Determination of the Disulfide Structure of Murine Meteorin, a

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Determination of the Disulfide Structure of Murine Meteorin, a Novel Neurotrophic Factor, by LC# MS and ETD-HCD Analysis of Proteolytic Fragments Dingyi Wen, Yongsheng Xiao, Malgorzata Monika Vecchi, Bang Jian Gong, Jana Dolnikova, and R. Blake Pepinsky Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04600 • Publication Date (Web): 28 Feb 2017 Downloaded from http://pubs.acs.org on March 1, 2017

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Analytical Chemistry

Determination of the Disulfide Structure of Murine Meteorin, a Novel Neurotrophic Factor, by LC‒MS and ETD-HCD Analysis of Proteolytic Fragments Dingyi Wen,* Yongsheng Xiao, Malgorzata M. Vecchi, Bang Jian Gong, Jana Dolnikova, R. Blake Pepinsky

Department of Protein Drug Discovery, Biogen Inc., 225 Binney Street, Cambridge, Massachusetts 02142

Corresponding Author *E-mail: [email protected]. Phone: (617) 679-2362. Fax: (617)-679-3208.

1 ACS Paragon Plus Environment


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ABSTRACT Meteorin and Cometin (Meteorin-like) are secreted proteins belonging to a newly discovered growth factor family. Both proteins play important roles in neural development and may have potential as therapeutic targets or agents. Meteorin and Cometin are homologs and contain ten evolutionarily conserved Cys residues across a wide variety of species. However, the status of the Cys residues has remained unknown. Here, we have successfully determined the disulfide structure for murine Meteorin by LC‒MS analysis of fragments generated by trypsin plus endoprotease-Asp-N. For proteolytic fragments linked by more than one disulfide bond, we used electron transfer dissociation (ETD) to partially dissociate disulfide bonds followed by highenergy collisional dissociation (HCD) to determine disulfide linkages. Our analysis revealed that the ten Cys residues in murine Meteorin form five disulfide bonds with Cys7 (C1) linked to Cys28 (C2), Cys59 (C3) to Cys95 (C4), Cys148 (C5) to Cys219 (C8), Cys151 (C6) to Cys243 (C9) and Cys161 (C7) to Cys266 (C10). Since the ten Cys residues are highly conserved in Meteorin and Cometin, it is likely that the disulfide linkages are also conserved. This disulfide structure information should facilitate structure-function relationship studies on this new class of neurotrophic factors and also assist in evaluation of their therapeutic potentials.


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Meteorin (Metrn) and Cometin (Metrnl, Meteorin-like, Subfatin, Interleukin 39) are relatively newly identified neurotrophic factors.1-3 Both Meteorin and Cometin are secreted 30kDa proteins. They are homologous (~40 percent identical amino acids) and belong to a novel, evolutionarily conserved growth factor family.3-6 Meteorin is expressed mainly in the central and peripheral nervous system with higher expression levels in neural and glial progenitors, while Cometin is widely distributed in the body with higher expression levels in white adipose tissue and barrier tissues6. Studies have shown that Meteorin is important for glial and neuronal cell differentiation during development and promotes neurogenesis1,6-9 and that Cometin promotes neurite outgrowth and migration, regulates immune-adipose interactions and plays an important role in the biology of white adipose tissue.3,6,10 Meteorin protein has shown potential as a drug for neurophathic pain.11 Meteorin exposure correlated well with dosing although it was no longer detectable after 24 hours.11 To study structure-function relationships and improve the half-life of the protein, we need to make various engineered forms, including different short forms and fusion proteins. Therefore knowledge of the disulfide linkages in Meteorin/Cometin is required for designing forms that have no unpaired or mispaired Cys residues as these are likely to cause scrambling or misfolding.12,13 Both Meteorin (Metrn) and Cometin (Metrnl) contain ten Cys residues that are conserved in all the species of Meteorin and Cometin analyzed so far (Figure 1). However, the disulfide linkages of Meteorin and Cometin are unknown because the Cys residues do not show a pattern similar to those of any proteins for which disulfide connectivity has been determined. Our efforts to obtain crystal structures of Meteorin and Cometin have not been successful.

To facilitate investigation of structure-function

relationships, to help evaluation of therapeutic potentials of Meteorin and Cometin, and to make a ‘gold’ standard protein for engineered Meteorin and Cometin proteins, we determined the



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disulfide structure of recombinant murine Meteorin (mMeteorin) by LC‒MS analysis with electron-transfer dissociation (ETD) and high-energy collisional dissociation (HCD) of peptides generated by digestion with a combination of trypsin and endoprotease-Asp-N (Asp-N). The ETD-HCD method we implemented for evaluation of Meteorin should be readily applicable to the analysis of the disulfide structures of other proteins, in cases where simple LC-MS analysis is insufficient.

EXPERIMENTAL PROCEDURES Expression of mMeteorin. Expression plasmid pACE197, with a cytomegalovirus promoter and encoding mMeteorin followed by Ires-mDHFR (Internal ribosome entry site-murine dihydrofolate reductase) for selection, was engineered for expression from CHO (Chinese Hamster Ovary) cells. Clones were isolated by gene amplification with methotrexate and singlecell cloning. Two clones were selected and scaled-up for purification. For production runs, cultures were expanded in serum-free media and grown at 37 ºC for 5 days to high density (5 x 106 cells/mL) with appropriate feeds, and then shifted to a reduced temperature of 28 °C. Cultures were held at this temperature for 13 days and then harvested by centrifugation and by passage through 0.45 micron capsule filters. Purification of mMeteorin. Four liters of clarified CHO culture medium containing expressed mMeteorin (estimated titer of 65 mg/L) was mixed with 1.2 L of 120 mM MES, pH 5.0. mMeteorin was sequentially purified on a Q Sepharose column (GE Healthcare) with a column volume (CV) of ~400 mL, a Butyl Sepharose, FF column (CV ~70 mL, GE 17-0980-01), a NiNTA FF column (CV ~70 mL, Qiagen), and a TMAE Fractogel column (CV ~70 mL, EMD


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Chemicals) as described in detail as follows. The medium was loaded on the Q Sepharose column equilibrated with 20 mM MES [2-(N-morpholino)ethanesulfonic acid], pH 5.0, 20 mM NaCl. The Q-Sepharose flow through fraction (5.2 L) containing mMeteorin was adjusted to 6.3 L final volume, 0.5 M (NH4)2SO4, by adding 1.13 L of 2.4 M (NH4)2SO4, and this solution was loaded onto the Butyl Sepharose FF column equilibrated in 20 mM sodium phosphate, pH 7.5, 0.1 M NaCl, 0.5 M (NH4)2SO4. The Butyl column was washed with 70 mL of the equilibrating buffer and then step-eluted with 3 CV of 0.2 M (NH4)2SO4 followed by 3 CV of 0.1 M (NH4)2SO4 in 20 mM sodium phosphate, pH 7.5. 0.1 M NaCl, and finally by 3 CV of 20 mM sodium phosphate, pH 7.2, 50 mM NaCl without (NH4)2SO4.

The fractions containing

mMeteorin, identified by SDS-PAGE (Sodium dodecyl sulphate-Polyacrylamide gel electrophoresis), were pooled and adjusted to 0.5 M NaCl and loaded onto the Ni-NTA FF column equilibrated in 20 mM sodium phosphate, pH 7.2, 0.5 M NaCl. The Ni-NTA column was washed with the equilibrating buffer followed by 20 mM sodium phosphate, pH 7.2, 50 mM NaCl and mMeteorin was then step-eluted with 3 CV of 5 mM imidazole, 20 mM sodium phosphate, pH 7.2, 50 mM NaCl.

The mMeteorin-containing fractions were pooled and

concentrated to about 120 mL using an Amicon Ultra-centrifugal filter unit with molecular weight cut off (MWCO) 10K and then loaded onto the TMAE Fractogel column equilibrated in 20 mM sodium phosphate, pH 7.2, 50 mM NaCl. The flow-through fractions were pooled, concentrated using an Amicon Ultra-centrifugal filter unit with MWCO 10K, and dialyzed into phosphate buffered saline (20 mM sodium phosphate, pH 7.2, 150 mM NaCl). The protein concentration was determined from absorbance at 280 nm using an extinction coefficient of 1.25 (0.1%, 1 cm). A total of 112 mg of mMeteorin, at 0.63 mg/mL was recovered.



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SDS-PAGE Analysis. The purity of mMeteorin from each chromatography purification step was checked by SDS-PAGE with Coomassie blue staining. For analysis of reduced proteins, samples were treated with Laemmli sample buffer14 containing 2.5 mM DTT (dithiothreitol), then heated at 95 °C for 2 min prior to SDS-PAGE analysis on 4-20% Tris-glycine gradient gels (Novex). For analysis of proteins under non-reducing conditions, samples were diluted with Laemmli non-reducing sample buffer and heated at 95 °C for 2 min. Gels were stained with SimplyBlue Safe stain from Novex. Size Exclusion Chromatography. A purified mMeteorin sample (~100 µg) was subjected to size exclusion chromatography (SEC) at room temperature on a Superdex 75 HR10/30 FPLC column (GE Healthcare) in PBS (Phosphate Buffered Saline).

The column effluent was

monitored for absorbance at 280 nm. Molecular weight standards were run as controls and their chromatograms overlaid on the test sample chromatogram. Intact Mass Measurement. mMeteorin was reduced with 40 mM DTT in PBS, pH 7.4, containing 4 M urea. The samples were then analyzed on an LC‒MS system comprised of a UPLC (ACQUITY, Waters Corp.), a TUV dual wavelength UV detector (Waters Corp.), and an LCT mass spectrometer (Waters Corp.).

A Vydac C4 cartridge was used for desalting.

Molecular masses were obtained by deconvolution of raw mass spectra using the MaxEnt 1 program embedded in MaxLynx 4.1 software (Waters Corp.). Tryptic Digestion, Endoprotease-Asp-N Digestion, Tryptic + Endoprotease-Asp-N Digestion, and Separation of Digests. About 0.4 µL of 4-vinylpyridine (4-vp) was added to a solution containing ~60 g of mMeteorin protein followed by immediately adding 95 mg of solid guanidine hydrochloride. The solution was held at room temperature in the dark for 35 min. The 4-vp-treated protein was recovered by precipitation with 40 volumes of cooled ethanol in 10


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vials (~6 µg/vial). The mixture was stored at -20 C for 1 h and then centrifuged at 14,000g for 8 min at 4 C. The supernatant was discarded and the precipitate was washed once with cold (– 20 ºC) ethanol. The protein pellet was redissolved with 100 µL of 50% acetonitrile and dried in a Speed-Vac concentrator (Savant). For tryptic digestion, about 6 µg of the 4-vp-treated protein was digested with 15% (w/w) trypsin (Catalog No. V5111, Promega) in 2 M urea, 10 mM CaCl2, 0.2 M Tris-HCl, pH 6.7, at room temperature for 8 h; the digestion volume was ~50 µL. Digestion with endoprotease-Asp-N was similar to that with trypsin described above, except that 15% of endoprotease-Asp-N (Catalog No. 11054589001, Roche) was used. For digestion with trypsin + endoprotease-Asp-N (tryptic/Asp-N digestion), after the protein had been digested with 10% (w/w) trypsin for 8 h, an additional 10% (w/w) trypsin was added to the solution and the solution was held at room temperature overnight. Then, 10% (w/w) of endoprotease-Asp-N was added to the tryptic digest, after which the solution was held at room temperature for 8 h; afterward, an additional 10% (w/w) of endoprotease-Asp-N was added to the solution and the digestion was allowed to proceed at room temperature overnight. The digestion reactions were stopped by acidification with 1 µL of 25% trifluoroacetic acid (TFA) and stored at –80 °C. LC‒MS Analysis. Prior to analysis of the digest on an LC‒MS system, the solution was split into two parts: one part was analyzed after reduction with

100 mM Tris-(2-

carboxyethyl)phosphine hydrochloride at room temperature for 1 h, and the other part was directly analyzed without reduction. The reduced and non-reduced digests were assessed using an LC‒MS system composed of an XEVO G2-S Q TOF mass spectrometer (Waters Corp.) or an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific) and a UPLC (ACQUITY, Waters Corp.), a TUV dual wavelength UV detector (Waters Corp.). Peptides from the digest were eluted from an ACQUITY HSS T3 C18 column (1.8-μm particle size, 2.1 mm x 150 mm; catalog



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no. 186003540, Waters Corp.) with a 75-min water/acetonitrile gradient (0-70% acetonitrile) containing 0.03% TFA at a flow rate of 0.07 mL/min at 30 ºC. About 0.6 µg of the digest was injected for the analysis. The LC-MS data acquired on the UPLC-XEVO G2-S Q TOF system were processed using BiopharmaLynx software version 1.3.3 (Waters Corp.). All tandem mass spectra [ETD, HCD and CID (collision-induced dissociation)] were acquired in the Orbitrap mass analyzer with a resolution of 15,000. The optimized reaction time for ETD was 70 ms with 30-45% HCD supplemental activation. The collision energy was 30-35 V for HCD and 35 V for CID. The automatic gain control (AGC) target was set to 4 x 105 with maximum injection time of 200 ms. Disulfide-linked peptide clusters containing Cys residues were identified by mass spectrometry, and disulfide linkages in a disulfide-linked peptide cluster containing more than one disulfide bond were determined by tandem mass spectrometry analysis as described below. Tandem mass spectrometry with ETD followed by HCD or CID. The disulfide-linked peptide cluster containing more than one disulfide bond was analyzed on an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific) equipped with a nanoUPLC (nanoACQUITY, Waters Corp.) and a nano-electrospray ionization source. The separation of peptides was achieved by using a 1.8-μm particle size 75-μm x 150-mm Acquity HSS T3 C18 column (Catalog no. 186005776, Waters Corp.) with Stainless Steel Emitters (Thermo Fisher Scientific, Catalog no. ES542) and a 100-min water/acetonitrile gradient (0-80% acetonitrile) containing 0.1% formic acid at a flow rate of 0.3 μL/min at 30 ºC. The nanospray voltage was set at 1.7 kV for stable spray. Targeted acquisition with a ±1.5 m/z isolation width was carried out to determine peptide sequences and disulfide linkages. All tandem mass spectra were acquired in the Orbitrap mass analyzer with a resolution of 30,000, except spectra for ETD-HCD MS3 of ion m/z 409.17 Da which were acquired in the ion trap mass analyzer. The optimized reaction time for ETD was


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70-75 ms with 30% CID or 38% HCD supplemental activation. The optimized collision energy was 35-39 V for HCD and 70 V for CID. The automatic gain control (AGC) target was set to 4 x 105 with a maximum injection time of 300 ms for ETD MS2 and AGC target 1 x 105 with maximum injection time of 200 ms for HCD MS3 and CID MS3 experiments.

RESULTS AND DISCUSSION Determination of Purity and Activity of Recombinant Murine Meteorin.

A construct

encoding murine Meteorin (mMeteorin) was expressed in CHO cells, and the expressed protein was purified from conditioned medium by sequential column chromatography. SDS-PAGE analysis of the purified product revealed a single band with a molecular mass of 30 kDa under both reducing (Figure 2A) and non-reducing conditions (data not shown).

Size exclusion

chromatography showed that the recombinant mMeteorin migrated as a single homogeneous peak with an apparent molecular weight of 30,000 (data not shown). Intact mass measurement of the product, under both reducing and non-reducing conditions, revealed only a single component with detected masses of 29,441 Da for the reduced mMeteorin and 29,431 Da for the native mMeteorin, which agree well with the respective predicted masses of 29,441.6 Da and 29,431.5 Da (assuming five disulfide bonds), respectively (Figure 2B,2C). These results also indicate that the protein does not contain any posttranslational modifications. Analysis of the biological activity, essentially as described by Jorgensen et al.,4 showed that mMeteorin promoted neurite outgrowth on P5 rat dissociated dorsal root ganglions in the presence of astrocytes (data not shown). All these results demonstrate that the mMeteorin used in this study was pure and active. Amino Acid Sequence Analysis and Determination of Disulfide-Linked Cys Residues in Murine Meteorin. As shown in Figure 1, all the species of Meteorin and Cometin discovered to 9 ACS Paragon Plus Environment


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date contain ten highly conserved Cys residues. Thus, resolving the disulfide connectivity for one of the proteins is likely to solve it for the other members of this novel growth factor family. We chose mMeteorin for this study because of the availability of high quality material. The easiest and most straightforward approach to determine disulfide linkages is to enzymatically cleave the protein between each Cys residue and then define the disulfide-linked peptides by mass spectrometry.

Therefore, we digested the mMeteorin with trypsin followed by

endoprotease-Asp-N. The digestion with a combination of trypsin and endoprotease-Asp-N will generate peptides containing a single Cys residue for all except C5 (Cys148) and C6 (Cys151) because the Arg149Pro150 sequence between them may not be cleaved by trypsin. LC‒MS analysis of the peptide map of the reduced tryptic/Asp-N digest resulted in identification of all significant components; the identified peptides accounted for 98% of the predicted sequence. Undetected in the tryptic/Asp-N map were small and hydrophilic peptides that presumably coelute with the solvent peak. Nevertheless, the sequences of these missing tryptic/Asp-N peptides could be deduced from individual tryptic and Asp-N peptide maps (Table 1). The mMeteorin used in this study is a recombinant protein which expression construct was confirmed by DNA sequencing. The peptide masses, detected using high resolution mass spectrometers, matched only peptide sequences predicted from the cleavage specificity of the proteases used (see Table 1) within an error range of ≤10 ppm or ≤0.02 Da. In addition, peptide sequences were further confirmed by tandem mass spectrometric analysis with CID, HCD, and/or ETD fragmentation. Thus, the identified peptides from a combination of tryptic, endoprotease-Asp-N, and tryptic/Asp-N peptide mapping accounted for 100% of the predicted mMeteorin sequence. Because the native protein was treated with 4-vinylpyridine prior to enzymatic cleavage, any Cys residues in the free thiol state should have been pyridylethylated, whereas Cys residues involved


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in disulfide bonds should have been detected as free cysteine after reduction. Peptide mapping analysis of the reduced tryptic/Asp-N digest identified all the Cys-containing peptides and no peptides containing a pyridylethylated Cys were observed, indicating that all ten Cys residues in the protein are either involved in disulfide bond formation or are otherwise modified. All identified peptides are listed in Table 1 with major peptides shown in bold and Cys residues in red fonts. Identification of Disulfide-Linked Peptide Clusters and Determination of Disulfide Linkages. LC‒MS analysis of the non-reduced tryptic/Asp-N digest led, by comparison of the map of the reduced digest, to identification of all disulfide-linked peptide clusters. Figure 3 shows tryptic/ Asp-N peptide maps for both the reduced and non-reduced digests with Cys-containing peptides marked. All identified disulfide-linked peptide clusters are unique to the peptide map of the nonreduced digest (Table 2; Figure 3B) and are absent in the map of the reduced digest (Table 1; Figure 3A), while all the corresponding Cys-containing peptides are seen in the reduced digest as unique components (Table 1; Figure 3A). As can be seen in Table 2, peptide TD3 (residues 7‒ 10, C1) is disulfide-linked to peptide TD5 (residues 27‒43, C2), TD6-7 (residues 44‒75, C3) linked to TD10 (residues 95‒97, C4), TD22 (residues 153‒163, C7) linked to TD33 (residues 253‒269, C10) and TD21 (residues 145‒152, C5 & C6) linked to two peptides, TD30 (residues 219‒235, C8) and TD31 (residues 236-246, C9). The identities of the disulfide-linked peptide clusters listed in Table 2 are the only possible assignments based on the protease specificities, accurate mass measurements (≤10 ppm or ≤0.02 Da), and the corresponding Cys-containing peptides detected after the reduction. Because each of the peptides TD3, TD5, TD6-7, TD10, TD22 and TD33 contains only a single Cys residue, the disulfide linkages are clearly C1 to C2, C3 to C4, and C7 to C10.

The identities and linkages of these peptide clusters were further



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confirmed by CID, HCD, or ETD fragmentation experiments. For example, Figure 4 indicates how these techniques are used for confirming the identities of the components. ETD MS2 analysis of the quadruply charged precursor ion, m/z 852.71 Da, for peptide TD6-7 and HCD MS2 analysis of singly charged precursor ion, m/z 409.17 Da, for peptide TD10 in the reduced digest confirmed the identities of the two peptides (Figures 4A and 4B, respectively). As shown in Figure 4C, ETD MS2 analysis of quadruply charged precursor ion, m/z 954.24 Da for disulfide-linked peptide cluster, TD6-7/TD10, revealed ETD-dissociated peptide ions, TD6-72+ and TD6-73+ (detected m/z = 1704.40 Da and calculated m/z = 1704.404 Da) and TD10 (detected m/z = 409.17 Da and calculated m/z = 409.169 Da), confirming that the cluster consists of the two peptides. The detected fragment ions of TD6-7 in the spectrum not only confirmed the identity of peptide TD6-7, but also demonstrated that C3 in TD6-7 is disulfide-linked to C4 in TD10 because all detected TD6-7 fragment ions containing C3, are linked to peptide TD10 which contains C4, e.g., ions TD10/TD6-7 c202+, TD10/TD6-7 c272+, TD10/TD6-7 c302+, TD10/TD6-7 c312+, TD10/TD6-7 z262+, TD10/TD6-7 z282+ and TD10/TD6-7 z302+ shown in Figure 4C.

Furthermore, ETD-HCD MS3 analysis of ETD-dissociated peptide ion, TD10+

(m/z=409.17 Da), in ETD MS2 spectrum (Figure 4C) confirmed the sequence of TD10 (Figure 4D). Other disulfide-linked peptide clusters were also confirmed by tandem mass spectrometric analysis. The CID MS2 spectrum for analysis of the peptide cluster TD3/TD5 is shown in Figure S-1; the HCD MS2 spectrum for the reduced peptide TD3 and CID MS2 spectrum for the reduced peptide TD5 are shown in Figure S-2. The ETD MS2 spectra for analysis of the peptide clusters, TD22/TD33 and TD22/TD33-34, are shown in Figure S-3, while Figures S-4 and S-5 show the HCD MS2 and CID MS2 spectra for the corresponding reduced peptides, TD22, TD33 and TD33-34, respectively. The identities of the reduced Cys-containing peptides, TD21, TD30 and


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TD31, were also confirmed by tandem mass spectrometry (Figure S-6). However, three peptides in the peptide cluster, TD21/TD30/TD31, are linked by two interpeptide disulfide bonds because TD21 contains two Cys residues, C5 and C6; thus additional work was required to determine the respective linkages of C5 or C6 to C8 or C9. In past studies, we used a strategy that utilized a partial reduction-alkylation method followed by LC-MS/MS analysis, to determine disulfide linkages for peptide clusters containing more than one disulfide bond.12,15

Although the approach works well to resolve complex disulfide

structures for proteins, the method not only requires fractionation of the disulfide-linked peptide clusters prior to partial reduction but may also require separation of partially reduced/alkylated products, which is inconvenient, sometimes difficult and requires much more material. In 2009, Wu et al.16 developed an online LC‒MS method utilizing ETD, based on the observation that ECD (electron capture dissociation) and ETD preferentially cleave disulfide bonds.17,18 This method uses ETD for partial dissociation of disulfide bonds followed by collision-induced dissociation (CID) for disulfide linkage determination. Using this method they successfully confirmed disulfide linkages in peptide clusters containing more than one disulfide bond for recombinant tissue plasminogen activator16,19 and for recombinant human arysulfatase A.20 The strategy was also successfully utilized by Nili et al. to resolve five unknown disulfide linkages in insulin-like growth factor-binding protein-5.21 To simplify procedures for disulfide linkage determination of TD21/TD30/TD31, we implemented a similar multistage fragmentation strategy to the cluster. We used ETD MS2 to partially dissociate disulfide linkages in TD21/TD30/TD31, which serves the function of partial reduction, then used HCD MS3 to fragment peptide backbones of a partially dissociated (partially reduced) peptide cluster containing one of the disulfide bonds in order to determine the Cys residues that formed the disulfide linkage. The



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mass of the disulfide-linked peptide cluster, TD21/TD30/TD31, was 3861.741 Da, which closely matches the calculated peptide mass of 3861.7207 Da for the cluster with two disulfide bonds (Table 2). The most abundant ion detected for the peptide cluster TD21/TD30/TD31 was the quintuply charged ion m/z 773.35 Da [calculated m/z = 773.352 Da (z=5)], when using the nanoUPLC-Orbitrap Fusion MS system. Therefore we selected this ion for partial dissociation of the disulfide bonds by ETD. As shown in the ETD MS2 spectrum (Figure 5A), the two disulfide bonds in the TD21/TD30/TD31 cluster were successfully partially dissociated. The ions of interest were partially dissociated clusters TD21/TD312+ (detected m/z = 1005.93 Da and calculated m/z = 1005.929 Da, -S• form) and TD21/TD302+ (detected m/z = 1330.09 Da and calculated m/z = 1330.0934 Da, -SH form) because each contained one disulfide bond and a Cys residue with a broken disulfide bond (-S• form) or a reduced Cys (-SH form). To determine the exact linkages among these four Cys residues, the key was to break a backbone between C5 and C6 in peptide TD21 for one of the two partially dissociated clusters by HCD or CID. Figure 5B shows the HCD spectrum for the partially dissociated peptide cluster TD21/TD312+. As shown in Figure 5B, HCD broke the backbone between Arg149 and Pro150 in TD21 and produced a disulfide linked fragment ion (TD31/TD21y3)+ (detected m/z = 1509.66 Da and calculated m/z = 1509.662 Da), demonstrating that C6 in TD21 is linked to C9 in TD31. In addition, a high abundance disulfide-linked fragment ion (TD21y3/TD31y5)+, generated from further cleavage between Leu241 and Gly242 in TD31 was observed in the HCD spectrum (detected m/z = 806.29 Da and calculated m/z = 806.329 Da), confirming the disulfide linkage between C6 and C9. Furthermore, fragment ion, TD21 b5+, containing C5, was also detected (detected m/z = 503.20 Da and calculated m/z = 503.204 Da), indicating that C5 is free in the disulfide-linked TD31/TD21 cluster. Because C6 is disulfide-linked to C9, C5 in TD 21 must be linked to C8 in


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TD30 by a disulfide bond. Although CID fragmentation of the TD21/TD312+ also cleaved the backbone between Arg149 and Pro150 in TD21 and produced the abundant critical fragment ion (TD21y3/TD31y5)+ for determination of the disulfide linkage, it generated fewer types of fragment ions and provided less structural information, compared to the HCD fragmentation. For example, the intensity of the ion (TD31/TD21y3)+ was very low and the ion TD21 b5+ was not observed in the CID spectrum at all (Supplemental Figure S-7). We also tried to analyze the disulfide linkage in the partially dissociated disulfide-linked peptide cluster ion TD21/TD302+ (Figure 5A) by both HCD and CID with various fragmentation energies. However, although many fragment ions were obtained, no critical fragment ions generated by cleavage between C5 and C6 in TD21 were observed. Highly abundant ions were those either having a cleaved disulfide bond or containing all three Cys residues. Nevertheless, successful analysis of disulfide linkage in the partially disulfide dissociated TD31/TD21 cluster, resulted in complete disulfide structure determination for mMeteorin.

To summarize, the ten Cys residues formed five

disulfide bonds with C1 linked to C2, C3 to C4, C5 to C8, C6 to C9 and C7 to C10 (Figure 6). Our study also demonstrated that using ETD for partial dissociation followed by HCD fragmentation (ETD-HCD) is an efficient approach for determination of disulfide linkages in peptide clusters containing more than one disulfide bond. Because the ETD partial dissociation of disulfide bonds is done online using a LC-MS system with the ETD function, the approach does not require fractionation of the disulfide-linked peptide clusters of interest, therefore it is more convenient than the partial reduction and alkylation approach.12,15 However, the ETDHCD/CID approach may not work if it is unable to produce critical ions for disulfide linkage determination. For example, HCD or CID fragmentation often cannot cleave the backbone between two contiguous Cys residues if one of them was disulfide-linked to another peptide.12,21



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In our experience, the disulfide linkages in a peptide cluster with two contiguous Cys residues need to be determined by the partial reduction and alkylation method.15

CONCLUSIONS We have completely determined the disulfide linkages for all ten Cys residues in murine Meteorin, a member of a novel, evolutionarily conserved growth factor family, by LC―MS analysis and the ETD-HCD approach, showing that C1 is linked to C2, C3 to C4, C5 to C8, C6 to C9 and C7 to C10. Since the ten Cys residues are highly conserved in Meteorin and Meteorinlike proteins from all different species, the disulfide structure information will certainly help engineering, molecular modeling and studies of structure-function relationships for this novel family of growth factors. This work has also demonstrated that the sequential ETD-HCD fragmentation could be a useful, efficient approach for determining disulfide linkages in peptide clusters containing more than one disulfide bond.

ACKNOWLEDGEMENTS The authors thank Param Murugan, Nels Pederson and Thomas Cameron for cloning and expression of mMeteorin; Yuting Huang for intact mass measurement of the reduced protein and Anthone Dunah and Charles Banos for assessment of biological activity of the protein by means of a neurite outgrowth assay. SUPPORTING INFORMATION Tandem mass spectra for the disulfide-linked peptide clusters and reduced Cys-containing peptides (PDF)


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Jørgensen, J. R.; Fansson, A.; Fjord-Larsen, L.; Thompson, L. H.; Houchins, J. P.; Andrade, N.; Torp, M.; Kalkkinen, N.; Andersson, E.; Lindvall, O.; Ultendahl, M; Brunak, S.; Johansen, T. E.; Wahlberg, L. U. Exp. Neurol. 2012, 233, 172-181.

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Jørgensen, J. R.; Thompson, L.; Fjord-Larsen, L.; Krabbe, C; Torp, M.; Kalkkinen, N.; Hansen, C.; Wahlberg, L. J. Mol. Neurosci. 2009, 39, 104-116 .

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Lee, H. S.; Han, J.; Lee, S. H.; Park, J. A.; Kim, K. W. J. Cell Sci. 2010, 123, 1959-1968.

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Jørgensen, J. R.; Xu X. J.; Arnold, H. M.; Munro, G.; Hao, J. X.; Pepinsky, B.; Huang, C.; Gong, B. J.; Wiesenfeld-Hallin, Z.; Wahlberg, L. U. and Johansen, T. E. Exp. Neurol. 2012, 237, 260-266.

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Foley, S.; Sun, Y.; Zheng, T. S.; Wen, D. Anal. Biochem. 2008, 377, 95‒104. 17 ACS Paragon Plus Environment


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Figure Legends Figure 1. Amino acid sequences of Meteorin (Metrn) and Cometin (Metrnl) precursors of various species in the UniProtKB/Swiss-Prot database, aligned with Vector NTI Advance 11.5.1. Identical amino acids are marked in yellow; similarity is marked in light blue; Cys residues in mature proteins are in red fonts. Hyphens indicate positions of potential gaps in the sequences.

Figure 2. (A) SDS-PAGE of reduced mMeteorin. Lane 1, 2 µg nonreduced mMeteorin; lane 2, 5 µg nonreduced mMeteorin; lane 3, molecular weight markers (molecular weights are noted at the right of the panel). (B) Deconvoluted mass spectrum of reduced mMeteorin. (C) Deconvoluted mass spectrum of non-reduced mMeteorin. The 29555-Da component in (B) and 29545-Da component in (C) are proteins with TFA adducts. Figure 3. Total ion chromatograms of tryptic/Asp-N maps of mMeteorin. Cys-containing peptides are labeled in blue. (A) Reduced digest; (B) non-reduced digest. Identified peak characteristics are summarized in Tables 1 and 2. Figure 4. Tandem mass spectra for analyzing Cys-containing peptides TD6-7, TD10 and their disulfide linkages. (A) ETD MS2 spectrum of precursor ion (Pi4+) of the reduced peptide TD6-7, m/z 852.71 Da. Internal fragment ions are labeled with grey fonts and * are for precursor ions with neutral loss. (B) HCD MS2 spectrum of precursor ion (Pi+) of the reduced TD10, m/z 409.17 Da. (C) ETD-HCD MS2 spectrum of precursor ion (Pi4+) of the disulfide-linked peptide cluster TD6-7/TD10, m/z 954.24 Da; the inserted fragmentation scheme shows the peptide sequences of TD6-7/TD10 with annotation of the fragment ions; ions related to TD6-7 (P1) are in blue and ions related to TD10 (P2) are in red. (D) ETD-HCD MS3 spectrum of the ion TD10+, m/z 409.2 Da, from the ETD MS2 experiment shown in (C). Figure 5. Tandem mass spectra for analyzing disulfide linkages in peptide cluster TD21/TD30/TD31. (A) ETD MS2 spectrum of precursor ion (Pi5+), m/z 773.35 Da; the ions containing a disulfide bond and a free Cys are in red. (B) ETD-HCD MS3 spectrum of the ion TD21/TD312+, m/z 1005.93 Da, from the ETD MS2 experiment shown in (A); the inserted box shows the peptide sequences of TD21/TD31 with annotation of the fragment ions; ions related to TD21 (P1) are in red and ions related to TD31 (P2) are in blue. Figure 6. Schematic summary of the disulfide connectivity within the mMeteorin sequence. Brackets represent disulfide bonds determined in this study. The designations C1‒ C10 represent the relative positions of Cys residues from the N-terminus to the Cterminus.



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Page 20 of 28

Table 1. List of peptides detected on LC–MS analysis of the reduced tryptic/Asp-N digest. ≠ Peptide Label

Peptide Sequence

Residue Number

RT (Min)

TD1 TD2-3 TD3 TD4 TD5 TD6-7 TD6-7a TD6-7b TD6-7c TD8 TD8a TD8b D5* TD9 TD10 oxTD10 TD11 TD13-14 TD14 D6* TD15 TD17 TD17-18 TD18 T14^ TD20 TD21 TD22 TD23 TD24a TD24 TD24b TD25-26 TD26 TD27 TD28 TD29 TD30 oxTD30 TD31 TD32 TD33-34 TD33

GYSE DRCSWR CSWR GSGLTQEPGSVGQLTL DCTEGAIEWLYPAGALR LTLGGPDPGTRPSIVCLRPERPFAGAQVFAER LTLGGPDPGTRPSIVCLRPERPFAGAQVFA LTLGGPDPGTRPSIVCLRPERPFAGAQVF LTLGGPDPGTRPSIVCLRPER MTGNLELLLAEGP MTGNL ELLLAEGP DLAGGRCMRWGPRERRALFLQATPHR DLAGGR CMR CMR WGPR RALFLQATPHR ALFLQATPHR DISRRVAAFRFELHE DISR VAAFR VAAFRFELHE FELHE FELHEDQR AEMSPQAQGLGV DGACRPCS DAELLLAACTS DFVIHGTIHGVAH DTELQ DTELQESVITVVVAR ESVITVVVAR VIRQTLPLFK QTLPLFK EGSSEGQGR ASIR TLLR CGVRPGPGSFLFMGWSR CGVRPGPGSFLFMGWSR FGEAWLGCAPR FQEFSR VYSAALTTHLNPCEMALD VYSAALTTHLNPCEMAL

1-4 5-10 7-10 11-26 27-43 44-75 44-73 44-72 44-64 76-88 76-80 81-88 89-114 89-94 95-97 95-97 98-101 104-114 105-114 115-129 115-118 120-124 120-129 125-129 125-132 133-144 145-152 153-163 164-176 177-181 177-191 182-191 192-201 195-201 202-210 211-214 215-218 219-235 219-235 236-246 247-252 253-270 253-269

29.6 35.6 36.3 53.5 68.9 57.6 62.1 62.9 48.8 69.7 37.5 54.5 43.2 30.9 27.9 23.9 36.4 41.2 45.2 46.3 28.5 36.3 51.6 39.7 34.1 42.6 30.5 61.7 41.1 32.2 59.3 48.3 52.7 52.6 25.2 29.6 37.2 70.6 63.9 56.0 37.8 60.0 63.9

≠

Detected Mass (Da) 454.166 821.357 550.227 1542.785 1863.879 3406.777 3121.641 3050.605 2233.194 1356.691 534.242 840.459 3049.587 587.299 408.160 424.156 514.260 1308.735 1152.630 1844.976 489.250 562.318 1217.610 673.305 1072.498 1186.557 807.299 1105.524 1401.712 604.267 1657.887 1071.618 1213.748 845.496 905.378 445.261 501.322 1852.883 1868.877 1205.559 812.379 1947.903 1832.875

Calculated Peptide Mass (Da) 454.1700 821.3603 550.2322 1542.7889 1863.8825 3406.7932 3121.6495 3050.6124 2233.2001 1356.6959 534.2472 840.4593 3049.5828 587.3027 408.1613 424.1562 514.2652 1308.7415 1152.6404 1844.9646 489.2547 562.3227 1217.6193 673.3071 1072.4938 1186.5652 807.3004 1105.5325 1401.7153 604.2704 1657.8887 1071.6288 1213.7546 845.5011 905.3839 445.2649 501.3275 1852.8865 1868.8859 1205.5652 812.3817 1947.9070 1832.8801

Intensity (Counts) 44755 39880 33315 782284 1210327 1448638 274907 254310 138224 114406 72914 206897 198427 82376 31176 5316 72857 143546 634967 178608 64753 78695 14600 61814 317483 162373 30515 70485 310226 46088 38589 159921 11422 430625 83944 79021 149283 131689 61111 251015 332152 131807 62887

Note

A/E cleavage F/A cleavage R/P cleavage L/E cleavage L/E cleavage For residues 102-103

M oxidation

For residue 119

For residues 130-132

Q/E cleavage Q/E cleavage

M oxidation

Detected major peptides are in bold fonts; a, b, and c indicate peptides resulting from digestion of the corresponding peptide at different sites; for example, peptide TD6-7a was related to peptide TD6-7, but resulted from cleavage between A and E. * Peptides detected in endoprotease-Asp-N peptide map. ^ Peptide detected in tryptic peptide map.


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Table 2. List of disulfide-linked peptide clusters identified by tryptic/Asp-N disulfide mapping using an LC‒MS system.≠ Peptide Clusters TD3/TD5 with one disulfide bond TD2-3/TD5 with one disulfide bond TD1-3/TD4-5 with one disulfide bond TD6-7/TD10 with one disulfide bond TD6-7/oxTD10 with one disulfide bond TD6-7a/TD10 with one disulfide bond TD6-7b/TD10 with one disulfide bond TD6-7c/TD10 with one disulfide bond

≠

Sequences of Disulfide-linked Peptides CSWR=DCTEGAIEWLYPAGALR DRCSWR=DCTEGAIEWLYPAGALR GYSEDRCSWR=GSGLTQEPGSVGQL TLDCTEGAIEWLYPAGALR LTLGGPDPGTRPSIVCLRPERPFAGA QVFAER=CMR LTLGGPDPGTRPSIVCLRPERPFAGA QVFAER=CMR LTLGGPDPGTRPSIVCLRPERPFAGA QVFA=CMR LTLGGPDPGTRPSIVCLRPERPFAGA QVF=CMR LTLGGPDPGTRPSIVCLRPER=CMR

TD21/TD30/TD31 with two disulfide bonds

CGVRPGPGSFLFMGWSR=DGACR PCS=FGEAWLGCAPR

TD21/oxTD30/TD31 with two disulfide bonds

CGVRPGPGSFLFMGWSR=DGACR PCS=FGEAWLGCAPR

TD22/TD33-34 with one disulfide bond TD22/TD33 with one disulfide bond

DAELLLAACTS=VYSAALTTHLN PCEMALD DAELLLAACTS=VYSAALTTHLN PCEMAL

Residue Number 7-10 27-43 5-10 27-43

RT (Min)

Detected Mass (Da)

Calculated Peptide Mass (Da)

Intensity (Counts)

63.7

2412.113

2412.0991

1044464

Note

61.7

2683.243

2683.2271

285636

1-10 11-43

70.3

4644.178

4644.1650

28857

44-75 95-97

52.1

3812.952

3812.9387

1004557

44-75 95-97

50.7

3828.946

3828.9339

311420

M oxidation

44-73 95-97

55.5

3527.801

3527.7950

201651

A/E cleavage

44-72 95-97

56.1

3456.765

3456.7579

179730

F/A cleavage

44-64 95-97 219-235 145-152 236-246 219-235 145-152 236-246 153-163 253-270

43.3

2639.358

2639.3458

141437

R/P cleavage

66.1

3861.741

3861.7207

227971

61.8

3877.737

3877.7204

92631

67.1

3051.438

3051.4238

177075

153-163 253-269

69.7

2936.414

2936.3970

163428

M oxidation

Detected major disulfide-linked peptide clusters are in bold fonts; ‘=’ represents a disulfide linkage between two peptides; a, b, and c indicate peptides resulting from digestion of the corresponding peptide at different sites; for example, peptide TD6-7a was related to peptide TD6-7, but resulted from cleavage between A and E.



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Figure 1.

A1L2K1 METRL Frog A0A151NWN6 METRL Alligator Q5RJL6 METRL Rat Q8VE43 METRL Mouse Q641Q3 METRL Human H9F471 METRL Monkey Q7ZV46 METRL Zebrafish Q5Q0T9 METRN Rat Q8C1Q4 METRN Mouse Q9UJH8 METRN Human H9FZM8 METRN Monkey F1N4K0 METRN Bovin Q5M7Y1 METRN Zebrafish A0A151N6M2 METRN Alligator E2AEI6 METRN Ant E2BLQ6 METRL Ant

(1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1)

1 85 ------------------------MLRRGLLSFFMVILIDRGTSQLYSSDMCNWKGSGLTHEGHTKDVEQVYLRCSEGSVEWLYP -------------------MLRAPAPGRLPLVLGLLLQLWRGGAAQYSSDLCNWKGSGLTHESHKKDVEQVYLRCSEGSIEWMYP MRGVVWAARRRAGQQWPRSPGPGPGPPPPPPLLLLLLLLLGGASAQYSSDLCSWKGSGLTREAHSKEVEQVYLRCSAGSVEWMYP MRGAVWAARRRAGQQWPRSPGPGPGPPPPPPLLLLLLLLLGGASAQYSSDLCSWKGSGLTREARSKEVEQVYLRCSAGSVEWMYP MRGAARAAWGRAGQPWPRPPAPGPPPPPLPLLLLLLAGLLGGAGAQYSSDRCSWKGSGLTHEAHRKEVEQVYLRCAAGAVEWMYP ---------------------------PLLLLLLLPAVLLGGAGAQYSSDLCSWKGSGLTHETHRKEVEQVYLRCAAGAVEWMYP ------------------------MLSPFLAYLLSVVLLCRIARSQYSSDQCSWRGSGLTHEGHTRGVEQVYLRCAQGFLEWLYP ------------------------MLVAALLCALCCGLLAASARAGYSEDRCSWRGSGLTQEPGS--VGQLTLDCTEGAIEWLYP ------------------------MLVATLLCALCCGLLAASAHAGYSEDRCSWRGSGLTQEPGS--VGQLTLDCTEGAIEWLYP ----------------------MGFPAAALLCALCCGLLAPAARAGYSEERCSWRGSGLTQEPGS--VGQLALACAEGAVEWLYP ----------------------MGFPAPALLCALCCGLLAPAARAGYSEERCSWRGSGLTQEPGS--VGQLALACAEGAVEWLYP ------------------------MPTSALLCTLCFCLLAAAARAGYSEDRCSWRGSGLTQEPGS--VGQLALACADGKIEWLYP -------------------------------------------MASYSEDQCSWRGSGLSQAVKN--VEQVWLRCAEGSVEWLYP ---------------------------MPLVLWALCLGVLDTVLCSYSEDQCSWRGSGLSQESGS--VEQISLHCAEGSLEWLYP -------------------------MLRAIIVVVSLIFVSVSAYEH-IVDQCDWSGSGENESGG---VRAVYLRCTRGTVLWSYP -------------------------MLHKTVTTFVLFFTLVSAYEHTTADQCDWSGSGGGEHGG---VRPVYLRCARGTVLWRYP


(62) (67) (86) (86) (86) (59) (62) (60) (60) (62) (62) (60) (41) (57) (57) (58)

86 170 TGAMVINLRPNTLTSA--------YKHLTVCIKPFKDSKG-ANIYSEKTG-ELKLVVPDGENNPH---KVYCFGLDRG----GLY TGALIVNLRPNMFSSS--------SKHLTVCIKPFKDSTG-ANIYLEKTG-ELKLLVRDGDRSPN---KVYCFGYDQG----GLF TGALIVNLRPNTFSP---------AQNLTVCIKPFRDSSG-ANIYLEKTG-ELRLLVRDVRGEPG---QVQCFSLEQG----GLF TGALIVNLRPNTFSP---------AQNLTVCIKPFRDSSG-ANIYLEKTG-ELRLLVRDIRGEPG---QVQCFSLEQG----GLF TGALIVNLRPNTFSP---------ARHLTVCIRSFTDSSG-ANIYLEKTG-ELRLLVPDGDGRPG---RVQCFGLEQG----GLF TGALIVNLRPNTFSP---------ARHLAVCIKPFRDSSG-ANIYLEKTG-ELRLLVPDGDGRPG---RVQCFGLEQG----GLF TGAIIVNLRPNTLSPA--------ASLLSVCIKPSKESSG-THIYLDRLG-KLRLLLSEGDQAEG---KVHCFNIQDG----ALF AGALRLTLGGSDP----------GTRPSIVCLRPTRPFAG-AQVFAERMAGNLELLLAEGQGLAG----GRCMRWGPRER-RALF AGALRLTLGGPDP----------GTRPSIVCLRPERPFAG-AQVFAERMTGNLELLLAEGPDLAG----GRCMRWGPRER-RALF AGALRLTLGGPDP----------RARPGIACLRPVRPFAG-AQVFAERAGGALELLLAEGPGPAG----GRCVRWGPRER-RALF AGALRLTLGGPDP----------SARPSIACLRPVRPFAG-AQVFAERAGGALELLLAEGPGPAG----GRCVRWGPRER-RALF AGALRLTLGGSEP----------SAQPGIVCLRPTRPFAG-AQVFVERTGGGLELLLAEGQGPAG----ARCARWGPRER-RALF AGALRLTLSPRLP-WSAMGPGESSRSPVSVCVKPDPHWGG-AQLYLERDG-VLELLVGDETSTTPGPAHVRCFSALPGER-PALF TGALRLSLSPRLP-TGTAGKGKSPRQ-VTTCIKPSSTFRG-AQIYLERDG-ILELLLSEADASLR--PRVRCFNWLPKEK-VALF RGAMRMVLSFPTSSMQ-RCPLDLTKLGLRTCVKI----SGPVQVFLETNH-MLRPIYSPSDGKHENS—-HRCFRWQKR---VALF RGALRVVLSFPASSSE-NSIPGRSNLGFRACVKI SGPVRVFLEGNG-KLRPLYSPSDGKHELS—-HRCFHSRKLIA--ALY


(130) (135) (153) (153) (153) (126) (130) (129) (129) (131) (131) (129) (122) (135) (131) (133)

171 255 IEATPQQ--DISRKITGFQYELISQRTL---SDLHTVS--DPCRPCSDTEVLLAVCISDFVVKGTISAVTNDEELQESLINVTVD IEATPQQ--DISRKITGFQYELISKGIA---SDLHTVS--APCRPCSDTEVLLAVCTNDFVVRGSIQDVTNEVEQQESIIDVSVS VEATPQQ--DISRRTTGFQYELMSGQRG---LDLHVLS--APCRPCSDTEVLLAICTSDFVVRGFIEDVTHVPEQQVSVIHLRVS VEATPQQ--DISRRTTGFQYELMSGQRG---LDLHVLS--APCRPCSDTEVLLAICTSDFVVRGFIEDVTHVPEQQVSVIYLRVN VEATPQQ--DIGRRTTGFQYELVRRHRA---SDLHELS--APCRPCSDTEVLLAVCTSDFAVRGSIQQVTHEPERQDSAIHLRVS VEATPQQ--DIGRRTTGFQYELIRRHRA---SDLHELS--APCRPCSDTEVLLAVCTSDFAVRGSIQEVTHEPERQDSAIHLRVS IEAVPQR--DISRKITAFQYELVNHRPG---ADPQSLS--APCQPCTDAEVLLAVCTSDFVARGRILGVSEEDEQTS--VTVSLS LQATPHR--DISRRVAAFQFELHEDQRAEMSPQAQGFGVDGACRPCSDAELLLTACTSDFVIHGTIHGVVHDMELQESVITVVAT LQATPHR--DISRRVAAFRFELHEDQRAEMSPQAQGLGVDGACRPCSDAELLLAACTSDFVIHGTIHGVAHDTELQESVITVVVA LQATPHQ--DISRRVAAFRFELREDGRPELPPQAHGLGVDGACRPCSDAELLLAACTSDFVIHGIIHGVTHDVELQESVITVVAA LQATPHR--DISRRVAAFRFELREDGRPELPPQAHGLGADGACRPCSDAELLLAACTSDFVIHGIIHAVAHDVDLQESVITVMAA LQATPHP--DLSRRLASFRFQLREDGRPELPPQARSLGADAACRPCSDAELLLAVCTSDFVIYGTILGVAHNAELQESVITVAAA LQATPHR--DISRRIAAFRYELRGDWTAQPAVNTDPVSSEGACRPCNNTEILMAVCTSDFVVRGNIRSVGTDSNLNAAVIKVSAT LQSTLHQ--DISRRIAAFRYELRGDWNSRLSLPSSNLSMEGACRPCNDTEILMAICTSDFVVRGNIRSVSNDIELQESIISVSAT MEAEDDY--SFKNNKVKLQYEIESASSK--GCVLHVSDEEEECRPCSMEELANAYCQSDLVARGTITAVEEQIYLDTAELVLNVN MEAEDDY--SYKREKVRLQYDLEPNSLK--GGALHIPEEEEECRPCSMEELAKAYCQSDLVARGTVSAVQQRFDLEAEELVLRVT


(208) (213) (231) (231) (231) (204) (206) (212) (212) (214) (214) (212) (205) (218) (212) (214)

256 340 KLYR---QKSKIFLP---KDNGGWEGMIRTPLECGVKTGMGSFLFTGRMHFGEPRLGCTPRYKDFKRIYLEAKKQGLNPCEISTD RLYR---QKSKVFQPS--EESGSWRGQIKTLLECGVKPGDGDFLFTGRMHFGEARLGCAPRFKDFQRMYKEAKDKGLNPCEIGPD RLHR---QKSRVFQPA-PEDSGHWLGHVTTLLQCGVRPGHGEFLFTGHVHFGEAQLGCAPRFSDFQKMYRKAEERGINPCEINME RLHR---QKSRVFQPA-PEDSGHWLGHVTTLLQCGVRPGHGEFLFTGHVHFGEAQLGCAPRFSDFQRMYRKAEEMGINPCEINME RLYR---QKSRVFEPV-PEGDGHWQGRVRTLLECGVRPGHGDFLFTGHMHFGEARLGCAPRFKDFQRMYRDAQERGLNPCEVGTD RLYR---QKSRVFEPV-PEGDGHWQGRVRTLLECGVRPGHGDFLFTGHMHFGEARLGCAPRFKDFQRMYRDAQERGLNPCEVGMD HLYR---QKTQVFVSG-GGRAKRWTGFVKMSRQCGVKPGDGEFLFTGTVRFGEAWLSCAPRYKDFLRVYQDARQQGTNPCHLETD RVIR---QTLPLFQEG--SSEGRGQASVRTLLRCGVRPGPGSFLFMGWSRFGEAWLGCAPRFQEFSRVYSAALAAHLNPCEVALD RVIR---QTLPLFKEG--SSEGQGRASIRTLLRCGVRPGPGSFLFMGWSRFGEAWLGCAPRFQEFSRVYSAALTTHLNPCEMALD RVLR---QTPPLFQAG--RSGDQGLTSIRTPLRCGVHPGPGTFLFMGWSRFGEARLGCAPRFQEFRRAYEAARAAHLHPCEVALH RVLR---QTLPLFGRR--GSGEEGLTSIRTPLRCGVRPGPGTFLFMGWSRFGEAWLGCAPRFQEFRRVYEAARTAHLHPCEVALH RVLR---QTLPVFRVG--GPGGQGQASIRTPLHCGVRPGPGTFLFMGWNRFGEAWLGCAPRLQEFSRAYAAAHADHLHPCEVVLD RVYR---QKFALFPES--GRLTR-SGEIRTPLQCGVRPGAGSFLFTGRVHFGEAWLGCAPRYKDFLKAYEQAKQSLMIPCTLVND RIHR---QKFTLFQPI--GKYGKSTGNIHTLLRCGVKPGPGSFLFTGWLHFGEAWLSCAPRYKDFRRIYEDAHQAHENPCEFPLD KILR-HVQETENNENT-NITASKKNIRVRVPLMCKARHGPGEFIIMAKRRLGDLVLICAPTLETWKETIQEIDN---APCVLTSKILRQVVQETEGNE----TVSAKKSVRVRVPAACDARHGLGEFVIMAKRRLGDLILVCAPRLEAWANAVREMDT---APCVLNS-


Page 22 of 28

Page 23 of 28

Figure 2.

1

2

3

100

B

29441

100

29431

C

250k 148k 98k 64k 50k 36k

% Relative Abundance %

A

Abundance % Relative %

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


29545

29555 16k

0 28000

29000

30000

mass 31000


0 28000

29000

30000

mass 31000


Figure 3.

TD6-7 57.61

A

TD5 68.88

37.74

45.20

TD22

53.49 41.05

TD6-7a TD6-7b

%

Relative Abundance (%)

36.27

TD21

TD10

29.62 25.19

28.86

30.89 32.70

27.88

52.64

39.74

TD31

54.53

42.58

38.86

TD2-3

56.02

35.59

TD33-34 62.06 62.84

48.28

58.04

TD6-7c

trypsin related

66.04

60.01

TD34 oxTD30

60.00

65.00

68.57 67.52

TD8 TD30 69.74 70.58

14 25.00

30.00

35.00

40.00

45.00

50.00

55.00

70.00

TD3/TD5

B

63.63

37.74

TD6-7/TD10 52.04

45.21

TD6-7c/TD10 41.07 40.00 36.30 29.64

TD6-7/oxTD10 42.57

38.87

48.31

50.62

25.20 28.47

30.89 32.71

TD21/oxTD30/TD31

53.48

%

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 28

TD22/TD33-34

TD2-3/TD5 TD6-7a/TD10 54.53

TD22/TD33 69.74

61.70

TD6-7b/TD10 55.44

43.30

TD21/TD30/TD31

66.10 67.08

68.57

58.02

14

Time 25.00

30.00

35.00

40.00

45.00

50.00

55.00

Time (min)


60.00

65.00

70.00

Page 25 of 28

Figure 4. Pi2+

x2

1704.90

Relative Abundance

100

A

80

z5+

z262+

606.32

1561.34

c272+ z4+ 507.25

40

z6+ 734.38

y1+

+

z3+

a3 359.18 + y3 300.17 375.20

175.12

200

1402.75

y253+

y4+

y5+

522.27

621.33

0 400

LPERPFAGA

+

*

z282+ 1504.79

Pi z302+ 918.83 z9+ z222+ z12 c292+ 1067.58 1136.61 1242.691333.70 1589.84 + 2+ 933.48 z10 c28 + y232+ z292+ 805.42 z8 1080.55 2+ * * c12+ c252+ z13+ y292+ c31 * 862.44 TLGGPDPGTR * z232+ z242+ 1179.65 PDPGTRPSIV c14+ + c16+ z312+ y252+ y272+ y11+ c13 y172+ *

600

3+

z7+

800

1000 m/z

y1+

1200

1400

1600

1800

175.12

100

Relative Abundance

c302+

1427.76

60

20

B

80

Pi+ 409.17

a2+ 207.06 60

40

Pi+-NH3

z2+ 20

392.14

289.13

b2+

a2+-NH3

y2+

235.06

190.04

Pi+-NH3-H2O

306.16

374.13

0 160

180

200

220

240

260

280

300 m/z

c12 c14

C

320

340

360

380

c27

c20

z30

c30 c31

z28 z26

z9 z8 z7 z6 z5 z4 y3

z13 z12

Relative Abundance

100

80 70 60

175.16

80 60

D

a2+ 207.10

Pi+ 1908.98

40

b2+

20

Pi+-NH3

z2+

289.18 y2+ 306.19

235.07

P2+ P1y3+ 409.17

200

250

375.18

392.17

300 m/z

350

400

P2/P1z282+

P1z4+

1271.99

606.32

507.26

P1z3+

P2/P1c202+

P1z9+ P1z8+ 933.48

P1z6+ 734.38

359.18

1707.86

Pi3+

P1z5+

20

Pi2+

409.22

0

30

10

P12+

y1+

90

40

420

1704.40

100

50

400

P1 (TD6-7) LT―LG―GP―DPGTRP―SI―VCLRP―E―RPF―A―G―A―Q―V―F―A―ER P2 (TD10) CMR

Relative Abundance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1242.15

862.44

P1c12+

P1z7+

1179.65

P2/P1z262+ 1630.82

P1z12+ 1333.70

P2/P1c272+ P1z13+

P2/P1c312+ 1828.92

P2/P1z302+ 1792.89

P2/P1c302+

Pi2+-NH3

1764.40

1605.80

P1c14+ 1462.72

1379.74

1949.38

0 400

600

800

1000

1200

1400

m/z


1600

1800

2000


Figure 5.

TD31+ 100

A

1205.57

90

TD302+

Relative Abundance %

80

926.94

70

Pi2+ TD30+ 1933.88

TD21/TD302+

TD21/TD312+

60

1330.09

1005.93

1853.88

50

TD30, z9+

TD30-SH2+

40 30

Pi3+

TD21/TD31/ TD30, c3+2 +1 1171.54

TD21/TD303+ 20

TD21+

910.45 806.29 887.06

TD21/TD30 +SH2+2

TD30, z8 1027.49

TD21/TD31/ TD30, c9+2

1288.92

TD21/TD31/ TD30, c12+2

TD30, z14+1 1622.74 1578.78

1347.09 1419.13

1143.48

10

TD30-SH+ 1819.90

0 800

900

1000

1100

1200

1300

1700

1800

1900

b5

b7

2000

D―G―A―C―R―P―C―S y7

y3 b4

P2/P1y72+ 2+ 948.92 Pi

1006.43

80

P2 F―G―E―A―W―L―G―C―A―P―R P2-SH2+

y9 y8

1172.58

z6 y5

P1-SH+ P2b4 +

P2+SH+

774.32

P1/P2y92+

P2b7+H+

405.17

762.30

50

P1b5+

P1y7+SH+

40

503.20

P1y7+

30

1600

806.29

90

60

1500

P1y3/P2y5+

100

70

1400 m/z

P1

B

Relative Abundance %

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 28

P2b4-17+

P2/P1y3+

P2y8-SH+ 1204.55

691.26

EAWLG+

905.40

563.26

1509.66

P2-H+

840.28

P1a5+ 557.25 P1y5+

20 388.17 475.20

1238.54

P1b7/P22+

P1/P2z5+ + 1291.49 P1/P2z6 1404.59

962.91

725.25

10 0 400

600

800

1000

1200 m/z

1400


1600

1800

2000

Page 27 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 6. 1

GYSEDRC1SWRGSGLTQEPGSVGQLTLDC2TEGAIEWLYPAGALRLTLGGPDPGTRP

56

SIVC3LRPERPFAGAQVFAERMTGNLELLLAEGPDLAGGRC4MRWGPRERRALFLQA

111

TPHRDISRRVAAFRFELHEDQRAEMSPQAQGLGVDGAC5RPC6SDAELLLAAC7TSD

165

FVIHGTIHGVAHDTELQESVITVV VARVIRQTLPLFKEGSSEGQGR ASIRTLLR

219

C8GVRPGPGSFLFMGWSRFGEAWLGC9APRFQEFSRVYSAALTTHLNPC10EMALD



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

TOC Graphic

GYSEDRC1SWRGSGLTQEPGSVGQLTLDC2TEGAIEWLYPAGALRLTLGGPDPGTRP SIVC3LRPERPFAGAQVFAERMTGNLELLLAEGPDLAGGRC4MRWGPRERRALFLQA TPHRDISRRVAAFRFELHEDQRAEMSPQAQGLGVDGAC5RPC6SDAELLLAAC7TSD FVIHGTIHGVAHDTELQESVITVV VARVIRQTLPLFKEGSSEGQGR ASIRTLLR C8GVRPGPGSFLFMGWSRFGEAWLGC9APRFQEFSRVYSAALTTHLNPC10EMALD


Page 28 of 28

Determination of the Disulfide Structure of Murine Meteorin, a

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