Characterization of Freezing Processes in Drug Substance Bottles by Ice Core Sampling.

Freezing of biological drug substance (DS) is a critical unit operation that may impact product quality, potentially leading to protein aggregation and sub-visible particle formation. Cryo-concentration has been identified as a critical parameter to impact protein stability during freezing and should therefore be minimized. The macroscopic cryo-concentration, in the following only referred to as cryo-concentration, is majorly influenced by the freezing rate, which is in turn impacted by product independent process parameters such as the DS container, its size and fill level, and the freezing equipment. (At-scale) process characterization studies are crucial to understand and optimize freezing processes. However, evaluating cryo-concentration requires sampling of the frozen bulk, which is typically performed by cutting the ice block into pieces for subsequent analysis. Also, the large amount of product requirement for these studies is a major limitation. In this study, we report the development of a simple methodology for experimental characterization of frozen DS in bottles at relevant scale using a surrogate solution. The novel ice core sampling technique identifies the axial ice core in the center to be indicative for cryo-concentration, which was measured by osmolality, and concentrations of histidine and polysorbate 80 (PS80), whereas osmolality revealed to be a sensitive read-out. Finally, we exemplify the suitability of the method to study cryo-concentration in DS bottles by comparing cryo-concentrations from different freezing protocols (-80°C vs -40°C). Prolonged stress times during freezing correlated to a higher extent of cryo-concentration quantified by osmolality in the axial center of a 2 L DS bottle.

Postassembly Modification of Peptides by Histidine-Directed ß-C(sp3)-H Arylation of Alanine at the Internal Positions: Overcoming the Inhibitory Effect of Peptide Bonds.

Peptide modification by C(sp3)-H functionalization of residues at the internal positions remains underdeveloped due to the inhibitory effect of backbone amides. In this study, using histidine (His) as an endogenous directing group, we developed a novel method for the ß-C(sp3)-H functionalization of alanine (Ala) at diverse positions of peptides. Through this approach, a wide range of linear peptides were modified on the side-chain of Ala adjacent to His to afford the functionalized peptides in moderate to good yield and excellent position selectivity. Furthermore, conjugation of peptides with functional molecules such as glucuronide, oleanolic acid, dipeptide, and fluorophore derivatives was achieved.

Protein purification via consecutive histidine-polyphosphate interaction.

While nickel-nitrilotriacetic acid (Ni-NTA) has greatly advanced recombinant protein purification, its limitations, including nonspecific binding and partial purification for certain proteins, highlight the necessity for additional purification such as size exclusion and ion exchange chromatography. However, specialized equipment such as FPLC is typically needed but not often available in many laboratories. Here, we show a novel method utilizing polyphosphate (polyP) for purifying proteins with histidine repeats via non-covalent interactions. Our study demonstrates that immobilized polyP efficiently binds to histidine-tagged proteins across a pH range of 5.5-7.5, maintaining binding efficacy even in the presence of reducing agent DTT and chelating agent EDTA. We carried out experiments of purifying various proteins from cell lysates and fractions post-Ni-NTA. Our results demonstrate that polyP resin is capable of further purification post-Ni-NTA without the need for specialized equipment and without compromising protein activity. This cost-effective and convenient method offers a viable approach as a complementary approach to Ni-NTA.

Affinity Purification of a 6X-His-Tagged Protein using a Fast Protein Liquid Chromatography System.

Functional characterization of proteins requires them to be expressed and purified in substantial amounts with high purity to perform biochemical assays. The Fast Protein Liquid Chromatography (FPLC) system allows high-resolution separation of complex protein mixtures. By adjusting various parameters in FPLC, such as selecting the appropriate purification matrix, regulating the protein sample's temperature, and managing the sample's flow rate onto the matrix and the elution rate, it is possible to ensure the protein's stability and functionality. In this protocol, we will demonstrate the versatility of the FPLC system to purify 6X-His-tagged flap endonuclease 1 (FEN1) protein, produced in bacterial cultures. To improve protein purification efficiency, we will focus on multiple considerations, including proper column packing and preparation, sample injection using a sample loop, flow rate of sample application to the column, and sample elution parameters. Finally, the chromatogram will be analyzed to identify fractions containing high yields of protein and considerations for proper recombinant protein long-term storage. Optimizing protein purification methods is crucial for improving the precision and reliability of protein analysis.

Molecular two-point recognition of fructosyl valine and fructosyl glycyl histidine in water by fluorescent Zn(II)-terpyridine complexes bearing boronic acids.

Selective recognition of fructosyl amino acids in water by arylboronic acid-based receptors is a central field of modern supramolecular chemistry that impacts biological and medicinal chemistry. Fructosyl valine (FV) and fructosyl glycyl histidine (FGH) occur as N-terminal moieties of human glycated hemoglobin; therefore, the molecular design of biomimetic receptors is an attractive, but very challenging goal. Herein, we report three novel cationic Zn-terpyridine complexes bearing a fluorescent N-quinolinium nucleus covalently linked to three different isomers of strongly acidified phenylboronic acids (ortho-, 2Zn; meta-, 3Zn and para-, 4Zn) for the optical recognition of FV, FGH and comparative analytes (D-fructose, Gly, Val and His) in pure water at physiological pH. The complexes were designed to act as fluorescent receptors using a cooperative action of boric acid and a metal chelate. Complex 3Zn was found to display the most acidic -B(OH)2 group (pKa = 6.98) and exceptionally tight affinity for FV (K = 1.43 × 105 M-1) with a strong quenching analytical response in the micromolar concentration range. The addition of fructose and the other amino acids only induced moderate optical changes. On the basis of several spectroscopic tools (1H, 11B NMR, UV-Vis, and fluorescence titrations), ESI mass spectrometry, X-ray crystal structure, and DFT calculations, the interaction mode between 3Zn and FV is proposed in a 1 : 1 model through a cooperative two-point recognition involving a sp3 boronate-diol esterification with simultaneous coordination bonding of the carboxylate group of Val to the Zn atom. Fluorescence quenching is attributed to a static complexation photoinduced electron transfer mechanism as evidenced by lifetime experiments. The addition of FGH to 3Zn notably enhanced its emission intensity with micromolar affinity, but with a lower apparent binding constant than that observed for FV. FGH interacts with 3Zn through boronate-diol complexation and coordination of the imidazole ring of His. DFT-optimized structures of complexes 3Zn-FV and 3Zn-FGH show a picture of binding which shows that the Zn-complex has a suitable (B⋯Zn) distance to the two-point recognition with these analytes. Molecular recognition of fructosyl amino acids by transition-metal-based receptors has not been explored until now.

Fluorescence Sensors for the Detection of L-Histidine Based on Silver Nanoclusters Modulated by Copper Ions.

In this study, Cu2+ modulated silver nanoclusters were constructed for the turn-on, label-free detection of L-histidine. Six Ag NCs protected by oligonucleotides (DNA-Ag NCs) were tested in a series of experiments. Finally, A-DAN-Ag NCs were chosen as the best candidate due to their excellent fluorescent properties. The fluorescence of A-DAN-Ag NCs was quenched using Cu2+ through energy or electron transfer. However, quenched fluorescence could be restored dramatically in the presence of L-histidine due to Cu2+ liberation from A-DAN-Ag NCs and because of the chelation between the imidazole group of L-histidine and Cu2+. The proposed sensor exhibited high selectivity towards L-histidine over other amino acids, with a limit of detection (LOD) of 0.096 µM ranging from 0 to 8 µM. The proposed sensor succeeded in detecting L-histidine in diluted human urine. Therefore, the sensor has promising practical applications in biological systems.

Copper(II), Nickel(II) and Zinc(II) Complexes of Peptide Fragments of Tau Protein.

Copper(II), nickel(II) and zinc(II) complexes of various peptide fragments of tau protein were studied by potentiometric and spectroscopic techniques. All peptides contained one histidyl residue and represented the sequences of tau(91-97) (Ac-AQPHTEI-NH2), tau(385-390) (Ac-KTDHGA-NH2) and tau(404-409) (Ac-SPRHLS-NH2). Imidazole-N donors of histidine were the primary metal binding sites for all peptides and all metal ions, but in the case of copper(II) and nickel(II), the deprotonated amide groups were also involved in metal binding by increasing pH. The most stable complexes were formed with copper(II) ions, but the presence of prolyl residues resulted in significant changes in the thermodynamic stability and speciation of the systems. It was also demonstrated that nickel(II) and especially zinc(II) complexes have relatively low thermodynamic stability with these peptides. The copper(II)-catalyzed oxidation of the peptides was also studied. In the presence of H2O2, the fragmentation of peptides was detected in all cases. In the simultaneous presence of H2O2 and ascorbic acid, the fragmentation of the peptide is less preferred, and the formation of 2-oxo-histidine also occurs.

Histidine-Covalent Stapled Alpha-Helical Peptides Targeting hMcl-1.

Several novel and effective cysteine targeting (Cys) covalent drugs are in clinical use. However, the target area containing a druggable Cys residue is limited. Therefore, methods for creating covalent drugs that target different residues are being looked for; examples of such ligands include those that target the residues lysine (Lys) and tyrosine (Tyr). Though the histidine (His) side chain is more frequently found in protein binding locations and has higher desirable nucleophilicity, surprisingly limited research has been done to specifically target this residue, and there are not many examples of His-targeting ligands that have been rationally designed. In the current work, we created novel stapled peptides that are intended to target hMcl-1 His 252 covalently. We describe the in vitro (biochemical, NMR, and X-ray) and cellular design and characterization of such agents. Our findings further suggest that the use of electrophiles to specifically target His residues is warranted.

Turn-off enzyme activity of histidine-rich peptides for the detection of lysozyme.

An assay that integrates histidine-rich peptides (HisRPs) with high-affinity aptamers was developed enabling the specific and sensitive determination of the target lysozyme. The enzyme-like activity of HisRP is inhibited by its interaction with a target recognized by an aptamer. In the presence of the target, lysozyme molecules progressively assemble on the surface of HisRP in a concentration-dependent manner, resulting in the gradual suppression of enzyme-like activity. This inhibition of HisRP's enzyme-like activity can be visually observed through color changes in the reaction product or quantified using UV-visible absorption spectroscopy. Under optimal conditions, the proposed colorimetric assay for lysozyme had a detection limit as low as 1 nM and exhibited excellent selectivity against other nonspecific interferents. Furthermore, subsequent research validated the practical applicability of the developed colorimetric approach to saliva samples, indicating that the assay holds significant potential for the detection of lysozymes in samples derived from humans.

Active droplets through enzyme-free, dynamic phosphorylation.

Life continuously transduces energy to perform critical functions using energy stored in reactive molecules like ATP or NADH. ATP dynamically phosphorylates active sites on proteins and thereby regulates their function. Inspired by such machinery, regulating supramolecular functions using energy stored in reactive molecules has gained traction. Enzyme-free, synthetic systems that use dynamic phosphorylation to regulate supramolecular processes have not yet been reported, to our knowledge. Here, we show an enzyme-free reaction cycle that consumes the phosphorylating agent monoamidophosphate by transiently phosphorylating histidine and histidine-containing peptides. The phosphorylated species are labile and deactivate through hydrolysis. The cycle exhibits versatility and tunability, allowing for the dynamic phosphorylation of multiple precursors with a tunable half-life. Notably, we show the resulting phosphorylated products can regulate the peptide's phase separation, leading to active droplets that require the continuous conversion of fuel to sustain. The reaction cycle will be valuable as a model for biological phosphorylation but can also offer insights into protocell formation.

Developing Isomeric Peptides for Mimicking the Sequence-Activity Landscapes of Enzyme Evolution.

Enzymes catalyze almost all material conversion processes within living organisms, yet their natural evolution remains unobserved. Short peptides, derived from proteins and featuring active sites, have emerged as promising building blocks for constructing bioactive supramolecular materials that mimic native proteins through self-assembly. Herein, we employ histidine-containing isomeric tetrapeptides KHFF, HKFF, KFHF, HFKF, FKHF, and FHKF to craft supramolecular self-assemblies, aiming to explore the sequence-activity landscapes of enzyme evolution. Our investigations reveal the profound impact of peptide sequence variations on both assembly behavior and catalytic activity as hydrolytic simulation enzymes. During self-assembly, a delicate balance of multiple intermolecular interactions, particularly hydrogen bonding and aromatic-aromatic interactions, influences nanostructure formation, yielding various morphologies (e.g., nanofibers, nanospheres, and nanodiscs). Furthermore, the analysis of the structure-activity relationship demonstrates a strong correlation between the distribution of the His active site on the nanostructures and the formation of the catalytic microenvironment. This investigation of the sequence-structure-activity paradigm reflects how natural enzymes enhance catalytic activity by adjusting the primary structure during evolution, promoting fundamental research related to enzyme evolutionary processes.

The effects of histidine substitution of aromatic residues on the amyloidogenic properties of the fragment 264-277 of nucleophosmin 1.

Histidine (His) plays a key role in mediating protein interactions and its unique side chain determines pH responsive self-assembling processes and thus in the formation of nanostructures. In this study, To identify novel self-assembling bioinspired sequences, we analyzed a series of peptide sequences obtained through the point mutation of aromatic residues of 264-277 fragment of nucleophosmin 1 (NPM1) with single and double histidines. Through several orthogonal biophysical techniques and under different pH and ionic strength conditions we evaluated the effects of these substitutions in the amyloidogenic features of derived peptides. The results clearly indicate that both the type of aromatic mutated residue and its position can have different effect on amyloid-like behaviors. They corroborate the crucial role exerted by Tyr271 in the self-assembling process of CTD of NPM1 in AML mutated form and add novel insights in the accurate investigation of how side chain orientations can determine successful design of innovative bioinspired materials.

Identification of histidine residues that affect the T/R-state conformations of human hemoglobin using constant pH molecular dynamics simulations.

Human hemoglobin (Hb) is a tetrameric protein consisting of two α and two ß subunits that can adopt a low-affinity T- and high-affinity R-state conformations. Under physiological pH conditions, histidine (His) residues are the main sites for proton binding or release, and their protonation states can affect the T/R-state conformation of Hb. However, it remains unclear which His residues can effectively affect the Hb conformation. Herein, the impact of the 38 His residues of Hb on its T/R-state conformations was evaluated using constant-pH molecular dynamics (CpHMD) simulations at physiological pH while focusing on the His protonation states. Overall, the protonation states of some His residues were found to be correlated with the Hb conformation state. These residues were mainly located in the proximity of the heme (α87 and ß92), and at the α1ß2 and α2ß1 interfaces (α89 and ß97). This correlation may be partly explained by how easily hydrogen bonds can be formed, which depends on the protonation states of the His residues. Taken together, these CpHMD-based findings provide new insights into the identification of titratable His residues α87, α89, ß92, and ß97 that can affect Hb conformational switching under physiological pH conditions.

Heterogeneous and Cooperative Rupture of Histidine-Ni2+ Metal-Coordination Bonds on Rationally Designed Protein Templates.

Metal-coordination bonds, a highly tunable class of dynamic noncovalent interactions, are pivotal to the function of a variety of protein-based natural materials and have emerged as binding motifs to produce strong, tough, and self-healing bioinspired materials. While natural proteins use clusters of metal-coordination bonds, synthetic materials frequently employ individual bonds, resulting in mechanically weak materials. To overcome this current limitation, we rationally designed a series of elastin-like polypeptide templates with the capability of forming an increasing number of intermolecular histidine-Ni2+ metal-coordination bonds. Using single-molecule force spectroscopy and steered molecular dynamics simulations, we show that templates with three histidine residues exhibit heterogeneous rupture pathways, including the simultaneous rupture of at least two bonds with more-than-additive rupture forces. The methodology and insights developed improve our understanding of the molecular interactions that stabilize metal-coordinated proteins and provide a general route for the design of new strong, metal-coordinated materials with a broad spectrum of dissipative time scales.

Substitution of both histidines in the active site of yeast alcohol dehydrogenase 1 exposes underlying pH dependencies.

Histidine residues 44 and 48 in yeast alcohol dehydrogenase (ADH) bind to the coenzymes NAD(H) and contribute to catalysis. The individual H44R and H48Q substitutions alter the kinetics and pH dependencies, and now the roles of other ionizable groups in the enzyme were studied in the doubly substituted H44R/H48Q ADH. The substitutions make the enzyme more resistant to inactivation by diethyl pyrocarbonate, modestly improve affinity for coenzymes, and substantially decrease catalytic efficiencies for ethanol oxidation and acetaldehyde reduction. The pH dependencies for several kinetic parameters are shifted from pK values for wild-type ADH of 7.3-8.1 to values for H44R/H48Q ADH of 8.0-9.6, and are assigned to the water or alcohol bound to the catalytic zinc. It appears that the rate of binding of NAD+ is electrostatically favored with zinc-hydroxide whereas binding of NADH is faster with neutral zinc-water. The pH dependencies of catalytic efficiencies (V/EtKm) for ethanol oxidation and acetaldehyde reduction are similarly controlled by deprotonation and protonation, respectively. The substitutions make an enzyme that resembles the homologous horse liver H51Q ADH, which has Arg-47 and Gln-51 and exhibits similar pK values. In the wild-type ADHs, it appears that His-48 (or His-51) in the proton relay systems linked to the catalytic zinc ligands modulate catalytic efficiencies.

A Water-Soluble Wavy Coordination Polymer of Cu(II) as a Turn-On Luminescent Probe for Histidine and Histidine-Rich Proteins/Peptides.

Histidine plays an essential role in most biological systems. Changes in the homeostasis of histidine and histidine-rich proteins are connected to several diseases. Herein, we report a water-soluble Cu(II) coordination polymer, labeled CuCP, for the fluorimetric detection of histidine and histidine-rich proteins and peptides. Single-crystal structure determination of CuCP revealed a two-dimensional wavy network structure in which a carboxylate group connects the individual Cu(II) dimer unit in a syn-anti conformation. The weakly luminescent and water-soluble CuCP shows turn-on blue emission in the presence of histidine and histidine-rich peptides and proteins. The polymer can also stain histidine-rich proteins via gel electrophoresis. The limits of quantifications for histidine, glycine-histidine, serine-histidine, human serum albumin (HSA), bovine serum albumin, pepsin, trypsin, and lysozyme were found to be 300, 160, 600, 300, 600, 800, 120, and 290 nM, respectively. Utilizing the fluorescence turn-on property of CuCP, we measured HSA quantitatively in the urine samples. We also validated the present urinary HSA measurement assay with existing analytical techniques. Job's plot, 1H NMR, high-resolution mass spectrometry (HRMS), electron paramagnetic resonance (EPR), fluorescence, and UV-vis studies confirmed the ligand displacement from CuCP in the presence of histidine.

The rotamer of the second-sphere histidine in AA9 lytic polysaccharide monooxygenase is pH dependent.

Lytic polysaccharide monooxygenases (LPMOs) catalyze a reaction that is crucial for the biological decomposition of various biopolymers and for the industrial conversion of plant biomass. Despite the importance of LPMOs, the exact molecular-level nature of the reaction mechanism is still debated today. Here, we investigated the pH-dependent conformation of a second-sphere histidine (His) that we call the stacking histidine, which is conserved in fungal AA9 LPMOs and is speculated to assist catalysis in several of the LPMO reaction pathways. Using constant-pH and accelerated molecular dynamics simulations, we monitored the dynamics of the stacking His in different protonation states for both the resting Cu(II) and active Cu(I) forms of two fungal LPMOs. Consistent with experimental crystallographic and neutron diffraction data, our calculations suggest that the side chain of the protonated and positively charged form is rotated out of the active site toward the solvent. Importantly, only one of the possible neutral states of histidine (HIE state) is observed in the stacking orientation at neutral pH or when bound to cellulose. Our data predict that, in solution, the stacking His may act as a stabilizer (via hydrogen bonding) of the Cu(II)-superoxo complex after the LPMO-Cu(I) has reacted with O2 in solution, which, in fine, leads to H2O2 formation. Also, our data indicate that the HIE-stacking His is a poor acid/base catalyst when bound to the substrate and, in agreement with the literature, may play an important stabilizing role (via hydrogen bonding) during the peroxygenase catalysis. Our study reveals the pH titration midpoint values of the pH-dependent orientation of the stacking His should be considered when modeling and interpreting LPMO reactions, whether it be for classical LPMO kinetics or in industry-oriented enzymatic cocktails, and for understanding LPMO behavior in slightly acidic natural processes such as fungal wood decay.

Comparison of the Backbone Dynamics of Dehaloperoxidase-Hemoglobin Isoenzymes.

Dehaloperoxidase (DHP) is a multifunctional hemeprotein with a functional switch generally regulated by the chemical class of the substrate. Its two isoforms, DHP-A and DHP-B, differ by only five amino acids and have an almost identical protein fold. However, the catalytic efficiency of DHP-B for oxidation by a peroxidase mechanism ranges from 2- to 6-fold greater than that of DHP-A depending on the conditions. X-ray crystallography has shown that many substrates and ligands have nearly identical binding in the two isoenzymes, suggesting that the difference in catalytic efficiency could be due to differences in the conformational dynamics. We compared the backbone dynamics of the DHP isoenzymes at pH 7 through heteronuclear relaxation dynamics at 11.75, 16.45, and 19.97 T in combination with four 300 ns MD simulations. While the overall dynamics of the isoenzymes are similar, there are specific local differences in functional regions of each protein. In DHP-A, Phe35 undergoes a slow chemical exchange between two conformational states likely coupled to a swinging motion of Tyr34. Moreover, Asn37 undergoes fast chemical exchange in DHP-A. Given that Phe35 and Asn37 are adjacent to Tyr34 and Tyr38, it is possible that their dynamics modulate the formation and migration of the active tyrosyl radicals in DHP-A at pH 7. Another significant difference is that both distal and proximal histidines have a 15-18% smaller S2 value in DHP-B, thus their greater flexibility could account for the higher catalytic activity. The distal histidine grants substrate access to the distal pocket. The greater flexibility of the proximal histidine could also accelerate H2O2 activation at the heme Fe by increased coupling of an amino acid charge relay to stabilize the ferryl Fe(IV) oxidation state in a Poulos-Kraut "push-pull"-type peroxidase mechanism.

Histidine tag modified magnetic beads for analysis of arsenic binding proteins.

BACKGROUND: Many proteins with thiol groups can bind with trivalent arsenic which are termed as arsenic binding proteins, thus change their physiological functions. Therefore, it is vital to analyze the arsenic binding proteins in cells. The Pull-Down strategy based on biotinylated phenylarsenic acid (Bio-PAO(III)) probes is an effective way for analysis of arsenic binding proteins. In this strategy, streptavidin magnetic beads (SA-MBs) was applied to capture the arsenic binding proteins conjugating with Bio-PAO(III) probe. However, strong interaction between SA and biotin makes the elution of arsenic binding proteins not easy. RESULTS: We developed a novel affinity separation strategy to address the challenge of eluting arsenic binding proteins, a key issue with the existing Bio-PAO(III) Pull-Down method. By employing magnetic beads modified with Nα-Bis(carboxymethyl)-l-lysine (NTA-Lys), polyhistidine-tag (His6-Tag), and SA (MB-NTA(Ni)-His6-SA), we established a more efficient purification process. This innovative approach enables selective capture of arsenic binding proteins in HepG2 cells labeled by Bio-PAO(III) probes, facilitating gentle digestion by trypsin for precise identification through capillary high performance liquid chromatography (Cap HPLC)-electrospray ionization (ESI)-tandem mass spectrometry (MS/MS). What is more, the magnetic beads can be regenerated by using imidazole as the eluent, and the obtained MB-NTA(Ni) can be reloaded with His6-SA for next use. Our method successfully identified 41 arsenic binding proteins, including those involved in cytoskeletal structure, heat shock response, transcriptional regulation, DNA damage repair, redox state regulation, mitochondrial dehydrogenase function, and protein synthesis and structure. SIGNIFICANCE: This work contributes to a more comprehensive understanding of the toxic mechanisms of arsenic, potentially providing valuable insights for the prevention or treatment of arsenic-related diseases.

m-nitrobenzyl alcohol supercharging reagent enhances the chromatographic separation and the charging of disulfide bond linked and His-tag peptides.

The linkages of disulfide bond (DSB) play important roles in protein stability and activity. Mass spectrometry-based (MS-based) techniques become accepted tools for DSB analysis in the recent decade. In the bottom-up approach, after enzyme digestion, the neighbouring amino acids of cysteines have great impacts on the physicochemical properties of resulting disulfide bond peptides, determining their retention behaviour on liquid chromatography (LC) and their MS ionization efficiency. In this study, the addition of supercharging reagent in LC mobile phase was used to examine the impact of supercharging reagent on the charge states of disulfide-bond peptides. The results showed that 0.1 % m-nitrobenzyl alcohol (m-NBA) in LC mobile phase increased the sensitivity and charge states of DSB peptides from our model protein, equine Interleukin-5 (eIL5), as well as the resolution of reversed-phase chromatography. Notably, also the sensitivity of C-terminal peptide with His-tag significantly improved. Our findings highlight the effectiveness of employing m-NBA as a supercharging reagent when investigating disulfide-linked peptides and the C-terminal peptide with a His-tag through nano-liquid chromatography mass spectrometry.