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PubMed1996

A stable mixed disulfide between thioredoxin reductase and its substrate, thioredoxin: preparation and characterization.

The flavoenzyme thioredoxin reductase (TrR) catalyzes the reduction of the small redox protein thioredoxin (Tr) by NADPH. It has been proposed that a large conformational change is required in catalysis by TrT in order to visualize a complete pathway for reduction of equivalents. The proposal is based on the comparison of the crystal structures of TrR and glutathione reductase, the latter being a well-understood member of the enzyme family [Waksman, G., et al. (1994) J. Mol. Biol. 236, 800-816]. Bound NADPH is perfectly positioned for electron transfer to the FAD in glutathione reductase, but in TrR, these two components are 17 angstroms apart. In order to provide evidence for the proposed conformational change, a complex between TrR and its substrate Tr involving a mixed disulfide between TrR and Tr was prepared. The redox active disulfide of TrR is composed of Cys135 and Cys138, and the redox active disulfide of Tr is made up of Cys32 and Cys35. The complex C135S-C32S is prepared from forms of TrR and Tr altered by site-directed mutagenesis where Cys138 and Cys35 are remaining in TrR and Tr, respectively. The purified C135S-C32S presents a band on a nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis responding to a molecular weight sum of one subunit of TrR and one of Tr. Several observations indicate that C135S-C32S can adopt only one conformation. It was reported previously that TrR C135S can form a charge transfer complex in the presence of ammonium cation in which the donor is the remaining thiolate of Cys138 [Prongay, A.J., et al., (1989) J. Biol. Chem. 264, 2656-2664], while titration of C135S-C32S with NH4Cl does not induce charge transfer, presumably because Cys138 is participating in the mixed dissulfide. Reduction of C135S-C32S with dithiothreitol (DTT) results in a decrease of epsilon454 to a value similar to that of TrR C135S, and subsequent NH4Cl titration leads to charge transfer complex formation in the nascent TrR C135S. Reductive titrations show that approximately 1 equiv of sodium dithionite or NADPH is required to fully reduce C135S-C32S, and treatment with NH4Cl and DTT demonstrates that the mixed disulfide between Cys138 of TrR C135S and Cys35 of TrC32S that locks the structure in a conformation where FAD can be reduced by NADPH, but electrons cannot flow from FADH2 to the mixed disulfide bond.

Disulfides↗
PubMed1996

Electrostatic interactions in the active site of the N-terminal thioredoxin-like domain of protein disulfide isomerase.

Proteins with the thioredoxin fold have widely differing stabilities of the disulfide bond that can be formed between the two cysteines at their active site sequence motif Cys1-Xaa2-Yaa3-Cys4. This is believed to be regulated not by varying the disulfide bond itself, but by modulating the stability of the dithiol form of the protein through interactions with the ionized form of the Cys1 thiol group. A consistent relationship between disulfide bond stability and Cys1 thiol pKa value is found here for DsbA, thioredoxin, and the N-terminal thioredoxin-like domain of protein disulfide isomerase (PDI a), which has a very low thiol pKa value of 4.5. This thiolate anion is stabilized by 5.7 kcal/mol in the dithiol form, giving rise to the corresponding instability of the disulfide bond and the oxidizing properties of PDI a. Electrostatic interactions in the active site of the PDI a-domain have been characterized in order to understand the physical basis of this stabilization. Linkage with the ionization of the imidazole group of His3 in the active site demonstrates that this charge-charge interaction contributes 1.1 kcal/mol. The remainder of the stabilization is believed to be due primarily to interactions with the partial positive charges at the N-terminus of an alpha-helix, which are exceedingly sensitive to charges of surrounding residues.

Binding Sites↗
PubMed1998

Secretion efficiency in Saccharomyces cerevisiae of bovine pancreatic trypsin inhibitor mutants lacking disulfide bonds is correlated with thermodynamic stability.

Bovine pancreatic trypsin inhibitor (BPTI) has been widely used as a model protein to investigate protein structure and folding pathways. To study the role of its three disulfide bonds in folding, proofreading, and secretion of BPTI in an intact eucaryotic cell, BPTI was expressed and secreted from a synthetic gene in the yeast Saccharomyces cerevisiae. Site-directed mutagenesis was used to create all possible single and pairwise cysteine to alanine BPTI mutants, and the effect of these mutations on secretion efficiency was determined. The 5-55 disulfide bond is found to be essential for secretion-loss of either Cys5, Cys55, or both prevents secretion. Removal of the 14-38 disulfide bond results in a small reduction of secretion, but individual Cys14 or Cys38 replacements reduce secretion efficiency by 30%. Cys30 and Cys30-51 mutants are secreted at half the level of wild-type BPTI, while secretion of the Cys51 mutant is reduced by 90%. BPTI containing only a single disulfide bond (5-55) is not secreted. No relationship is observed between secretion efficiency and in vitro folding or unfolding rates, but mutant BPTI secretion is directly correlated with the in vitro unfolding temperature Tm and the free energy of stabilization provided by each of the three disulfides. These results indicate that structural fluctuations rather than the time-averaged structure observed by NMR or X-ray crystallography may determine recognition of a protein as misfolded and subsequent retention and degradation.

Amino Acid Sequence↗
PubMed2004

Determination and reoxidation of the disulfide bridges of a squash-type trypsin inhibitor from Sechium edule seeds.

The determination of the disulfide pairings of SETI-II, a trypsin inhibitor isolated from Sechium edule, is described herein. The inhibitor contains 31 amino acid residues per mol, 6 of which are cysteine. Forty-five nmol (160 microg) of SETI-II was hydrolyzed with 20 microg thermolysin for 48 hr at 45 degrees C, and peptides were separated by reverse phase high performance liquid chromatography (RP-HPLC). The major products were identified by amino acid composition, Edman degradation, and on the basis of the sequence of the inhibitor. The disulfide bridge pairings and (yields) are: Cys1-Cys4 (79%), Cys2-Cys5 (21%) and Cys3-Cys6 (43%). When the reduced inhibitor was reoxidized with glutathione reduced form (GSH)/glutathione oxidized form (GSSG) at pH 8.5 for 3 hr, full activity was recovered. These data show that disulfide bridge pairing and oxidation can be determined at nanomole levels and that sensitive and quantitative Edman degradation can eliminate the final time- and material-consuming step of disulfide determinations by eliminating the need to purify and cleave each peptide containing a disulfide bridge.

Amino Acid Sequence↗
PubMed2005

Critical role of a thiolate-quinone charge transfer complex and its adduct form in de novo disulfide bond generation by DsbB.

Recent studies have revealed numerous examples in which oxidation and reduction of cysteines in proteins are integrated into specific cascades of biological regulatory systems. In general, these reactions proceed as thiol-disulfide exchange events. However, it is not exactly understood how a disulfide bond is created de novo. DsbB, an Escherichia coli plasma membrane protein, is one of the enzymes that create a new disulfide bond within itself and in DsbA, the direct catalyst of protein disulfide bond formation in the periplasmic space. DsbB is associated with a cofactor, either ubiquinone or menaquinone, as a source of an oxidizing equivalent. The DsbB-bound quinone undergoes transition to a pink (lambdamax, approximately 500 nm, ubiquinone) or violet (lambdamax, approximately 550 nm, menaquinone)-colored state during the course of the DsbB enzymatic reaction. Here we show that not only the thiolate form of Cys-44 previously suggested but also Arg-48 in the alpha-helical arrangement is essential for the quinone transition. Quantum chemical simulations indicate that proper positioning of thiolate anion and ubiquinone in conjunction with positively charged guanidinium moiety of arginine allows the formation of a thiolate-ubiquinone charge transfer complex with absorption peaks at approximately 500 nm as well as a cysteinylquinone covalent adduct. We propose that the charge transfer state leads to the transition state adduct that accepts a nucleophilic attack from another cysteine to generate a disulfide bond de novo. A similar mechanism is conceivable for a class of eukaryotic dithiol oxidases having a FAD cofactor.

Arginine↗
PubMed1997

Respiratory chain is required to maintain oxidized states of the DsbA-DsbB disulfide bond formation system in aerobically growing Escherichia coli cells.

DsbA, the disulfide bond catalyst of Escherichia coli, is a periplasmic protein having a thioredoxin-like Cys-30-Xaa-Xaa-Cys-33 motif. The Cys-30-Cys-33 disulfide is donated to a pair of cysteines on the target proteins. Although DsbA, having high oxidizing potential, is prone to reduction, it is maintained essentially all oxidized in vivo. DsbB, an integral membrane protein having two pairs of essential cysteines, reoxidizes DsbA that has been reduced upon functioning. It is not known, however, what might provide the overall oxidizing power to the DsbA-DsbB disulfide bond formation system. We now report that E. coli mutants defective in the hemA gene or in the ubiA-menA genes markedly accumulate the reduced form of DsbA during growth under the conditions of protoheme deprivation as well as ubiquinone/menaquinone deprivation. Disulfide bond formation of beta-lactamase was impaired under these conditions. Intracellular state of DsbB was found to be affected by deprivation of quinones, such that it accumulates first as a reduced form and then as a form of a disulfide-linked complex with DsbA. This is followed by reduction of the bulk of DsbA molecules. These results suggest that the respiratory electron transfer chain participates in the oxidation of DsbA, by acting primarily on DsbB. It is remarkable that a cellular catalyst of protein folding is connected to the respiratory chain.

Aerobiosis↗
PubMed2001

Disulfide-dependent folding and export of Escherichia coli DsbC.

DsbC, a member of the Dsb family in the periplasm of Gram-negative bacteria, is not only a disulfide isomerase but also a chaperone. Five DsbC mutants with Cys in the active site sequence of Cys(98)-Gly-Tyr-Cys(101) and the nonactive site disulfide Cys(141)-Cys(163) replaced by Ser have been studied. The results show that the active site Cys residues are necessary for enzyme activities but not required for chaperone activity, while the lack of the nonactive site disulfide results in a decreased chaperone activity in assisting the reactivation of denatured d-glyceraldehyde-3-phosphate dehydrogenase but has no effect on enzyme activities. Wild-type DsbC was overexpressed and correctly processed as a soluble periplasmic protein. Mutation in one of these Cys residues results in aggregation or extracellular/membrane locations, but does not affect the proper processing. DsbC mutated in either Cys residue of nonactive site disulfide shows higher sensitivity to unfolding by guanidine hydrochloride and slower refolding compared with wild-type DsbC and the active site Cys mutants. The above results provide experimental evidence for structural role of the nonactive site disulfide in folding and biological activities of DsbC.

Amino Acid Substitution↗
PubMed2002

Thiol-disulfide oxidoreductases are essential for the production of the lantibiotic sublancin 168.

Thiol-disulfide oxidoreductases are required for disulfide bond formation in proteins that are exported from the cytoplasm. Four enzymes of this type, termed BdbA, BdbB, BdbC, and BdbD, have been identified in the Gram-positive eubacterium Bacillus subtilis. BdbC and BdbD have been shown to be critical for the folding of a protein required for DNA uptake during natural competence. In contrast, no function has been assigned so far to the BdbA and BdbB proteins. The bdbA and bdbB genes are located in one operon that also contains the genes specifying the lantibiotic sublancin 168 and the ATP-binding cassette transporter SunT. Interestingly sublancin 168 contains two disulfide bonds. The present studies demonstrate that SunT and BdbB, but not BdbA, are required for the production of active sublancin 168. In addition, the BdbB paralogue BdbC is at least partly able to replace BdbB in sublancin 168 production. These observations show the unprecedented involvement of thiol-disulfide oxidoreductases in the synthesis of a peptide antibiotic. Notably BdbB cannot complement BdbC in competence development, showing that these two closely related thiol-disulfide oxidoreductases have different, but partly overlapping, substrate specificities.

ATP-Binding Cassette Transporters↗
PubMedDate not supplied

Extracellular disulfide exchange and the regulation of cellular function.

An emerging concept is that disulfide bonds can act as a dynamic scaffold to present mature proteins in different conformational and functional states on the cell surface. Two examples are the conversion of the receptor, integrin alphaIIbbeta3, from a low affinity to a high affinity state, and the interaction of CD4 receptor with the HIV-1 envelope glycoprotein gp120 to promote virus-cell fusion. In both of these cases there is a remodeling of the protein disulfide bonding pattern. The formation and rearrangement of disulfide bonds is modulated by a family of enzymes known as the thiol isomerases, which include protein disulfide isomerase (PDI), ERp5, ERp57, and ERp72. While these enzymes were reported originally to be restricted in location to the endoplasmic reticulum, in some cells thiol isomerases are found on the cell surface. This may indicate a wider role for these enzymes in cell function. In platelets it has been shown that reagents that react with cell surface sulfhydryl groups are capable of blocking a number of functional responses, including integrin-mediated aggregation, adhesion, and granule secretion. Furthermore, the use of function blocking antibodies to either PDI or ERp5 causes inhibition of these functional responses. This review summarizes current knowledge of the extracellular regulation of disulfide exchange and the implications of this in the regulation of cell function.

Animals↗
PubMedDate not supplied

Conservation and diversity of the cellular disulfide bond formation pathways.

Two pathways for the formation of biosynthetic protein disulfide bonds have been characterized in the endoplasmic reticulum (ER) of eukaryotes. In the major pathway, the membrane-associated flavoprotein Ero1 generates disulfide bonds for transfer to protein disulfide isomerase (PDI), which is responsible for directly introducing disulfide bonds into secretory proteins. In a minor fungal-specific protein oxidation pathway, the membrane-associated flavoprotein Erv2 can catalyze disulfide bond formation via the transfer of oxidizing equivalents to PDI. Genomic sequencing has revealed an abundance of enzymes sharing homology with Ero1, Erv2, or PDI. Herein the authors discuss the functional, mechanistic, and potential structural similarities between these homologs and the core enzymes of the characterized ER oxidation pathways. In addition they speculate about the possible differences between these enzymes that may explain why the cell contains multiple proteins dedicated to a single process. Finally, the eukaryotic ER protein oxidation and reduction pathways are compared to the corresponding prokaryotic periplasmic pathways, to highlight the functional, mechanistic, and structural similarities that exist between the pathways in these two kingdoms despite very low primary sequence homology between the protein and small molecule components.

Bacterial Proteins↗
PubMed1995

Disulfide bond formation and secretion of Escherichia coli heat-stable enterotoxin II.

The Escherichia coli heat-stable enterotoxin II (STII) is a typical extracellular toxin consisting of 48 amino acid residues, of which 4 are cysteine. There are two disulfide bonds, one between Cys-10 and Cys-48 and one between Cys-21 and Cys-36. We examined the involvement of DsbA in the formation of the disulfide bonds of STII and the role of each in the secretion of STII. A dsbA mutant was transformed with a plasmid harboring the STII gene, and STII was not detected either in the cells or in the culture supernatant. Reducing the level of STII brought about the dsbA mutation restored by introducing the wild-type dsbA gene into the mutant strain. These results showed that DsbA is involved in forming the disulfide bonds of STII and that STII without these disulfide bonds is degraded during secretion. We substituted these four cysteine residues in vivo by oligonucleotide-directed site-specific mutagenesis. The amino acid sequence of the purified STII (C48S) and pulse-chase studies revealed that two intermolecular disulfide bonds must be formed to be efficiently secreted and that cleavage between amino acid residues 14 and 15 is probably the first step in the proteolytic degradation of STII.

Bacterial Toxins↗
PubMed2001

Disulfide bond formation in secreton component PulK provides a possible explanation for the role of DsbA in pullulanase secretion.

When expressed in Escherichia coli, the 15 Klebsiella oxytoca pul genes that encode the so-called Pul secreton or type II secretion machinery promote pullulanase secretion and the assembly of one of the secreton components, PulG, into pili. Besides these pul genes, efficient pullulanase secretion also requires the host dsbA gene, encoding a periplasmic disulfide oxidoreductase, independently of disulfide bond formation in pullulanase itself. Two secreton components, the secretin pilot protein PulS and the minor pseudopilin PulK, were each shown to posses an intramolecular disulfide bond whose formation was catalyzed by DsbA. PulS was apparently destabilized by the absence of its disulfide bond, whereas PulK stability was not dramatically affected either by a dsbA mutation or by the removal of one of its cysteines. The pullulanase secretion defect in a dsbA mutant was rectified by overproduction of PulK, indicating reduced disulfide bond formation in PulK as the major cause of the secretion defect under the conditions tested (in which PulS is probably present in considerable excess of requirements). PulG pilus formation was independent of DsbA, probably because PulK is not needed for piliation.

Bacterial Outer Membrane Proteins↗
PubMed2000

The thioredoxin boxes of thyroglobulin: possible implications for intermolecular disulfide bond formation in the follicle lumen.

Multimerization of thyroglobulin (TG) takes place extracellularly in the thyroid follicle lumen and is regarded as a mechanism to store TG at high concentrations. Human thyroglobulin (hTG) has been shown to multimerize mainly by intermolecular disulfide cross-links. We recently noted that TG of various mammalian species contains three highly conserved thioredoxin boxes (CXXC). This sequence is known to underlie the enzymatic activity of protein disulfide isomerase (PDI). As hTG formed intermolecular disulfide bonds in the absence of other proteins depending on the redox conditions and hTG concentration, the CXXC-boxes of TG might provide the structural basis for self-assisted intermolecular cross-linking. To test this hypothesis we prepared a recombinant TG fragment containing the three thioredoxin boxes. This fragment exhibited a redox activity amounting to about 10% of the activity of PDI at redox conditions supposed to be present in the extracellular space. This activity might be supplemented by the oxidizing system of the apical cell surfaces of thyrocytes facing the follicle lumen. Indeed, incubation of hTG with peroxidase and H202 resulted in intermolecular disulfide bridge formation. Our results suggest a combined mechanism of self-assisted and peroxidase-mediated disulfide bond formation leading to the intermolecular cross-linking of lumenal hTG.

Animals↗
PubMed1994

Influence of protein disulfide isomerase (PDI) on antibody folding in vitro.

The role of eucaryotic protein disulfide isomerase (PDI) in the folding and reoxidation of proteins in vitro was investigated using an antibody Fab fragment as a model substrate, since PDI is known to participate in the disulfide bond formation of immunoglobulins in vivo. PDI has no effect on the folding of the Fab fragment with intact disulfide bonds, suggesting that, at least in this system, PDI is not able to influence the folding process in a chaperone-like manner. Instead, the role of PDI is limited to disulfide bond formation as demonstrated for the folding of the denatured and reduced Fab fragment. Here, PDI influences the yield of reactivation enormously with a maximum effect at about stoichiometric amounts of PDI and Fab. Furthermore, PDI changes the redox dependence of the reaction. In the presence of PDI, formation of the correct disulfide bonds is possible at higher oxidizing conditions compared to the spontaneous reaction. The requirements both for stoichiometric amounts of PDI and for the presence of PDI during the first seconds of refolding suggest that there is a kinetic competition between rapid structure formation of the antibody domains and interaction of PDI with cysteine residues in the folding protein.

Animals↗
PubMed1994

Involvement of two sulfur atoms of protein disulfide isomerase and one sulfur atom of the DsbA/PpfA protein in the oxidation of mutant human lysozyme.

Protein disulfide isomerase (PDI) and the DsbA/PpfA protein catalyze the oxidation of mutant human lysozyme, L79CC81A, which has two native disulfide bonds, Cys6-Cys128 and Cys30-Cys116, a non-native Cys79-Cys95, and 2 free cysteine residues at positions 65 and 77. Oxidation of L79CC81A (R-form) yielded two isomers, L79CC81A-a (A-form) with tandem-linked Cys65-Cys77 and Cys79-Cys95, and L79CC81A-b (B-form) with cross-linked Cys65-Cys79 and Cys77-Cys95 (Kanaya, E., Ishihara, K., Tsunasawa, S., Nokihara, K., and Kikuchi, M. (1993) Biochem. J. 292, 469-476). PDI mainly enhanced the formation of the A- form in the absence of oxidized glutathione (GSSG); however, as the concentration of GSSG increased, it markedly accelerated the formation of the B-form. In contrast, the DspA/PpfA protein mainly enhanced the formation of the A-form, regardless of the presence or absence of GSSG. These results and the presumed spatial locations of Cys65, Cys77, and Cys79-Cys95 in the R-form suggest that 1 of the half-cystine residues in the active site of PDI and the DsbA/PpfA protein can react with 1 of the 2 free Cys residues of the R-form. The dependence on GSSG of the B-form formation with PDI can be explained by the formation of two transient intermolecular disulfide bonds between PDI and the R-form and the attack of GSSG by the resultant thiolate anion of Cys79 or Cys95. The independence of the reaction with the DsbA/PpfA protein from GSSG can be explained by the formation of one transient intermolecular disulfide bond. The possible formation of the two transient intermolecular disulfide bonds involving two sulfur atoms of PDI and 2 cysteine or half-cystine residues of the substrate could explain the high isomerase activity of PDI.

Binding Sites↗
PubMed1997

Redox active disulfides: the thioredoxin system as a drug target.

Thioredoxin, and particularly extracellular thioredoxin, presents an attractive target for developing novel agents to treat cancer. Our studies have involved the examination of a series of alkyl 2-imidazolyl disulfides as inhibitors of the growth-stimulatory activity of the thioredoxin system. We originally determined the disulfides to be weak reversible inhibitors of thioredoxin reductase. Subsequently, we have shown that alkyl 2-imidazolyl disulfides interact directly with thioredoxin, thioalkylating critical cysteine residues or causing dimerization of the protein leading to its loss of biological activity. One of the analogues that binds to thioredoxin, 1-methylpropyl 2-imidazolyl disulfide (IV-2), selectively inhibits the thioredoxin-dependent growth of tumor cells in culture and has antitumor activity against MCF-7 and HL-60 tumors in vivo. Our work involves the development of a parallel combinatorial synthetic method to produce a large number of disulfide analogues at one time. These analogues, which differ sterically, electronically, and physically, were produced in a 96-well plate. The biological activity of these analogues was evaluated, also in the 96-well plate format. This rapid method of evaluating biological activity is a means to identify agents with specificity for inhibition of the thioredoxin system, and may provide novel antitumor agents with activity against solid tumor cancers.

Animals↗
PubMed2000

Mass spectrometric mapping of disulfide bonds in recombinant human interleukin-13.

Interleukin 13 (IL-13), a member of the a-helical family of cytokines, has approximately 30% primary sequence homology with IL-4 and shares a common receptor component. The biologically active rhIL-13 is monomeric and non-glycosylated, and contains two disulfide bonds as determined by comparative electrospray mass spectrometric (MS) analysis of the protein before and after reduction with dithiothreitol-dithioerythritol. A trypsin-resistant core peptide of rhIL-13 was isolated and analyzed by plasma desorption (PD) MS, identifying a disulfide-linked core peptide. Subsequent digestion of this core peptide by pepsin, followed by PDMS analysis of the resulting cystine-containing peptic fragments, provided rapid determination of the existing disulfide bonds between cysteine residues 28-56 and 44-70. This disulfide arrangement is similar to that observed for the analogous four internal cysteine residues in hIL-4. The conservation of disulfide bond arrangements between hIL-13 and hIL-4, coupled with their alpha-helical structure and sequence homologies, confirms that IL-13 and IL-4 are structural homologues. It is also consistent with their reported similarities in biological function and receptor binding kinetics.

Amino Acid Sequence↗
PubMed1999

On-line sample clean-up and chromatography coupled with electrospray ionization mass spectrometry to characterize the primary sequence and disulfide bond content of recombinant calcium binding proteins.

We have used on-line sample clean-up, concentration, and chromatography with electrospray ionization mass spectrometry (ESI-MS), to characterize and determine the presence of disulfide bonds in recombinant full-length rat brain calbindin D28K and two deletion mutants of the protein, one lacking EF-hand 2 (calbindin delta 2) and the other lacking EF-hands 2 and 6 (calbindin delta 2,6). The molecular weights of the expressed proteins dissolved in biological buffers were determined with high accuracy using a low-flow, pressurized chamber infusion system, that allows on-line protein clean-up by removing buffers/salts incompatible with ESI-MS. The molecular weight determinations showed that the amino-terminal methionine residues had been cleaved during the expression and isolation of the recombinant proteins. Approximately 85-90% of the protein sequences were confirmed by on-line HPLC-ESI-MS analysis of peptides generated by a lysyl endoproteinase C digestion. Comparisons of ESI-MS spectra of native and reduced calbindin D28K and delta 2 show that the full length- and delta 2 mutant-protein contain one disulfide bond. Molecular mass determinations of calbindin delta 2,6 showed that this protein contains a highly active cysteine residue that covalently binds a mercaptoethanol group, or forms a homodimer via a disulfide bond. The results show surprising differences amongst the deletion mutants of calbindin D28K with respect to the formation of disulfide bonds. These differences are not readily detected by other techniques and show that ESI-MS is a powerful, rapid method by which to detect disulfide linkages for intact proteins.

Amino Acid Sequence↗