Structural characterization of an unusually stable cyclic peptide, kalata B2 from Oldenlandia affinis

Kalata peptides are isolated from an African medicinal plant, Oldenlandia affinis, an aqueous decoction of which can be ingested to accelerate uterine contraction during childbirth. The closely packed disulfide core of kalata peptides confers unusual stability against thermal, chemical, and enzymatic degradation. The molecular arrangement may hamper NMR-assisted disulfide connectivity assignment. We have combined NMR with high-resolution mass spectrometry (MS) and MS/MS of native and chemically derivatized kalata B2 to determine its amino acid sequence and disulfide connectivity. Infrared multiphoton dissociation establishes the disulfide bond linkages in kalata B2 as I-IV, II-V and III-VI.     

Development of disulfide peptide mapping and determination of disulfide structure of recombinant human osteoprotegerin chimera produced in Escherichia coli

Recombinant human osteoprotegerin chimera is a 90-kDa protein containing a human IgG Fc domain fused to human osteoprotegerin. The molecule is a dimer linked by two intermolecular disulfide bonds and contains eleven intramolecular disulfide bonds per monomer. A cysteine-rich region in osteoprotegerin contains nine disulfide bridges homologous to the cysteine-rich signature structure of the tumor necrosis factor receptor/nerve growth factor receptor superfamily. In this report, we have developed peptide mapping procedures suitable to generate disulfide-containing peptides for disulfide structure assignment of the fusion molecule. The methods employed included proteolytic digestion using endoproteinases Glu-C and Lys-C in combination followed by LC-MS analyses. Disulfide linkages of peptide fragments containing a single disulfide bond were assigned by sequence analysis via detection of (phenylthiohydantoinyl) cystine and/or by MS analysis. Disulfide bonds of a large, core fragment containing three peptide sequences linked by four disulfides were assigned after generation of smaller disulfide-linked peptides by a secondary thermolysin digestion. Disulfide structures of peptide fragments containing two disulfide bonds were assigned using matrix-assisted laser desorption ionization mass spectrometry with postsource decay. Both the inter- and intramolecular disulfide linkages of the chimeric dimer were confirmed.     

Formation of a covalent complex between methylguanine methyltransferase and DNA via disulfide bond formation between the active site cysteine and a thiol-containing analog of guanine.

DNA repair methyltransferases (MTases) remove methyl or other alkyl groups from the O6 position of guanine or the O4 position of thymine by transfering the group to an active site cysteine. In order to trap an MTase-DNA complex via a disulfide bond, 2'-deoxy-6-(cystamine)-2-aminopurine (d6Cys2AP) was synthesized and incorporated into oligonucleotides. d6Cys2AP has a disulfide bond within an alkyl chain linked to the 6 position of 2,6-diaminopurine, which disulfide can be reduced to form a free thiol. Addition of human MTase to reduced oligonucleotide resulted in a protein-DNA complex that was insensitive to denaturation by SDS and high salt, but which readily dissociated in the presence of dithiothreitol. Formation of this complex was prevented by methylation of the active site cysteine. Evidence that the active site cysteine is directly involved in disulfide bond formation was obtained by N-terminal sequencing of peptides that remained associated with DNA after proteolysis of the complex.     

Determination of the disulfide structure of sillucin, a highly knotted, cysteine-rich peptide, by cyanylation/cleavage mass mapping

The disulfide structure of sillucin, a highly knotted, cysteine-rich, antimicrobial peptide, isolated from Rhizomucor pusillus, has been determined to be Cys2--Cys7, Cys12--Cys24, Cys13--Cys30, and Cys14--Cys21 by disulfide mass mapping based on partial reduction and CN-induced cleavage enabled by cyanylation. The denatured 30-residue peptide was subjected to partial reduction by tris(2-carboxyethyl)phosphine hydrochloride at pH 3 to produce a mixture of partially reduced sillucin species; the nascent sulfhydryl groups were immediately cyanylated by 1-cyano-4-(dimethylamino)pyridinium tetrafluoroborate. The cyanylated species, separated and collected during reversed phase high-performance liquid chromatography, were treated with aqueous ammonia, which cleaved the peptide chain on the N-terminal side of cyanylated cysteine residues. The CN-induced cleavage mixture was analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry before and after complete reduction of residual disulfide bonds in partially reduced and cyanylated species to mass map the truncated peptides to the sequence. Because the masses of the CN-induced cleavage fragments of both singly and doubly reduced and cyanylated sillucin are related to the linkages of the disulfide bonds in the original molecule, the presence of certain truncated peptide(s) can be used to positively identify the linkage of a specific disulfide bond or exclude the presence of other possible linkages.     

A new level of conotoxin diversity,a non-native disulfide bond connectivity in alpha-conotoxin AuIB reduces structural definition but increases biological activity

alpha-Conotoxin AuIB and a disulfide bond variant of AuIB have been synthesized to determine the role of disulfide bond connectivity on structure and activity. Both of these peptides contain the 15 amino acid sequence GCCSYPPCFATNPDC, with the globular (native) isomer having the disulfide connectivity Cys(2-8 and 3-15) and the ribbon isomer having the disulfide connectivity Cys(2-15 and 3-8). The solution structures of the peptides were determined by NMR spectroscopy, and their ability to block the nicotinic acetylcholine receptors on dissociated neurons of the rat parasympathetic ganglia was examined. The ribbon disulfide isomer, although having a less well defined structure, is surprisingly found to have approximately 10 times greater potency than the native peptide. To our knowledge this is the first demonstration of a non-native disulfide bond isomer of a conotoxin exhibiting greater biological activity than the native isomer.     

A novel methodology for assignment of disulfide bond pairings in proteins.

A novel methodology is described for the assignment of disulfide bonds in proteins of known sequence. The denatured protein is subjected to limited reduction by tris(2-carboxyethyl)phosphine (TCEP) in pH 3.0 citrate buffer to produce a mixture of partially reduced protein isomers; the nascent sulfhydryls are immediately cyanylated by 1-cyano-4-dimethylamino-pyridinium tetrafluoroborate (CDAP) under the same buffered conditions. The cyanylated protein isomers, separated by and collected from reversed-phase HPLC, are subjected to cleavage of the peptide bonds on the N-terminal side of cyanylated cysteines in aqueous ammonia to form truncated peptides that are still linked by residual disulfide bonds. The remaining disulfide bonds are then completely reduced to give a mixture of peptides that can be mass mapped by MALDI-MS. The masses of the resulting peptide fragments are related to the location of the paired cysteines that had undergone reduction, cyanylation, and cleavage. A side reaction, beta-elimination, often accompanies cleavage and produces overlapped peptides that provide complementary confirmation for the assignment. This strategy minimizes disulfide bond scrambling and is simple, fast, and sensitive. The feasibility of the new approach is demonstrated in the analysis of model proteins that contain various disulfide bond linkages, including adjacent cysteines. Experimental conditions are optimized for protein partial reduction, sulfhydryl cyanylation, and chemical cleavage reactions.     

Mass spectrometric characterization of the isoforms in Escherichia coli recombinant DNA-derived interferon alpha-2b

The isoforms Iso-2, Iso-3, and Iso-4 of Escherichia coli-derived recombinant human interferon alpha-2b (rhIFN α-2b), generated by posttranslational modifications of the protein during fermentation, present a major problem in terms of purification and the yield of the drug substance. We report here the structural characterization of these isoforms by mass spectrometry (MS) methods. An extensive MS study was conducted on Iso-4, which is composed of up to 75% of the in-process IFN, and on the native rhIFN α-2b. The trypsin-digested peptide mixtures generated from the two samples were analyzed by liquid chromatography (LC)–MS, and targeted peptides were further studied by LC–tandem MS (triple quadrupole mass spectrometer), high-resolution MSⁿ (LTQ Orbitrap), and matrix-assisted laser desorption/ionization MS (MALDI–MS). The structure of Iso-4 was elucidated as a novel pyruvic acid ketimine derivative of the N-terminal cysteine (Cys1) of IFN α-2b, where the disulfide bond between Cys1 and Cys98 was fully reduced and the other disulfide bond pair, Cys29-ss-Cys138, was partially reduced. Similarly, Iso-2 was identified as a correctly disulfide-folded rhIFN α-2b with acetylation on Cys1, and Iso-3 was identified as an S-glutathionylated form (Cys98) of partially reduced rhIFN α-2b that was pyruvated on Cys1. Based on the characterization work, a reproducible conversion procedure was successfully implemented to convert Iso-4 to rhIFN α-2b.     

Assignment of disulfide bonds in proteins by fast atom bombardment mass spectrometry

A rapid and sensitive method for assignment of disulfide bonds using fast atom bombardment mass spectrometry is described for hen egg white lysozyme and bovine ribonuclease A. The protein is initially digested to a mixture of peptides using chemical and enzymatic methods under conditions which minimize disulfide bond reduction and exchange. The digested sample is analyzed directly by fast atom bombardment mass spectrometry before and after chemical reduction of cystine residues. An important feature of the method is that it is not necessary to completely resolve the peptides in the digest chromatographically prior to analysis. The disulfide-containing peptides are also characterized directly by prolonged exposure of the sample to the high energy xenon atom beam which results in the reduction of cystine residues. Intra- as well as interchain disulfide bond assignments are made on the basis of the mass difference between the molecular ions (MH+) of the oxidized and reduced peptides. Confirmation of the mass assignments may be obtained from the mass spectra of the digests after one cycle of manual Edman degradation. Although the quantity of protein required to unambiguously assign all of the disulfide linkages will depend on the ease with which the appropriate peptide fragments can be formed, results from these studies indicate that approximately 1 nmol of protein is usually sufficient.     

Solution structure of the recombinant penaeidin-3, a shrimp antimicrobial peptide

Penaeidins are a family of antimicrobial peptides of 47-63 residues isolated from several species of shrimp. These peptides display a proline-rich domain (N-terminal part) and a cysteine-rich domain (C-terminal part) stabilized by three conserved disulfide bonds whose arrangement has not yet been characterized. The recombinant penaeidin-3a of Litopenaeus vannamei (63 residues) and its [T8A]-Pen-3a analogue were produced in Saccharomyces cerevisiae and showed similar antimicrobial activity. The solution structure of the [T8A]-Pen-3a analogue was determined by using two-dimensional 1H NMR and simulated annealing calculations. The proline-rich domain, spanning residues 1-28 was found to be unconstrained. In contrast, the cysteine-rich domain, spanning residues 29-58, displays a well defined structure, which consists of an amphipathic helix (41-50) linked to the upstream and the downstream coils by two disulfide bonds (Cys32-Cys47 and Cys48-Cys55). These two coils are in turn linked together by the third disulfide bond (Cys36-Cys54). Such a disulfide bond packing, which is in agreement with the analysis of trypsin digests by ESI-MS, contributes to the highly hydrophobic core. Side chains of Arg45 and Arg50, which belong to the helix, and side chains of Arg37 and Arg53, which belong to the upstream and the downstream coils, are located in two opposite parts of this globular and compact structure. The environment of these positively charged residues, either by hydrophobic clusters at the surface of the cysteine-rich domain or by sequential hydrophobic residues in the unconstrained proline-rich domain, gives rise to the amphipathic character required for antimicrobial peptides. We hypothesize that the antimicrobial activity of penaeidins can be explained by a cooperative effect between the proline-rich and cysteine-rich features simultaneously present in their sequences.     

Synthesis of cystine peptides 21-25/70-73 and 35-39/56-59 of the beta-subunit of human choriogonadotropin.

Syntheses of two asymmetrical cystine peptides with the amino acid residues 21-25/70-73 and 35-39/56-59, based on the linear amino acid sequence and the disulfide bond assignment in the beta-subunit of human choriogonadotropin (hCG-beta), are described. S-trityl and S-acetamidomethyl peptide fragments of each cystine peptide were prepared in solution phase and were subjected to oxidation with I2/MeOH to form the disulfide bridge. The cystine peptides were characterized by their amino acid analyses and fast atom bombardment mass spectrometry. Immunological characterization by several homologous radioimmunoassay systems showed that peptide 21-25/70-73 had significant hCG, hCG-beta, and hLH activities while peptide 35-39/56-59 failed to reveal any immunoreactivity.     

Assignment of the three disulfide bonds of Selenocosmia huwena lectin-I from the venom of spider Selenocosmia huwena.

The positions of the disulfide bonds of Selenocosmia huwena lectin-I (SHL-I) from the venom of the Chinese bird spider S. huwena have been determined. The existence of three disulfide bonds in the native SHL-I was proved by matrix-assisted laser desorption ionization time-of-flight mass spectroscopic analysis. To map the disulfide bonds, native SHL-I was proteolytically digested. The resulting peptides were separated by reverse phase high-performance liquid chromatography. Matrix-assisted laser desorption ionization time-of-flight mass spectroscopic analysis indicated the presence of one disulfide bond Cys7-Cys19. The partially reduced peptides by using Tris-(2-carboxyethyl)-phosphine at pH 3.0 were purified by reverse phase high-performance liquid chromatography. Four M Guanidine-HCl was found to increase the yields of partially reduced peptides prominently. The free thiols were carboxamidomethlate by iodoacetamide. The specific location of another disulfide bond Cys2-Cys14 was proved by comparing N-terminal sequencing analysis of the partially reduced and alkylated SHL-I with that of the intact peptide. Finally, the three disulfide linkage of SHL-I could be assigned as Cys2-Cys14, Cys7-Cys19, Cys13-Cys26.     

Determination of disulfide bond assignments and N-glycosylation sites of the human gastrointestinal carcinoma antigen GA733-2 (CO17-1A, EGP, KS1-4, KSA, and Ep-CAM)

The GA733-2 antigen is a cell surface glycoprotein highly expressed on most human gastrointestinal carcinoma and at a lower level on most normal epithelia. It is an unusual cell-cell adhesion protein that does not exhibit any obvious relationship to the four known classes of adhesion molecules. In this study, the disulfide-bonding pattern of the GA733-2 antigen was determined using matrix-assisted laser desorption/ionization mass spectrometry and N-terminal sequencing of purified tryptic peptides treated with 2-[2'-nitrophenylsulfonyl]-3-methyl-3-bromoindolenine or partially reduced and alkylated. Numbering GA733-2 cysteines sequentially from the N terminus, the first three disulfide linkages are Cys1-Cys4, Cys2-Cys6, and Cys3-Cys5, which is a novel pattern for a cysteine-rich domain instead of the expected epidermal growth factor-like disulfide structure. The next three disulfide linkages are Cys7-Cys8, Cys9-Cys10, and Cys11-Cys12, consistent with the recently determined disulfide pattern of the thyroglobulin type 1A domain of insulin-like growth factor-binding proteins 1 and 6. Analysis of glycosylation sites showed that GA733-2 antigen contained N-linked carbohydrate but that no O-linked carbohydrate groups were detected. Of the three potential N-linked glycosylation sites, Asn175 was not glycosylated, whereas Asn88 was completely glycosylated, and Asn51 was partially glycosylated. These data show that the extracellular domain of the GA733-2 antigen consists of three distinct domains; a novel cysteine-rich N-terminal domain (GA733 type 1 motif), a cysteine-rich thyroglobulin type 1A domain (GA733 type 2 motif), and a unique nonglycosylated domain without cysteines (GA733 type 3 motif).     

Chemical synthesis of La1 isolated from the venom of the scorpion Liocheles australasiae and determination of its disulfide bonding pattern

La1 is a 73‐residue cysteine‐rich peptide isolated from the scorpion Liocheles australasiae venom. Although La1 is the most abundant peptide in the venom, its biological function remains unknown. Here, we describe a method for efficient chemical synthesis of La1 using the native chemical ligation (NCL) strategy, in which three peptide components of less than 40 residues were sequentially ligated. The peptide thioester necessary for NCL was synthesized using an aromatic N‐acylurea approach with Fmoc‐SPPS. After completion of sequential NCL, disulfide bond formation was carried out using a dialysis method, in which the linear peptide dissolved in an acidic solution was dialyzed against a slightly alkaline buffer to obtain correctly folded La1. Next, we determined the disulfide bonding pattern of La1. Enzymatic and chemical digests of La1 without reduction of disulfide bonds were analyzed by liquid chromatography/mass spectrometry (LC/MS), which revealed two of four disulfide bond linkages. The remaining two linkages were assigned based on MS/MS analysis of a peptide fragment containing two disulfide bonds. Consequently, the disulfide bonding pattern of La1 was found to be similar to that of a von Willebrand factor type C (VWC) domain. To our knowledge, this is the first report of the experimental determination of the disulfide bonding pattern of peptides having a single VWC domain as well as their chemical synthesis. La1 synthesized in this study will be useful for investigation of its biological role in the venom. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Structure-activity relationships of the intramolecular disulfide bonds in coprisin,a defensin from the dung beetle

Defensins, which are small cationic molecules produced by organisms as part of their innate immune response, share a common structural scaffold that is stabilized by three disulfide bridges. Coprisin is a 43-amino acid defensin-like peptide from Copris tripartitus. Here, we report the intramolecular disulfide connectivity of cysteine-rich coprisin, and show that it is the same as in other insect defensins. The disulfide bond pairings of coprisin were determined by combining the enzymatic cleavage and mass analysis. We found that the loss of any single disulfide bond in coprisin eliminated all antibacterial, but not antifungal, activity. Circular dichroism (CD) analysis showed that two disulfide bonds, Cys20-Cys39 and Cys24-Cys41, stabilize coprisin’s α-helical region. Moreover, a BLAST search against UniProtKB database revealed that coprisin’s α-helical region is highly homologous to those of other insect defensins. [BMB Reports 2014; 47(11): 625-630]     

The disulfide bond pattern of catrocollastatin C, a disintegrin-like/cysteine-rich protein isolated from Crotalus atrox venom

The disulfide bond pattern of catrocollastatin-C was determined by N-terminal sequencing and mass spectrometry. The N-terminal disintegrin-like domain is a compact structure including eight disulfide bonds, seven of them in the same pattern as the disintegrin bitistatin. The protein has two extra cysteine residues (XIII and XVI) that form an additional disulfide bond that is characteristically found in the disintegrin-like domains of cellular metalloproteinases (ADAMs) and PIII snake venom Zn-metalloproteinases (SVMPs). The C-terminal cysteine-rich domain of catrocollastatin-C contains five disulfide bonds between nearest-neighbor cysteines and a long range disulfide bridge between CysV and CysX. These results provide structural evidence for a redefinition of the disintegrin-like and cysteine-rich domain boundaries. An evolutionary pathway for ADAMs, PIII, and PII SVMPs based on disulfide bond engineering is also proposed.     

The disulfide bond pattern between fragments obtained by the limited proteolysis of bovine thyroglobulin.

The comparative analysis of the products of the limited proteolysis of bovine thyroglobulin with trypsin by SDS-polyacrylamide gel electrophoresis in non-reducing and reducing conditions revealed the presence of disulfide linkages between some of the fragments. In order to define the disulfide bond pattern between the proteolytic fragments of thyroglobulin, these were isolated by SDS-polyacrylamide gel electrophoresis in non-reducing conditions and electrophoretic transfer onto polyvinylidene difluoride membranes. Individual bands were desorbed from the membranes and re-analyzed by SDS-polyacrylamide gel electrophoresis in reducing conditions. The resulting peptides were identified by comparison with the peptides directly obtained by SDS-electrophoresis in reducing conditions, and characterized by amino-terminal peptide sequencing either in this study or in a previous investigation (Gentile F., Salvatore G., Eur. J. Biochem. 218 (1993) 603-621). The analysis revealed that several fragments, produced by cleavages within the context of various cysteine-rich repeats of type 1 and within cysteine-rich repeat 3b.1, did not separate in the absence of reduction. On the other hand, the products of the cleavages at the carboxy-terminal extremity of the linker between type 2 and type 3 cysteine-rich repeats, and in the middle of the acetylcholinesterase-similar domain of thyroglobulin separated freely, with no need for reduction. On the base of these data, a model is presented in which distinct subsets of cysteine-rich repeats and the carboxy-terminal, acetylcholinesterase-similar domain of thyroglobulin form sequentially aligned subdomains with internal disulfide linkages.     

Defining the disulfide bonds of insulin-like growth factor-binding protein-5 by tandem mass spectrometry with electron transfer dissociation and collision-induced dissociation

The six high-affinity insulin-like growth factor-binding proteins (IGFBPs) comprise a conserved family of secreted molecules that modulate IGF actions by regulating their half-life and access to signaling receptors, and also exert biological effects that are independent of IGF binding. IGFBPs are composed of cysteine-rich amino- (N-) and carboxyl- (C-) terminal domains, along with a cysteine-poor central linker segment. IGFBP-5 is the most conserved IGFBP, and contains 18 cysteines, but only 2 of 9 putative disulfide bonds have been mapped to date. Using a mass spectrometry (MS)-based strategy combining sequential electron transfer dissociation (ETD) and collision-induced dissociation (CID) steps, in which ETD fragmentation preferentially induces cleavage of disulfide bonds, and CID provides exact disulfide linkage assignments between liberated peptides, we now have definitively mapped 5 disulfide bonds in IGFBP-5. In addition, in conjunction with ab initio molecular modeling we are able to assign the other 4 disulfide linkages to within a GCGCCXXC motif that is conserved in five IGFBPs. Because of the nature of ETD fragmentation MS experiments were performed without chemical reduction of IGFBP-5. Our results not only establish a disulfide bond map of IGFBP-5 but also define a general approach that takes advantage of the specificity of ETD and the scalability of tandem MS, and the predictive power of ab initio molecular modeling to characterize unknown disulfide linkages in proteins.     

Determination of phosphorylation levels of tyrosine hydroxylase by electrospray mass spectrometry

A novel approach has been developed to quantify the extent of phosphorylation of tyrosine hydroxylase (TH). The strategy consists of a chemical cleavage and characterization of the products using electrospray mass spectrometry (ESMS). The chemical cleavage involves selective hydrolysis of the aspartyl-peptide bond. Of the peptides formed, an 8-kDa NH2-terminus fragment is found to accurately duplicate the phosphorylation of TH using standard mixtures of TH-P/TH. The calibration yields a straight line with an R2 of 0.996, which is valid within the 10-90% range. The ESMS protocol has been used to determine the extent of phosphorylation of TH in the presence of CaM-PKII. The experimental conditions were designed to produce low levels of phosphorylation. Nevertheless, the ESMS analysis yielded single, double, and nonphosphorylation forms of TH. With respect to in vivo measurements, this ESMS protocol may be a generic procedure for determining the extent of phosphorylation of proteins.     

Conformational landscape and pathway of disulfide bond reduction of human alpha defensin

Human alpha defensins are a class of antimicrobial peptides with additional antiviral activity. Such antimicrobial peptides constitute a major part of mammalian innate immunity. Alpha defensins contain six cysteines, which form three well defined disulfide bridges under oxidizing conditions. Residues C3-C31, C5-C20, and C10-C30 form disulfide pairs in the native structure of the peptide. The major tissue in which HD5 is expressed is the crypt of the small intestine, an anaerobic niche that should allow for substantial pools of both oxidized and (partly) reduced HD5. We used ion mobility coupled to mass spectrometry to track the structural changes in HD5 upon disulfide bond reduction. We found evidence of stepwise unfolding of HD5 with sequential reduction of the three disulfide bonds. Alkylation of free cysteines followed by tandem mass spectrometry of the corresponding partially reduced states revealed a dominant pathway of reductive unfolding. The majority of HD5 unfolds by initial reduction of C5-C20, followed by C10-C30 and C3-C31. We find additional evidence for a minor pathway that starts with reduction of C3-C31, followed by C5-C20 and C10-C30. Our results provide insight into the pathway and conformational landscape of disulfide bond reduction in HD5.     

2,2′‐Dipyridyl diselenide: A chemoselective tool for cysteine deprotection and disulfide bond formation

There are many examples of bioactive, disulfide‐rich peptides and proteins whose biological activity relies on proper disulfide connectivity. Regioselective disulfide bond formation is a strategy for the synthesis of these bioactive peptides, but many of these methods suffer from a lack of orthogonality between pairs of protected cysteine (Cys) residues, efficiency, and high yields. Here, we show the utilization of 2,2′‐dipyridyl diselenide (PySeSePy) as a chemical tool for the removal of Cys‐protecting groups and regioselective formation of disulfide bonds in peptides. We found that peptides containing either Cys(Mob) or Cys(Acm) groups treated with PySeSePy in trifluoroacetic acid (TFA) (with or without triisopropylsilane (TIS) were converted to Cys‐S–SePy adducts at 37 °C and various incubation times. This novel Cys‐S–SePy adduct is able to be chemoselectively reduced by five‐fold excess ascorbate at pH 4.5, a condition that should spare already installed peptide disulfide bonds from reduction. This chemoselective reduction by ascorbate will undoubtedly find utility in numerous biotechnological applications. We applied our new chemistry to the iodine‐free synthesis of the human intestinal hormone guanylin, which contains two disulfide bonds. While we originally envisioned using ascorbate to chemoselectively reduce one of the formed Cys‐S–SePy adducts to catalyze disulfide bond formation, we found that when pairs of Cys(Acm) residues were treated with PySeSePy in TFA, the second disulfide bond formed spontaneously. Spontaneous formation of the second disulfide is most likely driven by the formation of the thermodynamically favored diselenide (PySeSePy) from the two Cys‐S–SePy adducts. Thus, we have developed a one‐pot method for concomitant deprotection and disulfide bond formation of Cys(Acm) pairs in the presence of an existing disulfide bond.