Labeling of cysteine-containing peptides with 2-nitro-5-thiobenzoic acid

A method for specific labeling of cysteine-containing peptides has been developed using Ellman's reagent, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). Prior to cleavage with proteases or chemical reagents, proteins are reacted with DTNB, resulting in the formation of a mixed disulfide between the protein sulfhydryl group and 2-nitro-5-thiobenzoic acid (TNB). The formation of the mixed disulfide introduces a chromophore, with an absorbance peak at 328 nm. By monitoring peptide maps generated by HPLC at 210 and 328 nm, peptides containing cysteine residues are readily identified. The stability of the derivative was tested using glutathione-TNB as a model compound. Glutathione-TNB is stable to conditions used for CNBr cleavage, as well as those for tryptic cleavage. The TNB label may also increase the hydrophobicity of small peptides, which otherwise might not bind to reverse-phase matrices. This was the case for an oxidatively modified tetrapeptide isolated from Escherichia coli glutamine synthetase.     

Ga2O3 photocatalyzed on-line tagging of cysteine to facilitate peptide mass fingerprinting

β-Ga(2)O(3) is a wide-band-gap semiconductor having strong oxidation ability under light irradiation. Herein, the steel target plates modified with β-Ga(2)O(3) nanoparticles have been developed to carry out in-source photo-catalytic oxidative reactions for online peptide tagging during laser desorption/ionization mass spectrometry (LDI-MS) analysis. Under UV laser irradiation, β-Ga(2)O(3) can catalyze the photo-oxidation of 2-methoxyhydroquinone added to a sample mixture to 2-methoxy benzoquinone that can further react with the thiol groups of cysteine residues by Michael addition reaction. The tagging process leads to appearance of pairs of peaks with an m/z shift of 138.1Th. This online labelling strategy is demonstrated to be sensitive and efficient with a detection-limit at femtomole level. Using the strategy, the information on cysteine content in peptides can be obtained together with peptide mass, therefore constraining the database searching for an advanced identification of cysteine-containing proteins from protein mixtures. The current peptide online tagging method can be important for specific analysis of cysteine-containing proteins especially the low-abundant ones that cannot be completely isolated from other high-abundant non-cysteine-proteins.     

A new method for the selective isolation of cysteine-containing peptides. Specific labelling of the thiol group with a hydrophobic chromophore.

A new method for the selective isolation of cysteine-containing peptides was designed. The method is based on the specific labelling of thiol groups with a hydrophobic chromophore followed by enzymic fragmentation of the labelled protein and reversed-phase high-pressure liquid-chromatographic separation of the peptide mixture. This new method has several distinct advantages: (1) the hydrophobic-chromophore-labelled cysteine-containing peptides are easily separated from non-cysteine-containing peptides by reversed-phase high-pressure liquid chromatography; (2) only cysteine-containing peptides are detected in the visible region with sensitivity at the low picomole level; this high sensitivity allows isolation of nanogram amounts of pure cysteine-containing peptide; (3) during sequence determination of the chromophore-labelled cysteine-containing peptides, the cysteine residues are released as coloured anilinothiazolinone derivatives and can be detected directly in the picomole range; (4) with proteins bearing several disulphide groups, each disulphide group may undergo a different degree of reduction, and therefore the recovery of individual cysteine-containing peptides may be used to deduce the disulphide linkages present in the native protein. Two thiol-specific reagents, 4-dimethylaminoazobenzene-4'-iodoacetamide and 4-dimethylaminoazobenzene-4'-N-maleimide, were synthesized and characterized. The method was successfully used to isolate five cysteine-containing peptides from a completely reduced monoclonal-antibody kappa-light chain raised against the azobenzenearsonate determinant and six cysteine-containing peptides from a kappa-light chain raised against streptococcal group A polysaccharide. The principle of this method is applicable to the isolation of any peptide containing amino acid residues that can be specifically labelled with a hydrophobic chromophore.     

Reversed-phase high-pressure liquid chromatographic tryptic peptide mapping for the comparison and study of monoclonal antibodies.

We have examined and optimized several parameters to generate efficient, high-resolution, high-information tryptic peptide maps of monoclonal antibodies and their Fab fragments, without separating the H and L chains, using reversed-phase high-pressure liquid chromatography. Use of a high protease-to-substrate ratio with optimized digestion time and HPLC gradient conditions led to a reproducible mapping of the reduced, carboxymethylated Fab fragments of two antibodies. The technique was then used to screen Fab lots for batch-to-batch consistency, and for examining the effect of 10 mM cysteine on papain cleavage of whole antibody. The technique was modified by labeling cysteine with chromophoric analogues of iodoacetamide instead of radiolabeled iodoacetamide, resulting in a three-dimensional peptide map. With multiwavelength detection, this consisted of simultaneous observation of all chromophores at 214 nm, those with aromatic residues at 280 nm, and those with cysteine at 422 nm, without collecting and counting each peak to identify cysteine-containing peptides.     

毛细管电泳测定牛红细胞超氧化物歧化酶的巯基位点

本文介绍了毛细管电泳分析蛋白质酶解产物中含巯基多肽的方法。还原的及天然的牛红细胞超氧化物歧化酶(SOD)经4-乙烯吡啶修饰后,由TPCK-胰蛋白酶水解,在254nm检测到还原的SOD水解物中含3个巯基多肽,天然的SOD为1个疏基多肽且其毛细管电泳行为与上述3个多肽之一相一致。分析它们的氨基酸顺序,证实Cys-6为游离的巯基,Cys-55和Cys~(-144)形成二硫键。     

Alkylation of cysteine-containing peptides to mimic palmitoylation.

Numerous proteins that are involved in cell signaling and viral replication require post-translational modification by palmitoylation to function properly. The molecular details by which this palmitoyl modification affects protein function remain poorly understood. To facilitate in vitro biochemical and structural studies of the role of palmitoylation on protein function, a method was developed for alkylating peptides with saturated C16 groups at cysteine residues and demonstrated using peptides derived from the palmitoylated region of Sindbis virus E2 glycoprotein. The synthetic approach takes advantage of disulfide chemistry to specifically modify only the cysteine residues within peptides and covalently links C16 groups via disulfide bridges using a new thioalkylating reagent, hexyldexyldithiopyridine. The chemistry presented here takes place in solution under mild conditions without the need for protection of the peptide functional groups. A method for purifying these modified peptides is also described. This protocol can be of general use to investigators studying the role of palmitoylation in biological systems.     

The origin and control of ex vivo oxidative peptide modifications prior to mass spectrometry analysis

The origin and control of ex vivo sample handling related oxidative modifications of methionine-, S-alkyl cysteine-, and tryptophan-containing peptides obtained from typical "in-solution" or "in-gel" proteolytic digestion strategies, have been examined by capillary HPLC and MS/MS. The origin of increased oxidation levels were found to be predominantly associated with the extensive ex vivo sample handling steps required for gel electrophoresis and/or in-gel proteolytic digestion of proteins prior to analysis by MS. Conditions for deliberately controlling the oxidation state (both oxidation and reduction) of these peptides, as well as for those containing cysteine, have been evaluated using a series of model synthetic peptides and standard tryptic protein digests. Essentially complete oxidation of methionine- and S-alkyl cysteine-containing peptides was achieved by reaction with 30% hydrogen peroxide/5% acetic acid at room temperature for 30 min. Under these conditions, cysteine was also converted to cysteic acid, while only limited oxidation of tryptophan to oxindolylalanine, and methionine and S-alkyl cysteine sulfoxides to their respective sulfones, were observed. Efficient reduction of methionine- and S-alkyl cysteine sulfoxide-containing peptides was achieved by reaction in 1 M dimethylsulfide/10 M hydrochloric acid at room temperature for 10 and 45 min, respectively. None of the reduction conditions evaluated were found to result in the reduction of oxindolylalanine, cysteic acid, or methionine sulfone.     

Rotavirus VP4 and VP7-Derived Synthetic Peptides as Potential Substrates of Protein Disulfide Isomerase Lead to Inhibition of Rotavirus Infection

Rotavirus infection of MA104 cells has been shown to be inhibited by cell membrane-impermeant thiol/disulfide exchange inhibitors and anti-PDI antibodies. To characterise the amino acid sequences of rotavirus structural proteins potentially mediating cell surface PDI?Csubstrate interactions, rotavirus-derived peptides from VP4 and VP7 (RRV) and VP7 (Wa), and their modified versions containing serine instead of cysteine were synthesized. Cysteine-containing VP7 peptides corresponding to residues 189?C210 or 243?C263 caused an infectivity inhibitory effect of about 64 and 85?%, respectively, when added to cells. Changing cysteine to serine significantly decreased the inhibitory effect. A cysteine-containing peptide corresponding to VP4 residues 200?C219 and its scrambled version reduced infectivity by 92 and 80?%, respectively. A cysteine to serine change in the original VP4 200?C219 peptide did not affect its inhibitory effect. Non-rotavirus related sequences containing cysteine residues efficiently inhibited rotavirus infectivity. Antibodies against VP7 residues 189?C210 or 243?C263 significantly inhibited rotavirus infectivity only after virus attachment to cells had occurred, whereas those against VP4 200?C219 peptide inhibited infectivity irrespective of whether virus or cell-attached virus was antibody-treated. A direct PDI?Cpeptide interaction was shown by ELISA for cysteine-containing VP7 and VP4 peptides. Virus?Ccell attachment was unaffected by the peptides inhibiting virus infectivity. The results showed that even though cysteine residues in the peptides tested are important in both virus infectivity inhibition and in vitro PDI?Cpeptide interaction, the accompanying amino acid sequence also plays some role. As a whole, our findings further support our hypothesis that cell surface PDI from MA104 cells might be contributing to rotavirus entry at a post-attachment step.     

Polyionic fusion peptides function as specific dimerization motifs.

The de novo design of a molecular adapter for directed association and covalent linkage of two polypeptides is presented. Using peptides containing charged amino acid residues and an additional cysteine residue (AlaCysLys(8) and AlaCysGlu(8)) we demonstrate that the electrostatic interaction promotes the association of two synthetic peptides and, subsequently, disulfide bond formation. The reaction depends on both the redox potential and on the ionic strength of the buffer. Varying the redox potential, the interaction of the peptides was quantified by a Delta G(0') of 6.6 +/- 0.2 kcal/mol. Heterodimerization of the peptides is highly specific, a competition of association by other cysteine containing compounds could not be observed. Two proteins comprising cysteine-containing polyionic fusion peptides, a modified Fab fragment and an alpha-glucosidase fusion, could be specifically conjugated by directed association and subsequent disulfide bond formation. Both proteins retain their functional characteristics within the bifunctional conjugate: enzymatic activity of the alpha-glucosidase and antigen-binding capacity of the Fab fragment are equivalent to the non-conjugated components.     

Identification of the reactive sulfhydryl and sequences of cysteinyl-tryptic peptides from beef heart aconitase

In an accompanying paper (Kennedy, M. C., Spoto, G., Emptage, M. H., and Beinert, H. (1988) J. Biol. Chem. 263, 8190-8193), it was shown that one cysteine per mol of aconitase is modified by a variety of sulfhydryl reagents. We have identified the tryptic peptide that contains the iodoacetamide-reactive cysteine. We have also demonstrated that this cysteine is the primary site of modification by phenacyl bromide (2-bromoacetophenone), a spin label analogue of N-ethylmaleimide (HO-461) and iodoacetate in both the 3Fe and 4Fe forms of aconitase. The amino acid sequence of the peptide containing the reactive cysteine from beef heart aconitase shares no homology with the reactive cysteine-containing peptide reported for pig heart aconitase (Hahm, K.-S., Gawron, O., and Piszkiewicz, D. (1981) Biochim. Biophys. Acta 667, 457-461). We also report the amino acid compositions and sequences of seven other cysteine-containing tryptic peptides from beef heart aconitase. However, none of the cysteinyl peptides isolated were found to correspond to the reported pig heart reactive cysteinyl peptide. Evidence is also presented that no previously unreactive cysteine becomes exposed and reactive to sulfhydryl reagents in the conversion from the [4Fe-4S] cluster of the enzyme to the [3Fe-4S] cluster. We conclude from this that any potential cysteine ligand to the Fea site of the cluster must be inaccessible to solvent in the 3Fe form or, alternatively, that active 4Fe aconitase does not contain a cysteine ligand to the Fea site.     

Reversible labeling of cysteine-containing peptides allows their specific chromatographic isolation for non-gel proteome studies

We report upon a novel procedure to specifically isolate cysteine-containing peptides from a complex peptide mixture. Cysteines are converted to hydrophobic residues by mixed disulfide formation with Ellman's reagent. Proteins are subsequently digested with trypsin and the generated peptide mixture is a first time fractionated by reverse-phase high-performance liquid chromatography. Cysteinyl-peptides are isolated out of each primary fraction by a reduction step followed by a secondary peptide separation on the same column, performed under identical conditions as for the primary separation. The reducing agent removes the covalently attached group from the cysteine side chain, making cysteine-peptides more hydrophilic and, thereby, such peptides can be specifically collected during the secondary separation and are finally used to identify their precursor proteins using automated liquid chromatography tandem mass spectrometry. We show that this procedure efficiently isolates cysteine-peptides, making the sample mixture less complex for further analysis. This method was applied for the analysis of the proteomes of human platelets and enriched human plasma. In both proteomes, a significant number of low abundance proteins were identified next to extremely abundant ones. A dynamic range for protein identification spanning 4-5 orders of magnitude is demonstrated.     

Differential protein labeling with thiol-reactive infrared DY-680 and DY-780 maleimides and analysis by two-dimensional gel electrophoresis

Differential protein labeling with 2-DE separation is an effective method for distinguishing differences in the protein composition of two or more protein samples. Here, we report on a sensitive infrared-based labeling procedure, adding a novel tool to the many labeling possibilities. Defined amounts of newborn and adult mouse brain proteins and tubulin were exposed to maleimide-conjugated infrared dyes DY-680 and DY-780 followed by 1- and 2-DE. The procedure allows amounts of less than 5 microg of cysteine-labeled protein mixtures to be detected (together with unlabeled proteins) in a single 2-DE step with an LOD of individual proteins in the femtogram range; however, co-migration of unlabeled proteins and subsequent general protein stains are necessary for a precise comparison. Nevertheless, the most abundant thiol-labeled proteins, such as tubulin, were identified by MS, with cysteine-containing peptides influencing the accuracy of the identification score. Unfortunately, some infrared-labeled proteins were no longer detectable by Western blots. In conclusion, differential thiol labeling with infrared dyes provides an additional tool for detection of low-abundant cysteine-containing proteins and for rapid identification of differences in the protein composition of two sets of protein samples.     

Recognition of cysteine-containing peptides through prompt fragmentation of the 4-dimethylaminophenylazophenyl-4'-maleimide derivative during analysis by MALDI-MS

Under specified UV-MALDI conditions, the 4-dimethylaminophenylazophenyl-4'-maleimide (DABMI) derivative of cysteine-containing peptides undergoes prompt fragmentation to produce a characteristic mass spectral pattern. As reported previously by others, derivatization with chromophoric DABMI allows cysteine-containing peptides and proteins to be tracked during HPLC by absorbance at upper UV and visible wavelengths. Here, we describe methodology by which these same peptide derivatives can be recognized by their distinctive MALDI mass spectral fragmentation pattern, then mass mapped, allowing for easy, rapid identification of cysteine-containing peptides.     

Structural Characterization of the Recombinant P40 Heavy Chain Subunit Monomer and Homodimer of Murine IL-12

Interleukin-12 (IL-12) is a heterodimeric cytokine that consists of two structurally unrelated subunits, P35 and P40. However, when expressed alone in Chinese hamster ovary (CHO) cells, murine P40 showed two species of different molecular weights under nonreducing conditions, a monomeric form of 45 kDa and a homodimer of >97 kDa. Under reducing conditions the two forms migrated as an identical array of species of 40-45 kDa. The monomer was separated from the homodimer under nonreducing conditions by heparin affinity chromatography and the disulfide bond structures of both species were determined by peptide mapping, Edman sequencing, and mass spectrometry. The peptide maps of the two species were identical except for a single peak that changed retention time. Sequencing showed that this peak contained two peptides of identical sequences in both maps. Mass spectrometric analysis of the peak from the >97-kDa species revealed an ion of double the expected mass, thus indicating that the peptide pair had dimerized. Mass analysis of the peak from the 40- to 45-kDa species showed that the peptide pair contained a mass difference that corresponded to that of an extra cysteine and which disappeared upon reduction. Amino acid analysis confirmed that the monomeric form of rmP40 is modified by a reducible cysteine. Structural analysis of the remainder of the cysteine-containing peaks showed that both species of rmP40 contained the same set of intramolecular disulfide bonds. The murine P40 homodimer arises from formation of a single intermolecular disulfide bond at Cys¹⁷⁵. In the monomeric P40, however, this cysteine is capped by an additional cysteine. Purified rmP40 monomer and homodimer inhibited the IL-12-dependent induction of interferon-γ, but neither appeared capable of inducing IL-12-like biological activity.     

Use of N-(7-dimethylamino-4-methylcoumarinyl)maleinimide for the detection of cysteine-containing peptides in peptide maps

A fluorescent reagent N-[7-dimethylamino-4-methylcoumarinyl]maleinimide (DACM), which reacts selectively with protein thiols, was used in the detection of cysteine-containing peptides in peptide maps. Direct staining of peptide maps of glutathione and tryptic digested α₁-antitrypsin resulted in the finding of one and four cysteine-containing spots, respectively. All other peptides could be visualized after the DACM staining, by the use of fluorescamine. Amino acid analysis of all peptides showed that only the DACM fluorescent spots contained cysteine residues.     

The subunits of rabbit-muscle phosphofructokinase. A search for sequence repetition.

Rabbit muscle phosphofructokinase uniformly carboxymethylated with iodo[2-14C]acetate consists of subunits with a molecular weight of 80 000 +/- 5000. The subunit polypeptide chain contains 16 and 52 residues respectively of cysteine and arginine and, contrary to previous results, peptide mapping experiments gave no indication that phosphofructokinase chains yield fewer than the expected numbers of cysteine and arginine containing peptides. To test further for the possible occurrence of repeat sequences within a single subunit chain, cysteine-containing peptides were isolated and sequenced from tryptic and thermolytic digests of s-[2-14C]carboxymethylated phosphofructokinase. In all, 15 different cysteine sequences (comprising a total of 104 residues) were identified, showing that not more than one of an expected 16 cysteine-containing sequences is repeated, and that the subunits of phosphofructokinase are of unique sequence along their entire length. The near quantitative isolation of several cysteine-containing peptides shows further that all subunits are of similar if not identical sequence.     

Discovery of highly reactive peptide tag by ELISA-type screening for specific cysteine conjugation

We report the discovery of a highly reactive peptide tag for the specific cysteine conjugation of proteins. Screening of cysteine-containing peptides using ELISA-type screening yielded a 19-amino acid tag (DCPPPDDAADDAADDAADD), named DCP3 tag, which enabled the rapid and selective labeling of the tag-fused protein with a synthetic zinc complex on the surface of living cells.     

The N-hydroxysuccinimide ester of Boc-[S-(3-nitro-2-pyridinesulfenyl)]-cysteine: a heterobifunctional cross-linking agent

Synthetic cysteine-containing peptides were unidirectionally conjugated to albumin via disulfide bonds using the S-(3-nitro-2-pyridinesulfenyl) derivative of cysteine. This method employs the N-hydroxysuccinimide ester of Boc-[S-(3-nitro-2-pyridinesulfenyl)]-cysteine, a protected amino acid derivative used in peptide synthesis, as a heterobifunctional cross-linking agent. The disulfide bonds in the conjugates are formed by the reaction of free thiols with S-(3-nitro-2-pyridinesulfenyl) groups. Bovine albumin was conjugated in this manner to several synthetic peptides derived from human fibrin. Amino acid analysis of these conjugates demonstrated incorporations of from 6 to 11 peptide molecules per molecule of protein.     

A cysteine residue (cysteine-116) in the histidinol binding site of histidinol dehydrogenase

Salmonella typhimurium L-histidinol dehydrogenase (EC 1.1.1.23), a four-electron dehydrogenase, was inactivated by an active-site-directed modification reagent, 7-chloro-4-nitro-2,1,3-benzoxadiazole (NBD-Cl). The inactivation followed pseudo-first-order kinetics and was prevented by low concentrations of the substrate L-histidinol or by the competitive inhibitors histamine and imidazole. The observed rate saturation kinetics for inactivation suggest that NBD-Cl binds to the enzyme noncovalently before covalent inactivation occurs. The UV spectrum of the inactivated enzyme showed a peak at 420 nm, indicative of sulfhydryl modification. Stoichiometry experiments indicated that full inactivation was correlated with modification of 1.5 sulfhydryl groups per subunit of enzyme. By use of a substrate protection scheme, it was shown that 0.5 sulfhydryl per enzyme subunit was neither protected against NBD-Cl modification by L-histidinol nor essential for activity. Modification of the additional 1.0 sulfhydryl caused complete loss of enzyme activity and was prevented by L-histidinol. Pepsin digestion of NBD-modified enzyme was used to prepare labeled peptides under conditions that prevented migration of the NBD group. HPLC purification of the peptides was monitored at 420 nm, which is highly selective for NBD-labeled cysteine residues. By amino acid sequencing of the major peptides, it was shown that the reagent modified primarily Cys-116 and Cys-377 and that the presence of L-histidinol gave significant protection of Cys-116. The presence of a cysteine residue in the histidinol binding site is consistent with models in which formation and subsequent oxidation of a thiohemiacetal occurs as an intermediate step in the overall reaction.     

A simple and efficient maleimide-based approach for peptide extension with a cysteine-containing peptide phage library

Although peptide-based molecules are known to have therapeutic potential, the generation of phage focused libraries to optimize peptides is effort-consuming. A chemical method is developed to extend a maleimide-conjugated peptide with a cysteine-containing random-peptide phage display library. As a proof of concept, a 15-mer epidermal growth factor receptor (EGFR)-binding peptide was synthesized with a maleimide group at its C-terminus and then conjugated to the cysteine-containing library. After panning and screening, several extended peptides were discovered and tested to have a higher affinity to EGFR. This strategy can have broad utility to optimize pharmacophores of any modalities (peptides, unnatural peptides, drug conjugates) capable of bearing a maleimide group