 1527.8681 1386.8128 1386.8111 1965.9167

1527.8695

3

1386.8129

4

1965.9171

2, 4, 6

596.3363 (2+) 397.8936 (3+) 435.5870 (3+)

1190.6570 1190.6573 1303.7375

1190.6581

9

1303.7422

9

494.2465 (2+)

986.4774

986.4806

12

540.2609 (2+)

1078.5062

1078.5063

12

575.7789 (2+)

1149.5422

1149.5439

12

672.9885 (3+) 504.9932 (4+)

2015.9420 2015.9415

2015.9440

12

557.2678 (4+)

2225.0399

2225.0390

12

385.2252 (3+) 577.3347 (2+) 606.2804 (2+)

1152.6521 1152.6538 1210.5452

1152.6537

12

1210.5450

12

669.3013 (2+)

1336.5870

1336.5880

13

649.7955 (2+)

1297.5754

1297.5771

13

N-terminal amino acids derived from the a1 ions on each MS2 spectrum. bProtein index according to Table 1.

analyzed with trypsin digestion followed by dimethyl labeling and RADAR search. For the native CTX III, two disulfidelinked peptides were observed as listed in Table 4A, which suggests a perfect disulfide structure without any scrambling. For the CTX III isomer, 10 disulfide-linked peptides were identified, and several cysteines were found present in different disulfide bonds as listed in Table 4B. For example, all possible connections via C3, C14, and C21 including C3−C14, C3−C21, and C14−C21 were observed, which indicates that the CTX III isomer is a mixture of proteins with different disulfide structures. Interestingly, the absence of the disulfide-linked peptides “TCPAGK, GCIDVCPK, YVCCNTDR, CN (m/z 701.59, 4+)” suggests that the native CTX III does not exist in the recovered

manufacturing of protein isomer products can be optimized and unified by measuring their disulfide status with the proposed method. In this study, the venom protein CTX III (cytotoxin 3) as well as its scrambled isomer X-CTX III was analyzed. The scrambled isomer preparation was initiated with full reduction of the native CTX III followed by oxidative refolding under diluted denaturing condition. The resulting protein mixture was then subject to HPLC analysis. Figure 2 is a comparative chromatogram showing the fully reduced, scrambled and native forms of CTX III, which were detected at the retention time (tR) of 25.88, 27.18, and 28.82 min, respectively. Both the native CTX III and its scrambled form, X-CTX III, were further 8748

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Table 4. Identified Disulfide-Linked Peptides from CTX III and its Scrambled Isomer A. native CTX III index (1) (2)

disulfide-linked peptides

Cys location

N-term. A.Aa

observed m/z

experimental mass (Da)

calculated mass (Da)

*L*KCN*K *NLCY*K *TCPAG*K *GCIDVCP*K *YVCCNTDR *CN

C3 C21 C14 C38C42 C53C54 C59

L N T G Y C

468.2799(3+)

1401.8162

1401.9081

701.5897(4+)

2802.3275

2802.3849

B. X-CTX III index (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) a

disulfide-linked peptides

Cys location

N-term. A.Aa

observed m/z

experimental mass (Da)

calculated mass (Da)

447.9197 (3+)

1340.7356

1340.7888

431.2166 646.3505 446.9566 669.9427 468.2932

(3+) (2+) (3+) (2+) (3+)

1290.6263 1290.6854 1337.8463 1337.8698 1401.8561

1290.7218

896.4698 502.2139 480.4816 640.3105 633.6450

(1+) (2+) (4+) (3+) (3+)

895.4620 1002.4122 1917.8951 1917.9080 1897.9115

895.4747 1002.4201 1917.9625

465.1991 (4+)

1856.7651

1856.8432

481.2050 (4+)

1920.7887

1920.8745

*TCPAG*K *NLCY*K *TCPAG*KNLCY*K

C14 C21 C14C21

T N T

*L*KCN*K *TCPAG*K *L*KCN*K *NLCY*K *GCIDVCP*K *YVCCNTDR *YVCCNTDRCN *L*KCN*K *GCIDVCP*K *YVCCNTDR *YVCCNTDRCN *TCPAG*K *YVCCNTDRCN *NLCY*K

C3 C14 C3 C21 C38C42 C53C54 C53C54C59 C3 C38C42 C53C54 C53C54C59 C14 C53C54C59 C21

L T L N G Y Y L G Y Y T Y N

1337.8768 1401.9081

1897.8949

N-terminal amino acids derived from the a1 ions on each MS2 spectrum

X-CTX III possibly due to insufficient refolding time.40 This result is consistent with the HPLC profile, in which the native CTX III is not detected in X-CTX III. The identification of scrambled disulfide bonds used to be an arduous task because the traditional methods require the manual inspection of all possible m/z for all cysteine combinations. Now the intensive labor work can be avoided using dimethyl labeling combined with LC-MS/MS and RADAR. The structural information generated with this method is not only valuable for investigating protein oxidative folding but also helpful for producing potential antivenom with cross reactivity.

disulfide-linked peptides from trypsin or thermolysin digestion. This material is available free of charge via the Internet at http://pubs.acs.org.



*E-mail: [email protected]. Notes

The authors declare no competing financial interest.





ACKNOWLEDGMENTS This work was supported by grants from the National Health Research Institutes under project no. 03A1-IVpp23-014 and from the National Science Council of Taiwan under Contracts NSC 102-2113-M-003-001-MY3. The authors also thank Centers for Disease Control (Taiwan CDC) and the core facility of NHRI Bioproduction plant for their technical assistance.

CONCLUSIONS In summary, the use of dimethyl labeling coupled with mass spectrometry and RADAR search proves to be a simple and automatic tool to analyze the disulfide linkages in protein mixture. For crude snake venom, the disulfide patterns of PLA2 and several subtypes of cytotoxins were successfully identified without protein separation. For CTX III isomer, the scrambled disulfide bonds were solved and compared with the native CTX III. This platform does not require the complex sample purification procedure or time-consuming data interpretation. It is suitable for global disulfide profiling or disulfide mismatch analysis of various kinds of samples and thus can further contribute to venomic/antivenomic studies and novel drug discovery.



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ASSOCIATED CONTENT

S Supporting Information *

Experimental section and results of the cobra venom protein identification; the representative MS2 spectra for these 8749

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