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Complete Mapping of Complex Disulfide Patterns with Closely-Spaced Cysteines by In-Source Reduction and Data-Dependent Mass Spectrometry Christian Necip Cramer, Christian D Kelstrup, Jesper Velgaard Olsen, Kim F. Haselmann, and Peter Kresten Nielsen Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 28 Apr 2017 Downloaded from http://pubs.acs.org on May 1, 2017

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Analytical Chemistry

Complete Mapping of Complex Disulfide Patterns with CloselySpaced Cysteines by In-Source Reduction and Data-Dependent Mass Spectrometry Christian N. Cramer1,2, Christian D. Kelstrup2, Jesper V. Olsen2, Kim F. Haselmann1 & Peter Kresten Nielsen1* 1. Protein Engineering, Global Research, Novo Nordisk A/S, Novo Nordisk Park, 2760 Måløv, Denmark 2. Proteomics Program, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark * Corresponding author: Peter Kresten Nielsen, Ph.D. Protein Engineering, Global Research Novo Nordisk A/S Novo Nordisk Park 2760 Måløv, Denmark Email: [email protected] Tel.: (+45) 30 79 03 75 Notes: The authors declare no competing financial interest.

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Abstract: Mapping of disulfide bonds is an essential part of protein characterization to ensure correct cysteine pairings. For this, mass spectrometry (MS) is the most widely used technique due to fast and accurate characterization. However, MS-based disulfide mapping is challenged when multiple disulfide bonds are present in complicated patterns. This includes presence of disulfide bonds in nested patterns and closely-spaced cysteines. Unambiguous mapping of such disulfide bonds typically requires advanced MS approaches. In this study, we exploited in-source reduction (ISR) of disulfide bonds during the electrospray ionization process to facilitate disulfide bond assignments. We successfully developed a LC-ISR-MS/MS methodology to use as an online and fully automated partial reduction procedure. Postcolumn partial reduction by ISR provided fast and easy identification of peptides involved in disulfide-bonding from nonreduced proteolytic digests, due to the concurrent detection of disulfide-containing peptide species and their composing free peptides. Most importantly, intermediate partially reduced species containing only a single disulfide bond were also generated, from which unambiguous assignment of individual disulfide bonds could be done in species containing closely-spaced disulfide bonds. The strength of this methodology was demonstrated by complete mapping of all four disulfide bonds in lysozyme and all seventeen disulfide bonds in human serum albumin, including nested disulfide bonds and motifs of adjacent cysteine residues.

Introduction: Disulfide linkage assignment is an important task in protein characterization, as protein structure, stability, and function is dependent on correct cysteine pairings. Since protein therapeutics typically are enriched with disulfide bonds to support their extracellular functions, disulfide mapping is especially critical in the biopharmaceutical industry to ensure potent and safe drug products.1 The most commonly used technique in disulfide mapping is mass spectrometry (MS). The general approach in MS-based disulfide assignments is to analyze fragments containing a single disulfide bond connecting two peptides generated by enzymatic digestion under nonreducing conditions.2 Identification of these fragments can either be done by their mass alone or by MS/MS sequencing. Nowadays this is straightforward, and software exists able to perform high-throughput disulfide assignments based on the MS/MS fragmentation patterns of these species3,4. However depending on the protein sequence and structure, generating these simple species exclusively containing a single disulfide bond from the proteolytic digestion is often not possible. This particularly involves disulfide patterns lacking enzymatic cleavage sites between cysteines, such as nested disulfide bonds with closely-spaced or even adjacent cysteine residues. With more than a single disulfide bond present in the species to be analyzed, more advanced methods are the only option to unravelling the disulfide bond connectivities, such as multistage MS/MS fragmentations (MSn) and/or partial reduction procedures1,2,5. The principle of the partial reduction approach is to reduce some disulfide bonds while leaving others intact, allowing for assignment of disulfide bonds from the partially reduced species. This can be done on smaller intact proteins and peptides as it was initially introduced by Gray6. Since then, many variations of the methodology have been applied using MS characterization with chemical reduction by TCEP7-10, gas-phase reduction (electron capture/transfer dissociation (ECD/ETD)5,11-13 and ultraviolet photodissociation (UVPD)14) as well as electrochemical (EC) reduction15. The limiting steps in these approaches are the ability to generate partially reduced species, which involves time consuming screenings, and the ability to resolve these by the subsequent separation and characterization. The study by Massonnet et al, demonstrated the potential of using partial reduction by ETD, followed by ion mobility separation and CID characterization to localize disulfide bonds in peptides bearing two intrachain disulfide bonds in direct infusion experiments.13 However, with larger proteins and/or with


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increasing number of disulfide bonds present, protein digestion and LC separation are required to create smaller disulfide-bonded species. The partial reduction step should thus be performed either before16 or after17,18 the digestion, involving complex procedures typically needing purification of the species between the different techniques. In recent years methods for online disulfide reduction compatible with LC-ESI-MS have gained a lot of attention by using chemical reductants19,20 or electrochemistry15,21. Postcolumn reduction has been demonstrated by addition of TCEP20 or DTT19 from a syringe into mixing tees or by using EC flow cells in a LC-EC-MS setup.15,21 These approaches provide benefits in disulfide mapping, since free reduced peptides follow the same elution profile and thereby assist in identifying peptides involved in disulfide bonding. The presence of both the free reduced peptides and the intact disulfide-bonded fragment is obtained when the reduction is only partial19,20, and even intermediate partially reduced species are possible to observe when more than a single disulfide bond is present in a given fragment.15 The drawbacks of these setups are the non-automated modification of the LC-MS apparatus, the difficulty in controlling the reduction efficiency and the introduction of ion suppression from the reducing chemicals19,20 and working electrode material15. A potential alternative disulfide bond reduction mechanism has previously been observed under the electrospray ionization (ESI) process in a study by Nicolardi et al. evaluating EC reduction of antibodies (mAbs)22. In direct infusion-MS experiments with increased capillary voltages and low percentage of acetonitrile (ACN), they observed reduction of the interchain disulfide bonds in mAbs resembling what they obtained by infusion-EC-MS using EC reduction in a commercial flow cell. Furthermore, ESI induced disulfide cleavage has been described.23 In this study, we set out to identify parameters important for in-source reduction (ISR) of disulfide bonds during ESI and to exploit the ISR mechanism in disulfide mapping. We successfully established a LC-ISR-MS/MS platform to use as an online and fully automated partial reduction procedure in disulfide mapping of nonreduced protein digestions. Using this strategy we correctly assigned all four disulfide bonds in lysozyme and all seventeen disulfide bonds in human serum albumin (HSA), including nested disulfide bonds and motifs of adjacent cysteine residues.

Experimental:

Reagents: Lyophilized HSA (≥99%), lyophilized lysozyme from chicken egg white (≥90%) and formic acid (FA) were purchased from Sigma-Aldrich, a synthetic peptide (hereafter referred to as CCpep) containing an inter-chain disulfide bond linking two peptide chains with the sequences HKSNTCRAMER and RVEHCPKEGK was ordered at TAG Copenhagen (>90% purity), modified Trypsin was purchased from Promega, endoproteinases Lys-C and Asp-N from Roche Diagnostics, ACN, optima LC/MS grade 0.1% FA in water (LC buffer A) and 0.1% FA in ACN (LC buffer B) from Fisher Scientific and sodium di-hydrogen phosphate mono-hydrate (NaH2PO4∙H2O) and di-sodium hydrogen phosphate di-hydrate (Na2HPO4∙2H2O) used to make 100mM NaH2PO4/Na2HPO4 buffers (pH 6.8 and pH 7.4) from Merck. Sample preparation: Proteolytic digestion of nonreduced Lysozyme was performed by addition of Lys-C and Trypsin in 1:40 and 1:20 (w/w) enzyme to protein ratios, respectively, to Lysozyme in NaH2PO4/Na2HPO4 buffer (pH 7.4) with a final Lysozyme concentration of 0.5mg/mL. The reaction was incubated for 2 hours, followed by additional addition of Asp-N in 1:100 enzyme to protein ratio. The digest reaction was incubated over-night at 37°C and quenched by addition of FA to a final concentration of 1% (v/v). Tryptic digestion of nonreduced HSA was performed by addition of Lys-C and Trypsin in 1:75 and 1:20 enzyme to protein ratios, respectively, to HSA in NaH2PO4/Na2HPO4 buffer (pH 6.8) with a final HSA concentration of 0.5 mg/mL. The digest reaction was performed



at 37°C using pressure-cycling technology (Barocycler NEP2320, Pressure BioSciences Inc.) with alternating cycles of high and low pressure to induce protein unfolding. 90 cycles were applied, each consisting of 50 sec at 45,000 psi and 10 sec at ambient pressure. When additional Asp-N digestion was desired Asp-N, Lys-C and Trypsin in 1:35, 1:75 and 1:20 (w/w) enzyme to protein ratios respectively, was added to the digest, followed by a second round of 90 cycles. The reactions were quenched by addition of FA to a final concentration of 1% FA (v/v). The synthetic CCpep peptide was used as received and freshly dissolved in Milli-Q water (18.2 MΩ·cm) (Millipore), from where 2 µM infusion-MS solutions were prepared in either 10% or 35% (v/v) ACN with 0.1% (v/v) FA and degassed for 15 min. Mass spectrometry: All MS experiments were carried out using an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA). Ionization was performed using the H-ESI ion source operating in positive ion mode. Identification of parameters important for ISR of disulfide bonds during ESI was done by screening of the source settings (spray voltage, sheath, aux and sweep gas, ion transfer tube temperature, vaporizer temperature and distance between the capillary and counter electrode) while infusing the synthetic CCpep peptide from a sample syringe with a flow rate of 4 µL/min. Based on this, the ISR settings for LC-MS were; spray voltage of 3.5 kV, sheath, aux and sweep gasses of 15, 10 and 0 arbitrary units respectively, ion transfer tube temperature of 300°C, vaporizer temperature of 135°C and default capillary position. The infusion-MS experiments were recorded with full MS scans using orbitrap detection with resolving power of 60k in the m/z range of 300-1300 with quadrupole isolation. Data-dependent higher-energy collision induced dissociation (ddHCD)24 acquisition was used in all LC-ISR-MS experiments. In the ddHCD method, survey scans of precursors from 350 to 1550 m/z were performed at 120k resolution with AGC target of 4.0x105 and maximum injection time of 200ms. The MS/MS was performed by quadrupole precursor isolation of 3 m/z, normalized HCD fragmentation energy of 24% and orbitrap detection with 120k resolution. The MS/MS AGC target was set to 4.0x105 and maximum injection time of 240ms. The filters applied were; peptide monoisotopic precursor selection, charge states 2-12, intensity threshold of 1.0x105 and dynamic exclusion duration set to 8 sec with a 25 ppm tolerance. The method was run in top speed mode with cycles of 2 sec. Liquid Chromatography: All LC separations were performed using a Waters Acquity Classic UPLC connected to the Orbitrap MS instrument. The analytical column used was an Acquity UPLC CSH C18 reversed-phase column, 1.7 µm, 1.0 x 150 mm (Waters Company, UK) with a column temperature of 55°C. The mobile phase buffers A and B were water and ACN, respectively, both containing 0.1% FA. The flow rate used was 100 µL/min, and sample elution was performed with increasing ratio of buffer B, using a gradient starting with 1% B for the first 4 min and then linearly increasing to 30% B at 92 min. The amounts of protein material injected on the column were approximately 3 µg. Data analysis: All data was recorded and raw MS files were analyzed using Xcalibur 4.0 (Thermo Fisher Scientific). To assist data analysis GPMAW v. 9.50 (Lighthouse Data, DK), pLink-SS,4 Byonics and Byologics (Protein Metrics, San Carlos, CA, USA) were used.

Results and discussion Identification of Important Parameters for In-Source Reduction of Disulfide Bonds. Reduction of interchain disulfide bonds during the ESI process has previously been observed in analysis of mAbs in direct infusion ESI-MS experiments.22 In order to elucidate which parameters are responsible for driving this process, we screened different solvent and source parameters in direct infusion-MS of a synthetic peptide containing an interchain disulfide bond (CCpep in Figure 1, inset). The most prominent factors influencing the ISR process were found to be the organic solvent content and the


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capillary voltage. Keeping all other parameters constant, the effect of the organic solvent concentration is illustrated by the level of reduction between Figure 1A and 1B. As seen in the spectra, the amount of reduced free A- and B-chain increased from 11% to 48% when the ACN concentration was lowered from 35% to 10%. The level of ISR was found to increase according to higher capillary voltages, which also were evident when the capillary voltage was increased from 3.0kV to 3.5kV in the 10% ACN sample, resulting in a further increase of ISR efficiency to 56% (Figure 1B and 1C). The exact mass of the free A- and B-chains were found to correlate with protonated cysteines. However, low intensity peaks corresponding to the radical forms (i.e. S˙) of the free chains were also detected, similar to what previously has been reported with ETD reduction of disulfide bonds.13 The methionine residue located on the A-chain was checked for oxidation after the ISR process, and was found to be below 5% of total free A-chain, hence not significantly diluting the signal. This correlation of induced ISR at lower organic content and higher capillary voltages is consistent with the previous observations.22 In addition, other factors found to induce ISR were lower flow rates, lower sheath gas settings and lowering the distance between the capillary and the counter electrode. Although the underlying mechanism of the ISR process is beyond the scope of this article, we speculate that disulfide reduction is caused by a corona discharge field during the ESI process. All identified factors contributing to ISR are known to influence corona discharge, and the relationship between corona discharge and disulfide reduction has recently been highlighted.23 However, the exact mechanism behind ISR of disulfide bonds is a study on its own, and here we have chosen to focus on the applications of ISR within disulfide mapping. Establishment of an Online LC-ISR-ddHCD Platform to Solve Complex Disulfide Bonds. As described, the common procedure in MS-based disulfide mapping is to analyze fragments with a single disulfide bond connecting two peptide chains, generated by proteolytic digestion under nonreducing conditions. However, in many cases nested disulfide bonds with closely-spaced or even adjacent cysteine residues with no enzymatic cleavage site between two cysteines, result in species more challenging to characterize. An example hereof is proteolytic digestion of lysozyme (Figure 2A) containing the nested disulfides Cys64-Cys80 and Cys76-Cys94. No common enzymatic cleavage site is present between Cys76 and Cys80, resulting in a disulfide-containing specie with three peptides connected by two disulfide bonds (the ¤3 fragment) when digested with trypsin and AspN. In such cases, assignment of the precise Cys connectivities relies on observation of MS/MS fragments between the middle two cysteines (i.e. Cys76 and Cys80). With MS/MS fragmentation channels split out over six termini present in a three-chain specie (two per peptide), the statistical chance of observing these specific fragments is low, depending on the protein sequence. Partial reduction of such species would increase the chance of observing these specific MS/MS fragments manifold. We aimed at developing an online LC-ISR-ddHCD platform for disulfide mapping deploying; 1) LC separation of species from nonreduced protein digests, 2) generation of partially reduced and free peptides by ISR during ESI, and 3) specific MS/MS fragmentation of all generated species by ddHCD. Since the organic solvent content is dictated by LC elution of given disulfide-containing species, ISR was primarily optimized by increasing the capillary voltage and lowering the sheath gas flow, as described in the experimental section. In Figure 2B, a chromatogram of lysozyme digested with trypsin and Asp-N under nonreducing conditions is shown. The three disulfide-containing species listed in Figure 2A are annotated in the chromatogram. The ISR of the three species are shown in the full MS spectra in Figure 2C-2E. As seen in the spectra, the ISR efficiency was at the highest in the start of the gradient at low % ACN, where the ¤1 fragment elutes (Figure 2C) and lower at higher % ACN where the ¤3 fragment elutes (Figure 2E). The ¤1 and ¤2 fragments containing disulfide bonds Cys6-Cys127 and Cys30-Cys115, respectively, represents the straightforward case of disulfide assignments with one disulfide bond connecting two peptides and could thus easily be mapped by MS/MS fragmentation without the need for ISR (Supporting Figures S1A and S1B). In that aspect, ISR only represents an easy way of identifying which peptides constitute the



disulfide-containing fragment, since the free reduced peptides will be present and follow the same elution profile as the intact specie. The approach is similar to what has previously been shown using online reduction in electrochemical flow cells.15,21 The most beneficial aspect of ISR is the generation of partial reduced species, when more than a single disulfide bond is present in disulfide bond-containing proteolytic fragments. As demonstrated in Figure 2E, ISR of the ¤3 fragment generates two partial reduced species each having only one of the two disulfide bonds intact. From these partial reduced species an increased chance of assigning the disulfide bonds is present, since the complexity of the species subjected to downstream ion isolation and fragmentation in the ddHCD method is lowered. In addition, only one of the two partial reduced species needs to be assigned and then the other can be assigned based on the method of exclusion. As shown in the ddHCD MS/MS spectra in Figure 3A and 3B, the disulfide bonds could successfully be assigned from both partial reduced species. In both spectra, multiple fragments are present stemming from backbone dissociation between the two closelyspaced Cys76 and Cys80 residues, with the mass increase of the Cys64-containing partner peptide on the Cys80 in Figure 3A, and the mass increase of the Cys94containing partner peptide on the Cys76 in Figure 3B. The MS/MS spectra shown were single scans from the relatively low-intensity partial reduced species in Figure 2E, illustrating that as long as partial reduced species were generated, a sensitive and optimized downstream ddHCD method with sufficient ion accumulation times can compensate for low levels of ISR. No disulfide rearrangements were detected during the ISR process, which were validated by inspection on both the full MS level for potential generation of a Cys64-Cys94 specie and on the MS/MS level for scrambled species isobaric to the expected species (i.e. generation of the Cys64-Cys76 and/or the Cys80Cys94 species). This validation approach was performed with all disulfide bonded species. Complete Mapping of All Seventeen Disulfide Bonds of Human Serum Albumin. To challenge the LC-ISR-ddHCD methodology towards a heavily disulfide-bonded protein with closely-spaced cysteines, HSA was chosen as a model protein. HSA is a 66.5kDa protein containing 17 disulfide bonds with 8 occurrences of two adjacent cysteine residues in the sequence. In Figure 4 the theoretical disulfide-containing species from a tryptic digest of HSA is shown, illustrating the complexity of disulfide patterns to be unraveled when adjacent and closely-spaced cysteines are present in the protein structure. The complex disulfide patterns generated can be grouped into four different categories; 1) three-chain species containing two interchain disulfide bonds (#2, #3, #5 and #8), 2) >3 peptide-chain species (#7), 3) single-chain intrachain disulfide-containing peptides (#1), and 4) two-chain peptides containing an inter- and an intra-chain disulfide bond (#4 and #6). Characterization and disulfide assignment of these different classes will be handled separately in the following sections. Three-chain species. LC separation of the tryptic digest of nonreduced HSA is shown in Figure 5, with indicated elution of all the disulfide-containing species listed in Figure 4. As seen in the chromatogram, the elution of these species was distributed over the entire gradient and the specific elution of each species was at distinct organic solvent concentrations. The resulting full MS spectra from the ISR of all these species are shown in Figure 6A, Figure 7A and Supporting Figure S3A, S4A, S5A, S6 and S7A. In summary, the ISR efficiency follows the organic solvent concentration also observed with lysozyme, with higher reduction efficiency of the earliest eluting species, i.e. at low organic solvent content. Despite different ISR efficiencies, the pattern observed with all tryptic digested HSA species are the same; with intact, fully reduced and partially reduced species all being present, only differing by varying ratios between the intact species and reduced products. Following the identification of peptides involved in disulfide bonding, the next step in three-chain species, such as the #3 specie shown in Figure 6A, is to assign specifically which cysteine residues are linked together. This can be done from the partially reduced species generated by ISR, as it was demonstrated for the ¤3 specie from the lysozyme digest. Where the lysozyme ¤3 specie had three residues between the closely-spaced cysteine residues, including a proline residue, which is one of the most preferential CID/-HCD cleavage sites, distinct disulfide mapping involving adjacent


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cysteines requires either the N- or C-terminal fragment from a single fragmentation site, i.e. between the two cysteine residues. As seen in the spectrum of the #3 three-chain specie from the HSA digest in Figure 6A, both partial reduced forms with only a single disulfide intact were formed by ISR. As previously discussed, assignment of only one of these disulfide bonds is needed by MS/MS, and assignment of the other disulfide bond can be deduced by the exclusion method when only four cysteines are present. However, in case of ISR the #3 specie, disulfide assignment from both partial reduced species was possible by MS/MS as seen in Figure 6B and 6C. As seen in the spectra, the y6 ion with backbone dissociation between the adjacent cysteines of the T21 peptide is observed upon fragmentation of both partial reduced forms, but is present in free/ unmodified form in the [T21+T22] specie, and with the mass increase of the T14 peptide in the [T14+T21] specie. The distinct disulfide mapping from both partially reduced forms only increases the confidence of assigning the cysteine parings to be Cys124-Cys169 and Cys168Cys177. By following the same approach as with the #3 specie, distinct disulfide mapping could be performed on the three other three-chain species (Supporting Figures S3-S5). In two out of three of these species, exact disulfide-determining MS/MS fragments between the adjacent cysteines were observed in both partially reduced variants. In the third specie, MS/MS on only one of the partially reduced species resulted in backbone dissociation between the two adjacent cysteines. >3 peptide-chains species. As the most extreme example in the tryptic HSA digest, the #7 specie consists of five cysteine-containing peptide chains linked by four interchain disulfide bonds. In Figure 7A, the resulting full MS spectrum from the ISR of the #7 specie is shown. Even though the #7 contains the highest number of disulfide bonds and had the latest elution at highest organic solvent concentration in the chromatogram (Figure 5), all reduced peptides and all partially reduced forms were observed. Most importantly, high quality MS/MS spectra are possible to obtain on all ISR generated species using an optimized ddHCD method with ion accumulation of low intensity species. As the partially reduced forms with only two peptides connected by a single disulfide bond represent the highest chance of determining the cysteines connectivities, the MS/MS spectra of these species were investigated for disulfide-determining fragments. From the MS/MS spectrum of the partially reduced [T64+T65] specie (Figure 7B), the Cys476-Cys487 pairing can be assigned due to the observation of the y8-fragment between the adjacent cysteines in the T64 peptide with Cys477 being unlinked. The partially reduced [T61+T64] specie can thus be deduced to be connected at the Cys477. As seen by the MS/MS fragmentation of the [T61+T64] specie in Figure 7C, Cys461 can be identified to be the binding partner (Cys461-Cys477) by observation of a series of y-ion fragments from the T61 peptide between the cysteines with the mass increase of the T64 peptide linked to Cys461. By this, two out of four disulfide bonds of the complex #7 specie can be assigned to specific cysteine connectivities. Despite close to complete sequence coverage of the [T51+T58] and [T58+T61] species (Figure 7D and 7E), fragments corresponding to dissociation between the adjacent cysteines of the T58 peptides were not observed. This is possibly due to the small size of the CCK peptide sequence and the fragmentation channels mainly being directed to the much larger disulfide-binding partner peptides. The conclusion to be drawn from these spectra was only, that the Cys392 and Cys448 residues were linked to the Cys437 and Cys438 residues in one of the two possible patterns. The complexity of the #7 specie nicely illustrates the powerful combination of ISR together with a sensitive and optimized ddHCD method, but also addresses the limitations regarding sample complexity. If distinct assignment of binding partners for the Cys437 and Cys438 residues in the T58 peptide was needed, reducing the complexity of the disulfide-containing species to be characterized would be necessary. This can be done by either adding an additional protease to the digest, or by a complementary digestion design choosing other digestion specificities. In the case of tryptic digestion of HSA, the #7 species would be split into two disulfide-containing species by additional Asp-N digestion, both of which contain three peptides connected by two disulfide bonds



(species $7 and $8 in Figure S8). The lowered complexity of the disulfide-containing species represents an increased chance of observing the disulfide-assigning MS/MS fragments. ISR of the $7E specie ($7E having alternative Asp-N digestion N-terminal to a Glu residue) following LC separation (Figure S9) is shown in Figure 8A. From the partially reduced species generated, MS/MS fragmentation of the [TA80+TA83] specie identified the cysteine linkage to between the Cys437 and Cys448 (Figure 8B). The unravelling of the Cys437-Cys448 and Cys392-Cys438 connectivities by additional Asp-N cleavages illustrates the strength of combining information from different digestion designs to obtain complete information on all disulfide pairings. Single-chain peptides containing an intra-chain disulfide bond. Until now, only ISR of inter-chain disulfide bonds have been discussed. However, intra-chain disulfide bonds are also present in the #1, #4 and #6 species from the tryptic HSA digest (Figure 4). To investigate the efficiency of ISR towards reduction of intrachain disulfide bonds the #1 specie was examined. As the earliest eluting disulfide-containing specie and thus lowest in organic solvent concentration, the conditions for ISR should be optimal based on the trends observed for ISR of interchain disulfide bonds. Reduction of intrachain disulfide bonds result in a mass increase of +2H and will overlap with the isotopic distribution of the intact disulfide-bonded species. The resulting full MS spectrum containing the isotopic envelope upon ISR of #1 is shown and compared to the theoretical isotopic distribution of the intact disulfide-bonded specie in the inset in Figure 9. The small increase in relative intensity of the third isotope peak containing the reduced +2H peptide indicated that only a low amount of ISR is obtained on the intrachain disulfide bond. However, with a ddHCD isolation window of 3 m/z values, contribution from both the intact and reduced species would be present upon isolation and fragmentation. As seen from the sequence coverage in Figure 9, this results in observation of MS/MS fragments between the disulfide-forming cysteines. As the mass of the #1 corresponded to have an intact disulfide bond and only two cysteines present in the sequence, the disulfide linkage between Cys53 and Cys62 could be assigned from the intact mass measurement. In that aspect ISR represents a possibility of observing better sequence coverage between the cysteine residues allowing for increased confidence in peptide assignment. Location of an interchain disulfide-forming cysteine between two intrachain disulfideforming cysteines. Unambiguous mapping of cysteine pairings in patterns with a cysteine forming an interchain disulfide bond being located between two cysteines forming an intrachain disulfide bond is a central challenge. This is due to the hampered MS/MS fragmentations between the two intrachain-forming cysteines. Absence of MS/MS fragment ions between the outer disulfide-forming cysteines can be argued to indicate the location of the intrachain disulfide bond.3,4 Depending on the question asked in a specific study, this argumentation might be sufficient. Beneficial aspects of ISR in that regard mainly consist of fast and easy ways of identifying the two peptides forming the two disulfide bonds. This is exemplified by ISR of the #4 and #6 species from the tryptic HSA digest having this disulfide pattern (Figure 4), which are shown in Figure S6 and Figure S7A. However, direct evidence for the exact cysteine pairings requires MS/MS fragmentation between the cysteine residues in question. Theoretically a partially reduced specie with the intrachain disulfide reduced and the interchain intact would hold the potential for mapping such disulfide patterns. However, with a much lower ISR efficiency of intrachain than interchain disulfide bonds observed experimentally, only small amounts of these partially reduced species can be expected. The intact and such partially reduced species will only differ by 2H and have overlapping isotopic envelopes, with a slight shift to higher m/z values depending on the amount of partial reduction. With a ddHCD isolation window of 3 m/z values, contribution from both species would be present upon isolation and fragmentation of the intact precursor. As a result, MS/MS fragments were observed between the intrachain-forming cysteines when fragmenting the #6 specie (Figure S7B). From the fragmentation pattern, the binding partner of Cys316 can be narrowed down to be either the Cys360 or Cys361 residues. In order to obtain a disulfide-assigning MS/MS fragment between the adjacent cysteines, a complementary digestion design should be done. By the further digestion with Asp-N,


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described previously, the complex disulfide pattern of the #6 specie can be cleaved into the $6 three-chain specie with two interchain disulfide bonds (Figure S8). The disulfide patterns of this $6 specie can be assigned like the other three-chain species as described earlier, by ISR (Figure 10A) followed by MS/MS characterization of one of the partially reduced species (Figure 10B). The same approach, with additional Asp-N digestion of the #4 specie, can also be used to map the disulfide bonds of the $4 specie (Figure S10).

Concluding Remarks We successfully demonstrated the use of ISR during ESI as an online reduction approach facilitating assignment of disulfide bonds. In the LC-ISR-ddHCD setup, postcolumn partial reduction of disulfide-containing species from nonreducing proteolytic digestions was obtained. This allowed easy identification of peptides involved in disulfide bonding, with intact disulfide-bonded species and free reduced peptides having the same elution profiles. More importantly, partially reduced species were generated, opening the possibility of analyzing multi-chain species (i.e. more than two peptides connected by disulfide bonds) in an online and automated fashion. This category has not previously been possible to analyze,4 even though it inherently will be generated when nonreducing proteolytic digestions are performed on proteins with no enzymatic cleavage site between closely-spaced cysteines. Using this methodology, complete mapping of all disulfide bonds of lysozyme and HSA was done including nested disulfide bonds and occurrences of adjacent cysteine residues. Importantly, no disulfide rearrangement (i.e. disulfide scrambling) was observed during the ISR process. The setup is fully automated with no modification of hardware needed, thus avoiding introduction of dead volumes and ion suppression caused by e.g. EC flow cells and tee-in of chemical reductants.15,19,20 With the ability to characterize multi-chain species containing multiple disulfide bonds, a key point in the presented workflow is the digestion procedure. A starting point should be to choose enzymes, which theoretically should generate peptide fragments primarily consisting of inter-chain disulfide bonds from the protein in scope of a specific study. Depicted by the protein sequence, a single digestion procedure to generate only interchain disulfide-bonded species might not be possible. In such cases, complementary digestion design can be performed to get complete information of all disulfide linkages, as it was demonstrated in disulfide mapping of tryptic digested HSA without and with Asp-N digestion in the present study.



Supporting information: Additional information as mentioned in text, is available free of charge via the Internet at http://pubs.acs.org.

Acknowledgements: This work, a part of the industrial PhD project for Christian Cramer, was supported by The Danish Agency for Science, Technology and Innovation and the Novo Nordisk STAR program. The Novo Nordisk Foundation Center for Protein Research (CPR) is supported financially by the Novo Nordisk Foundation (Grant agreement NNF14CC0001).


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A

[CCpep]5+

CCpep:

[CCpep]6+

A B 35% ACN 3.0 kV

11%

[CCpep]4+

[CCpep]7+

B

48%

C

[A]4+

10% ACN 3.0 kV

56%

[A]3+ [B]3+

10% ACN 3.5 kV

[B]2+

[A]2+

Figure 1. Reduction of the interchain disulfide bond of the CCpep by ISR during ESI in direct infusion-MS. 2µM CCpep in 0.1% FA and 35% (A) or 10% (B and C) ACN was infused with capillary voltages of either 3.0kV (A and B) or 3.5kV (C). The disulfide bond reduction efficiencies by ISR of 11%, 48% and 56% in (A), (B) and (C) respectively, were calculated as a ratio between the intensities of the intact [CCpep]5+ and the [A]4+ ISR product. A

¤1: ¤2: ¤3:

PCys6 PCys127 PCys30 PCys115 PCys64 PCys76+80 PCys94

B

34-45

¤1

¤32

¤2

117-125

¤3

46-61

TIC

15-21 98-112

[PCys6]2+

C

MS

¤1:

[¤1]3+

[¤2]4+

[¤2]3+

PCys127 [PCys6]1+

[¤1]2+

D

PCys6

[PCys30]2+

MS

¤2:

PCys30 PCys115

[¤2]2+

E

¤3:

PCys64 PCys76+80 PCys94

[¤3]4+

MS

[PCys64+PCys76+80]2+ [PCys64]1+

3+ [PCys76+80+PCys94]3+ [¤3]

[PCys76+80]2+ [PCys94]2+

Figure 2. LC-ISR-MS of lysozyme digested with trypsin and Asp-N under nonreducing conditions. In (A), the disulfide-containing species generated by proteolytic digestion of lysozyme with trypsin and Asp-N are shown. (B) LC separation of the lysozyme peptide map with annotation of the disulfide-containing species marked with ¤, and linear peptides annotated with numbering according to spanning amino acid sequences. In (C-



E), the full MS spectra following partial reduction by ISR of the ¤1, ¤2 and ¤3 species are shown, respectively. Generation of partially reduced species of the ¤3 specie by ISR are highlighted with enlarged annotations in (E). A

b5 b4 a2 b2

B

[y8+PCys64]

PCys64

MS/MS

PCys76+80 [y8-y3+PCys64] [y8-y2+PCys64]

b3

[y8-y4+PCys64]

PCys76+80

[y8-y1+PCys64]

[b10+PCys94]2+ [b9+PCys94]2+

y2 a2

MS/MS

PCys94

b2

b4-H2O b3 b5-H2O b6 b4 b

y8

[b11+PCys94]2+

[b5+PCys94]

[b5+PCys94-b2]

5

Figure 3. ddHCD MS/MS fragmentation of the partially reduced [PCys64+PCys76+80]2+ (A) and [PCys76+80+PCys94]3+ (B) species from ISR of ¤3, as shown in Figure 2. Disulfide mapping fragments elucidating the Cys64-Cys80 and Cys76-Cys94 connections are annotated in the spectra. All identified fragments are summarized of in the peptide sequences as insets.

#1 : T7 T9

#2 : T10

T12

#3 :

T14 T21 T22 T28

#4 : T 36 T38

#5 : T40 T41 T42

#6 : T 49 T51 T58

#7 : T61 T64 T65 T66

#8 : T75 T77

Figure 4. Disulfide-containing species from a tryptic digestion of HSA under nonreducing conditions is listed named with #. The numbering of peptides and residues are according to an in silico digestion.


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#3

TIC

T50

#21 T6

#4

#51+KP

T55

#5KP

T52

#1 #6 T4

T13

#21

T16

T35 T48 T34

T23/ T24

#8

T54/ T55

#41

T31

T69/ T70

T79

QpE

#21

T70

Mox

T8 T50KP

#2

T74

#3

T50KP

#5KP

Mox

T45/ T46

QpE

#51+KP

T78/ T79

#81

#83

#7

#82

*

T70

Mox

T17/ T18

#82 1 #8 T18

#7 QpE

T44 *

Mox

Mox

Figure 5. LC separation of tryptic digest of HSA. Elution of disulfide-containing species is marked in the chromatogram. 1 and KP are used to indicate a missed cleavage site and unexpected digestion between K and P residues, respectively. Full size version of the annotated chromatogram can be found in supporting information (Figure S2). A

[#3]6+

T14

#3 : T21 T22

[T21+T22]4+ [T22]2+

B

[T14+T21]5+

[T14]4+ [T21]2+

[#3]5+ [T14+T21]4+

[#3]8+

[T14]3+

[T22]1+

T14

[y9+T14]4+

[#3]4+ [y10+T14

[y8+T14]4+ [y7+T14]4+

T21

[y6+T14

y2

C

y3

y4

y4

y92+ b

[y11+T14]4+ [y14+T21]3+

y112+

[y8+T22]2+

T22 [y9+T22]3+ y2

MS/MS

[y10+T22]3+

T21 y2

5

]4+

]4+

[P]5+ [P-b2]5+

y5

b3

b2

a2 a2 b2 b2

MS

[#3]7+

y3 y3

y4

y4

[y9+T22]2+

MS/MS

[y7+T22]2+

y6

[y10+T22]2+

Figure 6. Disulfide mapping of the HSA #3 specie by LC-ISR-ddHCD. (A) Full MS spectrum following LC separation and partial reduction by ISR of the #3 specie, with the generated partially reduced species highlighted. In (B) and (C), the ddHCD MS/MS fragmentation spectra of the partially reduced [T14+T21]5+ and [T21+T22]4+ species are shown, respectively. Enlarged annotations indicate fragments originating from backbone dissociation between the two adjacent cysteines of the T21 peptide. These fragments allowed unambiguous assignment of the disulfide linkages, as summarized by the sequence coverages in the insets. P: Precursor.



T51 T58

#7 : T61 T64 T65

A

B

[#7]8+

[#7]7+

MS

[T51+T58]3+ [T58+T61]4+ [T64+T65]4+ [T61+T64]4+

T64

[#7]6+

y7

[b8+T64]2+

MS/MS

T65

[y8]1+

y2

C

[Precursor]4+

T61

MS/MS

[y6+T64]2+

T64

[y9+T64]2+ [y13+T64]2+

[Precursor]4+

D

y6

T58

MS/MS

T61 y9

[Precursor]3+

E

MS/MS

T51 T58

y4

y7

Figure 7. Assignment of two out of four disulfide bonds of the HSA #7 specie by LC-ISRddHCD. (A) Full MS spectrum following LC separation and partial reduction by ISR of the #7 specie, with only the partially reduced species containing a single disulfide bond annotated. (B-E) show the ddHCD MS/MS fragmentation spectra of these partially reduced species. In the fragmentation spectra of the [T64+T65]4+ (B) and [T61+T64]4+ (C) species, fragments that allowed mapping of the cysteine connections between the T61, T64, and T61 peptides are annotated. No disulfide determining fragments were observed in fragmentation of [T58+T61]4+ and [T51+T58]3+ species. The sequence coverage obtained on all four species is shown as insets in the spectra. A

TA76E

$7E : TA83 TA86

[$7E]3+

MS

[TA83+TA86]2+ [$7E]4+

[TA76E]1+

[TA76E+TA83]2+ [TA83]1+

B

y42+

TA83 TA86 y1

[$7E]2+

y4

x2

MS/MS

[Precursor]2+ y3

y2

Figure 8. Mapping of the Cys437-Cys448 disulfide bond from the $7E specie by LC-ISRddHCD characterization of HSA digested with trypsin and Asp-N. (A) Full MS spectrum


Page 14 of 24

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following LC separation (Figure S9) and partial reduction by ISR of the $7E specie. The partially reduced species generated by ISR are highlighted in the spectrum, and ddHCD MS/MS fragmentation spectrum of the partially reduced [TA80+TA83]2+ specie is shown in (B). The fragments enabling mapping of the disulfide bond are annotated in the spectrum, and all identified fragments are summarized in the inset of the sequence coverage. [#1]2+

x2

MS

Observed

Theoretical

#1 :

MS/MS y9

y7

y10 y11

y8

Figure 9. Assignment of the intrachain disulfide bond of the #1 specie. The LC-ISRddHCD MS/MS fragmentation of the #1 peptide provided close to complete sequence coverage beyond the disulfide-forming cysteines. An inset shows the observed full MS spectrum of the #1 peptide compared to the theoretical isotopic envelope with an intact intrachain disulfide bond, indicating a low amount of intrachain disulfide reduction during ISR. [TA63+TA71]2+

A

TA63

[TA63]1+

B

[$6E]3+

a2

TA63

y2

TA71

MS

$6E : TA71

[TA71]1+

[TA72E]1+

TA72E

[TA71+TA72E]2+

[b3+TA63]1+

[b2+TA63]1+

x2

MS/MS

[y4+TA63]1+

y3

Figure 10. Mapping of the Cys316-Cys361 disulfide bond from the $6E specie (with alternative/non-specific Asp-N digestion N-terminal to the Glu residue in the TA72 peptide) by LC-ISR-ddHCD characterization of HSA digested with trypsin and Asp-N. (A) Full MS spectrum following LC separation (Figure S9) and partial reduction by ISR of the $6E specie. The partially reduced species generated by ISR are highlighted in the spectrum, and ddHCD MS/MS fragmentation spectrum of the partially reduced [TA63+TA71]2+ specie is shown in (B). The fragments enabling mapping of the disulfide bond is annotated in the spectrum, and all identified fragments are summarized as an inset of the sequence coverage.



For TOC only: Workflow

Step 1: Protein digestion

Step 2: LC separation

§2

Step 3: In-Source Reduction of Disulfides

Step 4: MS/MS (ddHCD)


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References: (1) Wiesner, J.; Resemann, A.; Evans, C.; Suckau, D.; Jabs, W. Expert review of proteomics 2015, 12, 115-123. (2) Goyder, M. S.; Rebeaud, F.; Pfeifer, M. E.; Kalman, F. Expert Rev. Proteomics 2013, 10, 489-501. (3) Liu, F.; van Breukelen, B.; Heck, A. J. Molecular & cellular proteomics : MCP 2014, 13, 2776-2786. (4) Lu, S.; Fan, S. B.; Yang, B.; Li, Y. X.; Meng, J. M.; Wu, L.; Li, P.; Zhang, K.; Zhang, M. J.; Fu, Y.; Luo, J.; Sun, R. X.; He, S. M.; Dong, M. Q. Nat. Methods 2015, 12, 329331. (5) Wu, S. L.; Jiang, H.; Hancock, W. S.; Karger, B. L. Anal Chem 2010, 82, 5296-5303. (6) Gray, W. R. Protein Sci. 1993, 2, 1732-1748. (7) Albert, A.; Eksteen, J. J.; Isaksson, J.; Sengee, M.; Hansen, T.; Vasskog, T. Anal Chem 2016, 88, 9539-9546. (8) Foley, S. F.; Sun, Y.; Zheng, T. S.; Wen, D. Anal. Biochem. 2008, 377, 95-104. (9) Nair, S. S.; Nilsson, C. L.; Emmett, M. R.; Schaub, T. M.; Gowd, K. H.; Thakur, S. S.; Krishnan, K. S.; Balaram, P.; Marshall, A. G. Anal Chem 2006, 78, 8082-8088. (10) Bingham, J. P.; Broxton, N. M.; Livett, B. G.; Down, J. G.; Jones, A.; Moczydlowski, E. G. Analytical biochemistry 2005, 338, 48-61. (11) Wu, S. L.; Jiang, H.; Lu, Q.; Dai, S.; Hancock, W. S.; Karger, B. L. Anal Chem 2009, 81, 112-122. (12) Ni, W.; Lin, M.; Salinas, P.; Savickas, P.; Wu, S. L.; Karger, B. L. Journal of the American Society for Mass Spectrometry 2013, 24, 125-133. (13) Massonnet, P.; Upert, G.; Smargiasso, N.; Gilles, N.; Quinton, L.; De Pauw, E. Anal Chem 2015, 87, 5240-5246. (14) Agarwal, A.; Diedrich, J. K.; Julian, R. R. Anal Chem 2011, 83, 6455-6458. (15) Cramer, C. N.; Haselmann, K. F.; Olsen, J. V.; Nielsen, P. K. Anal Chem 2016, 88, 1585-1592. (16) Jordan, J. B.; Poppe, L.; Haniu, M.; Arvedson, T.; Syed, R.; Li, V.; Kohno, H.; Kim, H.; Schnier, P. D.; Harvey, T. S.; Miranda, L. P.; Cheetham, J.; Sasu, B. J. The Journal of biological chemistry 2009, 284, 24155-24167. (17) Yen, T. Y.; Yan, H.; Macher, B. A. Journal of mass spectrometry : JMS 2002, 37, 15-30. (18) Haniu, M.; Horan, T.; Spahr, C.; Hui, J.; Fan, W.; Chen, C.; Richards, W. G.; Lu, H. S. Protein science : a publication of the Protein Society 2011, 20, 1802-1813. (19) Liu, H.; Lei, Q. P.; Washabaugh, M. Anal Chem 2016, 88, 5080-5087. (20) Li, X.; Wang, F.; Xu, W.; May, K.; Richardson, D.; Liu, H. Analytical biochemistry 2013, 436, 93-100. (21) Switzar, L.; Nicolardi, S.; Rutten, J. W.; Oberstein, S. A.; Aartsma-Rus, A.; van der Burgt, Y. E. J. Am Soc Mass Spectrom 2015. (22) Nicolardi, S.; Deelder, A. M.; Palmblad, M.; van der Burgt, Y. E. Anal Chem 2014, 86, 5376-5382. (23) Li, G.; Pei, J.; Yin, Y.; Huang, G. The Analyst 2015, 140, 2623-2627. (24) Olsen, J. V.; Macek, B.; Lange, O.; Makarov, A.; Horning, S.; Mann, M. Nature methods 2007, 4, 709-712.



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Complete Mapping of Complex Disulfide Patterns ... - ACS Publications

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