Assigning Peptide Disulfide Linkage Pattern Among Regio-Isomers via Methoxy Addition to Disulfide and Tandem Mass Spectrometry
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Pinpointing disulfide linkage pattern is critical in the characterization of proteins and peptides consisting of multiple disulfide bonds. Herein, we report a method based on coupling online disulfide modification and tandem mass spectrometry (MS/MS) to distinguish peptide disulfide regio-isomers. Such a method relies on a new disulfide bond cleavage reaction in solution, involving methanol as a reactant and 254 nm ultraviolet (UV) irradiation. This reaction leads to selective cleavage of a disulfide bond and formation of sulfenic methyl ester (–SOCH3) at one cysteine residue and a thiol (–SH) at the other. Under low energy collision-induced dissociation (CID), cysteine sulfenic methyl ester motif produces a signature methanol loss (–32 Da), allowing its identification from other possible isomeric structures such as S-hydroxylmethyl (–SCH2OH) and methyl sulfoxide (–S(O)-CH3). Since disulfide bond can be selectively cleaved and modified upon methoxy addition, subsequent MS2 CID of the methoxy addition product provides enhanced sequence coverage as demonstrated by the analysis of bovine insulin. More importantly, this reaction does not induce disulfide scrambling, likely due to the fact that radical intermediates are not involved in the process. An approach based on methoxy addition followed by MS3 CID has been developed for assigning disulfide linkage patterns in peptide disulfide regio-isomers. This methodology was successfully applied to characterizing peptide systems having two disulfide bonds and three disulfide linkage isomers: side-by-side, overlapped, and looped-within-a-loop configurations.
KeywordsDisulfide peptides Collision-induced dissociation Disulfide linkage Sulfenic methyl ester UV Photolysis
Formation of disulfide bond between two cysteine amino acid residues offers a possibility to covalently link segments within a polypeptide or among multiple peptides. It is one of the most common post-translational modifications in proteins, serving a vital role in defining and stabilizing three-dimensional structure in peptides and proteins [1, 2]. Peptides with multiple disulfide bonds are often encountered in biological systems, such as conotoxins (a group of neurotoxic peptides), defensins, and cyclotides [3, 4, 5]. Knotted disulfide bonds help them maintain a stable folded structure despite relatively small number of amino acid residues. Owing to different connection patterns of disulfide bonds, disulfide-rich peptides can have many disulfide regio-isomers, and usually only one specific isomer is biologically active .
Mass spectrometry (MS) has been demonstrated as a powerful tool for the identification and quantitation of peptides and proteins from complex mixtures. Characterization of disulfide-containing peptides requires information of both sequence and disulfide connectivity. The existence of disulfide bond, however, poses challenges in acquiring related information directly from mass spectrometry . For instance, cleavage of disulfide bonds is not generally observed under low energy CID conditions for positively charged ions with mobile protons [6, 7]. This is explained by Lioe et al. from the aspect that higher activation energy is required for disulfide bond cleavage (40–70 kcal/mol) compared with that required for amide bond cleavages . As a result, CID of the protonated peptide ions only produces sequence ions that fall outside of the disulfide covered region. To overcome this limitation, multiple methods have been developed with the aim of opening disulfide loops prior to MS analysis. The most commonly used approach is disulfide bond reduction followed by alkylation. This process, as well as electrolytic reduction of disulfide bonds, makes proteins more prone to subsequent enzymatic digestion and, therefore, renders increased sequence coverage in protein digests via MS/MS analysis [9, 10]. Consequently, disulfide bond linkage information is lost in this process. In order to preserve disulfide linkages, multi-enzyme digestion has been utilized to induce protein backbone cleavages within regions covered by one or multiple disulfide bonds. This method can release simple peptides connected by interchain disulfide bonds. Since there is no loop structure in these peptides, MS/MS via CID can be used to extract information on peptide sequences and disulfide bond linkages. It should be noted that delicate control of solution pH is key to avoiding disulfide bond scrambling, which can occur at pH over 8.3, the pKa of the cysteine side chain , while achieving relatively high enzyme activity. For highly knotted disulfide structures, partial disulfide bond reduction often needs to be coupled with multi-enzyme digestion. Dithiothreitol (DTT) and tris(2-carboxyethyl)phosphine (TCEP) followed by alkylation have been used in disulfide bond partial reduction . Achieving partial reduction rather than complete reduction, however, requires precise control of reaction conditions and can be a demanding task to accomplish.
In the gas phase different types of dissociation techniques have been developed to facilitate the analysis of disulfide-containing peptides in the last decade. These techniques include CID of deprotonated ions , peptide–metal complex (route 66 method) , and ultraviolet photodissociation (UVPD, at 157 nm or 266 nm) [15, 16, 17]. In electron-based dissociation, EXD, where X represents capture , transfer [19, 20, 21, 22], or detachment , backbone cleavage as well as disulfide bond cleavage can be observed, allowing rich sequence information to be acquired. Radicals are known to be highly reactive towards disulfide bonds; a number of radical approaches have been used recently to cleave disulfide bonds and enhance disulfide peptide analysis . These include distonic ion reaction approach , CID of TEMPO peptides , reactions with hydroxyl radical , sulfinyl radical , and hydroxyl methyl radical . Because radical intermediates are produced in EXD processes and radical reactions, disulfide bond scrambling may happen in a millisecond (ms)-long detection/analysis window . Consequently, the connectivity of multiple disulfide bonds in a peptide may not be accurately determined. For instance, Tan et al. studied ETD of peptide disulfide regio-isomers and found that the isomers all shared identical fragment ions resulting from radical cascades after initial electron transfer. Relative intensities of some fragment ions differed among isomers; however, the small differences could not be solely relied on for disulfide bond connectivity assignment .
The above discussed strategies, although have not fully addressed challenges in disulfide peptide/protein characterization, they clearly suggest a route based on either gas-phase or solution reaction to expand the capability of MS in their analysis, as long as the reaction is controllable and does not lead to disulfide rearrangement. Herein, we demonstrate an interesting disulfide bond cleavage pathway involving methanol upon 254 nm UV irradiation in solution. This new reaction results in the formation of sulfenic methyl ester (-S-O-CH3) and free thiol (-SH) at the pair of separated cysteine residues. Although exact mechanism is not clear, experimental evidence suggests that this reaction does not involve radical intermediates; therefore, it should not lead to disulfide bond scrambling. Based on this new reaction, we have developed an online reaction-MS/MS (via low energy CID) approach to further explore its utility in the analysis of peptide systems containing one or more disulfide bonds.
List of Disulfide Linked Peptides Studied
Synthesis of Disulfide Bonds
The oxidizing agent [Pt (en)2 (OH)2 Cl2] was synthesized from [Pt en2] Cl2 in-house following the procedure described by Heneghan and Bailar . [Pt en2 (OH)2 Cl2] was added to the dissolved peptide (1.0 mg/mL) in a molar ratio of 2:1 ~ 5:1 Pt (IV): peptide. The reaction was allowed to proceed at room temperature for 1–3 hours and the reaction progress was monitored via MS. Following complete oxidation, peptide disulfide bond isomers were separated via reverse phase-high performance liquid chromatography (RP-HPLC). The separation condition is described in Supporting Information (SI), and LC separation chromatogram of P3 regio-isomers is shown in SI Figure 1 as an example.
Methoxy Addition to Disulfide via 254 nm UV Irradiation and Mass Spectrometry
Reactions were initiated by UV irradiation from a low-pressure mercury (LP-Hg) lamp (20 mA, 2.54 cm lamp length, 0.64 cm diameter, double bore tubing with a synthetic quartz lamp casing) (model no 81-1057-5; BHK, Inc., Ontario, CA). The primary emission from the lamp is 254 nm. Peptides were introduced via nanoelectrospray ionization (nanoESI) in methanol/water (v:v = 1:1) solution. A photo of the reaction setup is shown in SI Figure 2. A similar schematic and description of the experimental setup was published elsewhere . A thin layer of aluminum foil was used to protect bulk part of the solution in tip from over-irradiation, with about 3 mm of taper exposed at the end. All experiments (except for insulin) were carried out on a 4000QTRAP tandem mass spectrometer (Applied Biosystems/SCIEX, Toronto, Canada). The triple quadrupole/linear ion trap configuration allowed for two types of collisional activation methods, (i.e., beam-type CID and ion trap CID). In beam-type CID, the precursor ions were isolated by Q1 and accelerated to q2 for collisional activation. The collision energy (CE), defined by the DC potential difference between Q0 and q2, was optimized and typically within the range of 5–10 V. Ion trap CID was carried out in Q3 linear ion trap, where a dipolar excitation was used for on-resonance collisional activation. The activation amplitudes were within the range of 20–60 mV and activation time of 200 ms was used. The characteristic parameters of the mass spectrometer during this study were set as follows: spray voltage, 1200–1800 V; curtain gas, 10 psi; and declustering potential, 20 V. Mass analysis was achieved by using Q3 as a linear ion trap at a scan rate of 1000 Da/s. Data acquisition, processing, and instrument control were performed using Analyst 1.5 software. All the insulin data were collected using a LTQ-Orbitrap (Thermo Fisher Scientific, San Jose, CA, USA) with a resolution of 30,000 Da. The data shown were typically an average of 50 scans.
In the context of this paper, peaks with a “SH” as a superscript indicate that the ion contains a free thiol group following disulfide cleavage. Labels containing “#” as a superscript suggests that the ion has been modified by the addition of the methoxy group to form the sulfenic methyl ester. Peaks labeled as “Ay n” or “Bb n” mean a sequence ion originating from that particular chain of a polypeptide, whereas in “ABb n” or BAy n”, the first letter indicates that the fragment contains either intact A or B chain, and the latter part (“Bb n” or “Ay n”) refers to amide bond cleavage from either B or A chain. Fragment ions containing an “SSH” superscript represent C–S bond cleavage yielding disulfohydryl at the cleavage site. Its complimentary fragment ion has a dehydroalanine structure and is represented by a superscript of “CH2”.
Results and Discussion
Sequence Coverage in Insulin
Distinguish Disulfide Regio-Isomers
There are several MS/MS techniques relying on radical mechanism (i.e., ETD) to analyze peptides or proteins with disulfide bond. Although they succeed in giving enhanced sequence coverage compared with CID, many fail to provide disulfide connectivity due to radical cascades that result in disulfide scrambling or consecutive disulfide cleavage [24, 41]. As our mechanistic studies have shown, methoxy addition reaction does not involve radical reactants or intermediates. Moreover, upon cleavage of a disulfide bond, methoxy and hydrogen each add on to cysteine sulfur atoms forming an even electron species. As a result, disulfide bond connectivity is well retained for subsequent analysis and disulfide regio-isomers can be unambiguously identified.
UV photolysis at 254 nm of disulfide linked peptides in the presence of methanol leads to cleavage of disulfide bond and formation of free thiol and sulfenic methyl ester at the pair of cysteine residues. MS2 CID of methoxy addition product produces a distinct neutral loss of 32 Da (CH3OH), allowing the use of tandem mass spectrometry to distinguish this product from other possible isomeric structures (S-hydroxyl methyl and methyl sulfoxide). Although the exact mechanism for methoxy addition is not clear, experimental evidences suggest that cleavage of disulfide bond is likely to go through nucleophilic attack by methanol under 254 nm UV excitation instead of radical pathways. Given its capability of cleaving disulfide bond, this reaction was coupled online with MS2 CID to provide enhanced sequence information for peptides and small proteins containing multiple disulfide bonds. For instance, CID of the methoxy addition product of bovine insulin almost doubled the detection of sequence ions compared with CID of the intact insulin ions. A strategy based on online methoxy addition –MS3 CID was developed and applied to the analysis of peptide disulfide regio-isomers containing two disulfide bonds. No disulfide bond scrambling was observed and distinctive fragment ions were obtained that allowed for the assignment of disulfide bond connecting patterns with high confidence. Beside the above advantages, relatively low reaction yield of methoxy addition reaction limits its immediate application to the analysis of more complicated disulfide peptide/protein systems. Our future studies will focus on reaction mechanism elucidation and reaction yield improvement.
Financial support from NSF CHE-1308114 is greatly appreciated. Y.X. acknowledges ASMS research award for supporting research on radical ion chemistry. K.L.D. thanks the Midwest Crossroads Alliance for Graduate Education and the Professoriate (AGEP) and the Purdue doctoral fellowship for support during the course of this research. The authors are grateful to Katelynn M. Smith for her contribution in disulfide peptide separation and Professor R. Graham Cooks for use of the LTQ-Orbitrap mass spectrometer.
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