毛细管等电聚焦和电渗泵驱动聚焦区带分离蛋白质

建立了一种利用电渗泵驱动毛细管内的聚焦区带,实现毛细管电泳等电聚焦分离蛋白质的方法。通过控制电压来调节泵的输出流量,从而调节聚焦区带的迁移速度。适用于毛细管电泳等电聚焦两步法分离蛋白质等两性物质。考察了对牛血清白蛋白和溶菌酶两种粗提蛋白质混合物的分离,迁移时间的RSD分别为1.6%和1.3%,峰面积的RSD均为1.6%,证明方法可行。     

Rapid determination of lysine in biological samples by isocratic liquid chromatography.

A simple, rapid, and sensitive method was developed for detection and quantitation of lysine (Lys) in various biological samples by isocratic liquid chromatography (LC). Samples containing Lys and other amino acids were derivatized with 9-fluorenylmethyl chloroformate (FMOC-CI). The mobile phase used for isocratic elution was 50 mmol/L sodium acetate buffer (pH 4.20)-acetonitrile (43 + 57, v/v). Lys was detected with a UV detector at 265 nm. The derivatized Lys eluted from a LiChrospher 100 RP-18 (150 x 4.0 mm id) column at a retention time of 5.6 min. The limit of detection was 0.73 mumol/L (signal-to-noise [S/N] ratio, 3:1), and the limit of quantitation was 2.37 mumol/L (S/N ratio, 10:1). Lys recoveries from fortified biological samples were > 97.5%. Average Lys contents found in rumen fluid samples collected before the morning feeding and at 2.0, 4.0, and 6.0 h after feeding were 4.26, 3.34, 3.58, and 3.82 mumol/L, respectively. The hydrolysate of a sample of mixed rumen microorganisms collected before the morning feeding was determined to contain 1.372 mumol/mg microbial nitrogen in the form of Lys. The Lys concentrations of human plasma, goat plasma, human urine, and goat urine were 140.0, 102.0, 58.0, and 32.0 mumol/L, respectively.     

高效弱阳离子交换色谱法对脲还原变性溶菌酶的折叠研究

用高效弱阳离子交换色谱(HPWCX)对脲还原变性溶菌酶(Lys)进行了复性研究. 在流动相中脲浓度固定为4.0 mol•L^－1和选用对天然态蛋白有稳定作用的硫酸铵为盐或置换剂时, 在蛋白浓度为15.0～50.0 mg•mL^－1时, HPWCX法比稀释法活性回收率高. 为了提高Lys的质量及活性回收率对所用色谱条件进行了优化研究, 当蛋白起始浓度为20.0 mg•mL^－1时, Lys的质量回收率和活性收率分别为97.8%和95.4%. 表明此种方法简便且有可能对其他还原变性蛋白的复性具有通用性.     

Characterization of N-palmitoylated human growth hormone by in situ liquid-liquid extraction and MALDI tandem mass spectrometry

Acylation is a common post-translational modification found in secreted proteins and membrane-associated proteins, including signal transducing and regulatory proteins. Acylation is also explored in the pharmaceutical and biotechnology industry to increase the stability and lifetime of protein-based products. The presence of acyl moieties in proteins and peptides affects the physico-chemical properties of these species, thereby modulating protein stability, function, localization and molecular interactions. Characterization of protein acylation is a challenging analytical task, which includes the precise definition of the acylation sites in proteins and determination of the identity and molecular heterogeneity of the acyl moiety at each individual site. In this study, we generated a chemically modified human growth hormone (hGH) by incorporation of a palmitoyl moiety on the N(epsilon) group of a lysine residue. Monoacylation of the hGH protein was confirmed by determination of the intact molecular weight by mass spectrometry. Detailed analysis of protein acylation was achieved by analysis of peptides derived from hGH by protease treatment. However, peptide mass mapping by MALDI MS using trypsin and AspN proteases and standard sample preparation methods did not reveal any palmitoylated peptides. In contrast, in situ liquid-liquid extraction (LLE) performed directly on the MALDI MS metal target enabled detection of acylated peptide candidates by MALDI MS and demonstrated that hGH was N-palmitoylated at multiple lysine residues. MALDI MS and MS/MS analysis of the modified peptides mapped the N-palmitoylation sites to Lys158, Lys172 and Lys140 or Lys145. This study demonstrates the utility of LLE/MALDI MS/MS for mapping and characterization of acylation sites in proteins and peptides and the importance of optimizing sample preparation methods for mass spectrometry-based determination of substoichiometric, multi-site protein modifications.     

Sequence Elucidation of an Unknown Cyclic Peptide of High Doping Potential by ETD and CID Tandem Mass Spectrometry

Identification of an unknown substance without any information remains a daunting challenge despite advances in chemistry and mass spectrometry. However, an unknown cyclic peptide in a sample with very limited volume seized at a Pennsylvania racetrack has been successfully identified. The unknown sample was determined by accurate mass measurements to contain a small unknown peptide as the major component. Collision-induced dissociation (CID) of the unknown peptide revealed the presence of Lys (not Gln, by accurate mass), Phe, and Arg residues, and absence of any y-type product ion. The latter, together with the tryptic digestion results of the unusual deamidation and absence of any tryptic cleavage, suggests a cyclic structure for the peptide. Electron-transfer dissociation (ETD) of the unknown peptide indicated the presence of Gln (not Lys, by the unusual deamidation), Phe, and Arg residues and their connectivity. After all the results were pieced together, a cyclic tetrapeptide, cyclo[Arg-Lys-N(C₆H₉)Gln-Phe], is proposed for the unknown peptide. Observations of different amino acid residues from CID and ETD experiments for the peptide were interpreted by a fragmentation pathway proposed, as was preferential CID loss of a Lys residue from the peptide. ETD was used for the first time in sequencing of a cyclic peptide; product ions resulting from ETD of the peptide identified were categorized into two types and named pseudo-b and pseudo-z ions that are important for sequencing of cyclic peptides. The ETD product ions were interpreted by fragmentation pathways proposed. Additionally, multi-stage CID mass spectrometry cannot provide complete sequence information for cyclic peptides containing adjacent Arg and Lys residues. The identified cyclic peptide has not been documented in the literature, its pharmacological effects are unknown, but it might be a “designer” drug with athletic performance-enhancing effects.     

茚三酮与赖氨酸荧光反应的研究

茚三酮与赖氨酸的显色反应已广泛用于各种样品的测定,但高灵敏的氨基酸荧光测定法的研究还很不够。尽管茚三酮与某些氨基酸的荧光反应已有报道,但反应条件苛刻,反应时间长达60～120min,对赖氨酸的专一测定法尚未见报道^[1~3]。     

Pressure-induced isomerization of retinal on bacteriorhodopsin as disclosed by fast magic angle spinning NMR

Bacteriorhodopsin (bR) is a retinal protein in purple membrane of Halobacterium salinarum, which functions as a light-driven proton pump. We have detected pressure-induced isomerization of retinal in bR by analyzing 15N cross polarization-magic angle spinning (CP-MAS) NMR spectra of [zeta-15N]Lys-labeled bR. In the 15N-NMR spectra, both all-trans and 13-cis retinal configurations have been observed in the Lys N(zeta) in protonated Schiff base at 148.0 and 155.0 ppm, respectively, at the MAS frequency of 4 kHz in the dark. When the MAS frequency was increased up to 12 kHz corresponding to the sample pressure of 63 bar, the 15N-NMR signals of [zeta-15N]Lys in Schiff base of retinal were broadened. On the other hand, other [zeta-15N]Lys did not show broadening. Subsequently, the increased signal intensity of [zeta-15N]Lys in Schiff base of 13-cis retinal at 155.0 ppm was observed when the MAS frequency was decreased from 12 to 4 kHz. These results showed that the equilibrium constant of [all-trans-bR]/[13-cis-bR] in retinal decreased by the pressure of 63 bar. It was also revealed that the structural changes induced by the pressure occurred in the vicinity of retinal. Therefore, microscopically, hydrogen-bond network around retinal would be disrupted or distorted by a constantly applied pressure. It is, therefore, clearly demonstrated that increased pressure induced by fast MAS frequencies generated isomerization of retinal from all-trans to 13-cis state in the membrane protein bR.     

Mapping the glycation sites in the neoglycoconjugate from hexasaccharide antigen of Vibrio cholerae,serotype Ogawa and the recombinant tetanus toxin C‐fragment carrier

We report herein the glycation sites in a vaccine candidate for cholera formed by conjugation of the synthetic hexasaccharide fragment of the O‐specific polysaccharide of Vibrio cholerae, serotype Ogawa, to the recombinant tetanus toxin C‐fragment (rTT–Hc) carrier. Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry analysis of the vaccine revealed that it is composed of a mixture of neoglycoconjugates with carbohydrate : protein ratios of 1.9 : 1, 3.0 : 1, 4.0 : 1, 4.9 : 1, 5.9 : 1, 6.9 : 1, 7.9 : 1 and 9.1 : 1. Liquid chromatography tandem mass spectrometry (LC‐MS/MS) analysis of the tryptic and GluC V8 digests allowed identification of 12 glycation sites in the carbohydrate–protein neoglycoconjugate vaccine. The glycation sites are located exclusively on lysine (Lys) residues and are listed as follows: Lys 22, Lys 61, Lys 145, Lys 239, Lys 278, Lys 318, Lys 331, Lys 353, Lys 378, Lys 389, Lys 396 and Lys 437. Based on the 3‐D representation of the rTT–Hc protein, all the glycation sites correspond to lysines located at the outer surface of the protein. Copyright © 2013 John Wiley & Sons, Ltd.     

Study on open tubular capillary affinity liquid chromatography

A novel mode of affinity chromatography (AC) based on an open tubular capillary column (OTAC) is demonstrated. The OTAC column is prepared by immobilizing Cibacron blue F3GA onto the inner surface of a 50-microm-i.d. capillary column. The AC experiment is performed on a capillary electrophoresis instrument by using its pressure system as the driving force. Bovine serum albumin and lysozyme (Lys) are successfully separated with stepwise gradient elution. The relative standard deviation (RSD) for the elution time of the retained Lys is 0.08%, and good repeatability of its peak area and peak height with an RSD value lower than 2.12% for 10 consecutive runs is observed. The loading capacity and detection limit for the retained Lys are approximately 36 ng and 8.6 ng, respectively. It is also found that the amount of protein adsorbed is unaffected by the flow rate of the loading buffer, and OTAC can be used for the fast determination of biopolymers. Some of the advantages of OTAC over conventional modes of open tubular capillary liquid chromatography are that the detection sensitivity and loading capacity of a sample can be greatly improved, because the relatively large inner diameter of the capillary can be adopted and the whole capillary column can be used to adsorb the solute in OTAC.     

Protein release behavior from porous microparticle with lysozyme/hyaluronate ionic complex

Porous microparticles (PMs) with a low density (<0.4 g/cm3) for pulmonary protein delivery were prepared by the water-in-oil-in-water (W1/O/W2) multi-emulsion method using a cyclodextrin derivative as a porogen. The complexation of positively charged lysozyme (Lys) and negative-charged hyaluronate (HA) was investigated for long-term protein release from PMs. The interaction of Lys and HA not only increased protein encapsulation efficiency but also stabilized Lys against a denaturing organic solvent (dichloromethane). Furthermore, PMs with Lys/HA complexes increased the Lys release period up to 7 days, as opposed to a 4h Lys release time from PMs without Lys/HA complexes. In particular, PMs containing 10mg of HA and 50mg of Lys showed almost zero-order Lys release kinetic for 7 days and preserved the bioactivity of Lys more than 98% during its entire release period. This result suggests that PMs with Lys/HA complexes may be applied in long-term pulmonary administration of protein or peptide drugs, including those that require particles to arrive at a deep lung epithelium with the help of low density (high porosity) of PMs.     

端基含甘露糖的聚赖氨酸的合成及其聚集行为

设计合成了2种含炔基的甘露糖衍生物(A-Man和A-DiMan)及2-[2-(2-叠氮乙氧基)-乙氧基]-乙胺(N3-TEG-NH2).以N3-TEG-NH2为引发剂引发Nε-苄氧羰基-L-赖氨酸酸酐(Lys(Z)-NCA)的开环聚合,得到2种分子量不同的端基含叠氮基团的聚赖氨酸,N3-P(Lys(Z))15和N3-P(Lys(Z))35,分别与A-Man和A-DiMan进行点击化学反应得到4种端基含甘露糖的聚合物.用透射电镜研究了4种聚合物在水溶液中的聚集行为,结果表明,基于P(Lys(Z))15的2种糖聚合物聚集形成囊泡,而基于P(Lys(Z))35的2种糖聚合物则聚集形成胶束.     

Minimization of side reactions during Lys Tag derivatization of C-terminal lysine peptides

Several issues need to be considered concerning chemical labeling strategies in proteomics. Some of these are labeling specificity, possible side reactions, completeness of reaction, recovery rate, conserving integrity of sample, hydrolysis of peptide bonds at high pH, and signal suppression in mass spectrometry (MS). We tested the effects of different reaction conditions for 2-methoxy-4,5-dihydro-1H-imidazole (Lys Tag) derivatization of the ?-amine group of lysine (K) residues. By using nanoflow LC–electrospray ionization-MS (LC–ESI-MS) and MS/MS in combination with MSight 2-D image analysis, we found that standard Lys Tag derivatization processes and conditions induce side reactions such as (i) Lys Tag labeling of the N-terminus, (ii) methylation of internal aspartic acid (D), glutamic acid (E) and C- and N-peptide termini and (iii) deamidation of asparagine (N) and glutamine (Q). We found temperature and pH to be the main variables to control side reactions. Lowering the reaction temperature from 55 °C to room temperature reduced deamidation from 22.8 ± 1.4% (SEM) to 7.7 ± 5.5% (SEM) and almost totally blocked methylation (7.0 ± 1.2% (SEM) to 0.4 ± 0.4% (SEM) of the internal acidic amino acids (D and E) at high pH. We conclude that lowering the reaction temperature minimizes undesired side reactions during Lys Tag derivatization in solution.     

Structural model for an alkaline form of ferricytochrome C

An (15)N-enriched sample of the yeast iso-1-ferricytochrome c triple variant (Lys72Ala/Lys79Ala/Cys102Thr) in an alkaline conformation was examined by NMR spectroscopy. The mutations were planned to produce a cytochrome c with a single conformer. Despite suboptimal conditions for the collection of spectra (i.e., pH approximately equal to 11), NMR remains a suitable investigation technique capable of taking advantage of paramagnetism. 76% of amino acids and 49% of protons were assigned successfully. The assignment was in part achieved through standard methods, in part through the identification of groups maintaining the same conformation as in the native protein at pH 7 and, for a few other residues, through a tentative analysis of internuclear distance predictions. Lys73 was assigned as the axial ligand together with His18. In this manner, 838 meaningful NOEs for 108 amino acids, 50 backbone angle constraints, and 203 pseudocontact shifts permitted the convergence of randomly generated structures to a family of conformers with a backbone RMSD of 1.5 +/- 0.2 A. Most of the native cytochrome c conformation is maintained at high pH. The NOE pattern that involves His18 clearly indicates that the proximal side of the protein, including the 20s and 40s loops, remains essentially intact. Structural differences are concentrated in the 70-80 loop, because of the replacement of Met80 by Lys73 as an axial ligand, and in the 50s helix facing that loop; as a consequence, there is increased exposure of the heme group to solvent. Based on several spectral features, we conclude that the folded polypeptide is highly fluxional.     

Study of peptides containing modified lysine residues by tandem mass spectrometry: precursor ion scanning of hexanal-modified peptides

Upon hexanal-modification in the presence of NaCNBH(3), the oxidized B chain of insulin becomes mono- and further dialkylated on both the N-terminal and Lys(29) residues. A pseudo-MS(3) study was performed with a triple-quadrupole mass spectrometer on the different modified lysine-containing species to gain further insights into the characteristic fragmentation pattern. These fragmentations, in good agreement with true MS(3) measurements obtained using an ion trap mass spectrometer, highlighted characteristic monoalkylated lysine (immonium-NH(3)) and protonated modified caprolactam ions at m/z 168 and 213, respectively. In contrast, no fragment ion derived from a modified lysine residue (immonium or caprolactam) was observed when dialkylation occurs on Lys(29). However, a fragment ion corresponding to a protonated dihexylamine was observed at m/z 186. This loss, characteristic of dialkylated lysine fragmentation, was also observed upon dialkylation of N(alpha)-acetyllysine with either hexanal or pentanal. On the other hand, acetylation and malondialdehyde-modification of the N(alpha)-acetyllysine side chain led mainly to the corresponding modified (immonium-NH(3)) fragment ions at m/z 126 and 138, respectively. Finally, it was demonstrated that precursor ion scanning for both m/z 168 and 213 ions led to specific and sensitive identification of peptides containing hexanal-modified lysine residues within an unfractionated tryptic digest of hexanal-modified apomyoglobin. Thus, Lys(42), Lys(45), Lys(62), Lys(63), Lys(77), Lys(87), Lys(96), Lys(98), Lys(145) and Lys(147) were found to be modified upon reaction with hexanal.     

Colorimetric detection of lysozyme based on electrostatic interaction with human serum albumin-modified gold nanoparticles

In this study, an aqueous solution of 13-nm gold nanoparticles (AuNPs) covalently bonded with human serum albumin (HSA) was used for sensing lysozyme (Lys). HSA molecules were good stabilizing agents for AuNPs in high-salt solution and exhibited the ability to bond with Lys electrostatically. The aggregation of HSA-AuNPs was achieved upon the addition of high-pI proteins, such as Lys, alpha-chymotrypsinogen A, and conalbumin. Not the same was achieved, however, when low-pI proteins such as ovalbumin, bovine serum albumin, and alpha-lactalbumin were added. Matrix-assisted desorption/ionization mass spectrometry was used to demonstrate the interaction between HSA-AuNPs and Lys. It was found that the sensitivity of HSA-AuNPs for Lys was highly dependent on the HSA concentration. The Lys-induced aggregation of HSA-AuNPs was suggested based on the London-van der Waals attractive force. We further improved the selectivity of the probe by adjusting the pH solution to 8.0. Under the optimum conditions, the selectivity of this system for Lys over other proteins in high-salt solutions was remarkably high, even when their pI was very close to the Lys. The lowest detectable concentration of Lys in this approach was 50 nM. The applicability of the method was validated through the analyses of Lys in chicken egg white.     

磷酸三乙胺-乙腈流动相体系反相分离2，4-二硝基氟苯-氨基酸衍生物

从两个方面改进了反相分离2,4-二硝基氟苯-氨基酸衍生物测定氨基酸的分析方法:一是使用高缓冲容量pH 2.75和6.50的磷酸三乙胺-乙腈流动相体系代替醋酸盐/乙腈流动相体系;另一个是强调了衍生反应的操作细节。以含精、丝、天冬、谷、苏、甘、丙、脯、组、蛋、缬、色、苯丙、亮、异亮、赖、酪氨酸注射液为目标试样,对方法进行认证,线性不低于0.9999(对谷氨酸、赖氨酸和酪氨酸不低于0.9998),准确度（回收率）为100±1%,精密度（RSD）低于0.5%,均优于以往的方法。方法适用于在一般液相色谱实验室进行氨基酸注射液和原料药的分析,无需专用氨基酸分析仪。     

Effect of pH on the interaction of baicalein with lysozyme by spectroscopic approaches

The interaction mechanism of baicalein and lysozyme (Lys) has been characterized by fluorescence, synchronous fluorescence, ultraviolet-vis absorbance, and three-dimensional (3D) fluorescence. The structural characteristics of baicalein and Lys were probed, and their binding affinities were determined under different pH conditions (pH 7.4, 4.5, and 2.5). The results showed that the binding abilities of the drug to Lys increased under lower pH conditions (pH 4.5 and 2.5) due to the alterations of the protein secondary and tertiary structures or the structural change of baicalein. The effect of baicalein on the conformation of Lys was analyzed using UV, synchronous fluorescence and three-dimensional (3D) fluorescence under different pH conditions. These results indicate that the binding of baicalein to Lys causes apparent change in the secondary and tertiary structure of Lys. In the presence of Cu(2+), the decrease of the binding constant in buffer solution of pH 2.5 may result from the competition of the metal ion and baicalein binding to Lys. In addition, the presence of Cu(2+) increased the binding constants of baicalein-Lys complex under higher pH conditions (pH 7.4 and 4.5). The possible site of binding of baicalein to Lys has been proposed to explain these observations.     

Infrared spectroscopy of cationized lysine and epsilon-N-methyllysine in the gas phase: effects of alkali-metal ion size and proton affinity on zwitterion stability

The gas-phase structures of protonated and alkali-metal-cationized lysine (Lys) and epsilon-N-methyllysine (Lys(Me)) are investigated using infrared multiple photon dissociation (IRMPD) spectroscopy utilizing light generated by a free electron laser, in conjunction with ab initio calculations. IRMPD spectra of Lys.Li(+) and Lys.Na(+) are similar, but the spectrum for Lys.K(+) is different, indicating that the structure of lysine in these complexes depends on the metal ion size. The carbonyl stretch of a carboxylic acid group is clearly observed in each of these spectra, indicating that lysine is nonzwitterionic in these complexes. A detailed comparison of these spectra to those calculated for candidate low-energy structures indicates that the bonding motif for the metal ion changes from tricoordinated for Li and Na to dicoordinated for K, clearly revealing the increased importance of hydrogen-bonding relative to metal ion solvation with increasing metal ion size. Spectra for Lys(Me).M(+) show that Lys(Me), an analogue of lysine whose side chain contains a secondary amine, is nonzwitterionic with Li and zwitterionic with K and both forms are present for Na. The proton affinity of Lys(Me) is 16 kJ/mol higher than that of Lys; the higher proton affinity of a secondary amine can result in its preferential protonation and stabilization of the zwitterionic form.     

Differentiation of Compact and Extended Conformations of Di-Ubiquitin Conjugates with Lysine-Specific Isopeptide Linkages by Ion Mobility-Mass Spectrometry

Modification of ubiquitin, a key cellular regulatory polypeptide of 76 amino acids, to polyubiquitin conjugates by lysine-specific isopeptide linkage at one of its seven lysine residues has been recognized as a central pathway determining its biochemical properties and cellular functions. Structural details and differences of distinct lysine-isopeptidyl ubiquitin conjugates that reflect their different functions and reactivities, however, are only partially understood. Ion mobility spectrometry (IMS) combined with mass spectrometry (MS) has recently emerged as a powerful tool for probing conformations and topology involved in protein interactions by an electric field-driven separation of polypeptide ions through a drift gas. Here we report the conformational characterization and differentiation of Lys63- and Lys48-linked ubiquitin conjugates by IMS–MS. Lys63- and Lys48-linked di-ubiquitin conjugates were prepared by recombinant bacterial expression and by chemical synthesis using a specific chemical ligation strategy, and characterized by high-resolution Fourier transform ion cyclotron resonance mass spectrometry, circular dichroism spectroscopy, and molecular modeling. IMS–MS was found to be an effective tool for the identification of structural differences of ubiquitin complexes in the gas phase. The comparison of collision cross-sections of Lys63- and Lys48-linked di-ubiquitin conjugates showed a more elongated conformation of Lys63-linked di-ubiquitin. In contrast, the Lys48-linked di-ubiquitin conjugate showed a more compact conformation. The IMS-MS results are consistent with published structural data and a comparative molecular modeling study of the Lys63- and Lys48-linked conjugates. The results presented here suggest IMS techniques can provide information that complements MS measurements in differentiating higher-order polyubiquitins and other isomeric protein linkages.     

Stereospecific capillary electrophoresis assays using pentapeptide substrates for the study of Aspergillus nidulans methionine sulfoxide reductase A and mutant enzymes

Stereospecific capillary electrophoresis‐based methods for the analysis of methionine sulfoxide [Met(O)]‐containing pentapeptides were developed in order to investigate the reduction of Met(O)‐containing peptide substrates by recombinant Aspergillus nidulans methionine sulfoxide reductase A (MsrA) as well as enzymes carrying mutations in position Glu99 and Asp134. The separation of the diastereomers of the N‐acetylated, C‐terminally 2,4‐dinitrophenyl (Dnp)‐labeled pentapeptides ac‐Lys‐Phe‐Met(O)‐Lys‐Lys‐Dnp, ac‐Lys‐Asp‐Met(O)‐Asn‐Lys‐Dnp and ac‐Lys‐Asn‐Met(O)‐Asp‐Lys‐Dnp was achieved in 50 mM Tris‐HCl buffers containing sulfated β‐CD in fused‐silica capillaries, while the diastereomer separation of ac‐Lys‐Asp‐Met(O)‐Asp‐Lys‐Dnp was achieved by sulfated β‐CD‐mediated MEKC. The methods were validated with regard to range, linearity, accuracy, limits of detection and quantitation as well as precision. Subsequently, the substrates were incubated with wild‐type MsrA and three mutants in the presence of dithiothreitol as reductant. Wild‐type MsrA displayed the highest activity towards all substrates compared to the mutants. Substitution of Glu99 by Gln resulted in the mutant with the lowest activity towards all substrates except for ac‐Lys‐Asn‐Met(O)‐Asp‐Lys‐Dnp, while replacement Asn for Asp134 lead to a higher activity towards ac‐Lys‐Asp‐Met(O)‐Asn‐Lys‐Dnp compared with the Glu99 mutant. The mutant with Glu instead of Asp134 was the most active among the mutant enzymes. Molecular modeling indicated that the conserved Glu99 residue is buried in the Met‐S‐(O) groove, which might contribute to the correct placing of substrates and, consequently, to the catalytic activity of MsrA, while Asp134 did not form hydrogen bonds with the substrates but only within the enzyme.