Binary Complex Research Articles

Isobaric crosslinking mass spectrometry technology for studying conformational and structural changes in proteins and complexes.

Dynamic conformational and structural changes in proteins and protein complexes play a central and ubiquitous role in the regulation of protein function, yet it is very challenging to study these changes, especially for large protein complexes, under physiological conditions. Here, we introduce a novel isobaric crosslinker, Qlinker, for studying conformational and structural changes in proteins and protein complexes using quantitative crosslinking mass spectrometry. Qlinkers are small and simple, amine-reactive molecules with an optimal extended distance of ~10 Å, which use MS2 reporter ions for relative quantification of Qlinker-modified peptides derived from different samples. We synthesized the 2-plex Q2linker and showed that the Q2linker can provide quantitative crosslinking data that pinpoints key conformational and structural changes in biosensors, binary and ternary complexes composed of the general transcription factors TBP, TFIIA, and TFIIB, and RNA polymerase II complexes.

Driving Forces in the Formation of Biocondensates of Highly Charged Proteins: A Thermodynamic Analysis of the Binary Complex Formation

A thermodynamic analysis of the binary complex formation of the highly positively charged linker histone H1 and the highly negatively charged chaperone prothymosin α (ProTα) is detailed. ProTα and H1 have large opposite net charges (−44 and +53, respectively) and form complexes at physiological salt concentrations with high affinities. The data obtained for the binary complex formation are analyzed by a thermodynamic model that is based on counterion condensation modulated by hydration effects. The analysis demonstrates that the release of the counterions mainly bound to ProTα is the main driving force, and effects related to water release play no role within the limits of error. A strongly negative Δcp (=−0.87 kJ/(K mol)) is found, which is due to the loss of conformational degrees of freedom.

Supramolecular architectures, Hirshfeld surfaces analysis, spectroscopic, computational and photovoltaic properties of nickel(II) binary and ternary complexes of N-phenethyl-iminodiacetate (2-) chelate

The bluish-green color nickel(II) binary and/or ternary compounds have been isolated from the solution of stoichiometric reaction between Ni(II) hydroxy-carbonate and N-phenethyl-iminodiacetic acid (H2pheida) chelating ligand in aqueous media with compound (2); [Ni(pheida)(Him)3]·4H2O and without imidazole compound (1); [Ni(pheida)(H2O)3]. The crystal pattern of binary compound [Ni(pheida)(H2O)3] (1) differs from the related tetra-hydrated ternary complex of [Ni(pheida)(Him)3]·4H2O (2) can be attributed to the presence of stronger N-(Him) spacer of imidazole ligand. The compounds reported herein, have been characterized by means of elemental analysis, FTIR, UV–vis, TGA and X-ray crystallography. Moreover, the distinctive inter-molecular forces particularly H-bonding interactions, 2D fingerprint plots and supramolecular architecture have been studied using Hirshfeld surface analysis. The nickel(II) binary and ternary complexes (1) and (2) show distorted octahedral geometry around the Ni(II) ion of the type 1 + 2 + 2 + 1 imposed by 3d8 electronic configuration with degenerate fully and half occupied t2g6 and eg2 orbitals respectively. The higher John-Teller distortion has been observed for complex (2) than that of complex (1). The IDA arm of chelate adopted fac-NO2 conformation in both complexes (1) and (2). Moreover, the photovoltaic properties as semiconducting nature for ligand and their corresponding Ni(II) complexes were estimated using Tauc’s equation, αhν = A(hν-Eg)r, where r = ½ for indirect and 2 for direct electronic transitions through UV–vis absorption data. The indirect and direct electronic transition bandgap (Eg) calculated for complex (1) is 2.0 and 3.07 eV respectively. Similarly, 2.55 and 4.39 eV respectively measured for complex (2).

Functional modulation of RAGE activation by multimeric S100B using single-domain antibodies.

S100B is a multifunctional protein primarily found in the brain, where it plays crucial roles in cell proliferation, differentiation, and survival. It has intra- and extracellular functions and, depending on S100B levels, can exhibit both neurotrophic and neurotoxic activities, both mediated by the receptor for advanced glycation end products (RAGE). Here, we report the discovery and characterization of nanobodies (Nbs) targeting dimeric and tetrameric S100B, which are the two most abundant oligomeric functional forms of the protein, aiming to modulate S100B-mediated RAGE activation. Two Nbs were selected for detailed structural and functional studies, and found to bind tetrameric S100B with high affinity, as determined by biolayer interferometry analysis and SEC-stable binary complex formation. Structural and docking analyses revealed preferential contact sites of Nbs with S100B regions implicated in interactions with RAGE, namely residues at the interfacial cleft on dimeric S100B and the at hydrophobic cleft formed by the association of two homodimeric units in the tetramer. In accordance, assays in SH-SY5Y cells showed that Nbs modulate the RAGE-mediated neurotrophic activity of S100B by hindering its functional interactions with the receptor. Biolayer interferometry competition assays between tetrameric S100B and the RAGE-VC1 domain, confirmed that Nbs selectively block S100B-mediated RAGE engagement, in agreement with cell activation experiments. These findings highlight Nbs as powerful tools for elucidating molecular and cellular mechanisms through the modulation of S100B and RAGE functions, inspiring potential therapeutic applications.

Single-Molecule-Based, Label-Free Monitoring of Molecular Glue Efficacies for Promoting Protein-Protein Interactions Using YaxAB Nanopores.

Modulating protein-protein interactions (PPIs) is an attractive strategy in drug discovery. Molecular glues, bifunctional small-molecule drugs that promote PPIs, offer an approach to targeting traditionally undruggable targets. However, the efficient discovery of molecular glues has been hampered by the current limitations of conventional ensemble-averaging-based methods. In this study, we present a YaxAB nanopore for probing the efficacy of molecular glues in inducing PPIs. Using YaxAB nanopores, we demonstrate single-molecule-based, label-free monitoring of protein complex formation between mammalian target of rapamycin (mTOR) and FK506-binding proteins (FKBPs) triggered by the molecular glue, rapamycin. Owing to its wide entrance and adjustable pore size, in combination with potent electro-osmotic flow (EOF), a single funnel-shaped YaxAB nanopore enables the simultaneous detection and single-molecule-level quantification of multiprotein states, including single proteins, binary complexes, and ternary complexes induced by rapamycin. Notably, YaxAB nanopores could sensitively discriminate between the binary complexes or ternary complexes induced by rapamycin and its analogues, despite the subtle size differences of ∼122 or ∼116 Da, respectively. Taken together, our results provide proof-of-concept for single-molecule-based, label-free, and ultrasensitive screening and structure-activity relationship (SAR) analysis of molecular glues, which will contribute to low-cost, highly efficient discovery, and rational design of bifunctional modality of drugs, such as molecular glues.

Elucidating Phosphate and Cadmium Cosorption Mechanisms on Mineral Surfaces with Direct Spectroscopic and Modeling Evidence.

The simultaneous sorption of cations and anions at the mineral-water interface can substantially alter their individual sorption characteristics; however, this phenomenon lacks a mechanistic understanding. Our study provides direct spectroscopic and modeling evidence of the molecular cosorption mechanisms of the cadmium ion (Cd2+) and phosphate (P) on goethite and layered manganese (Mn) oxide of birnessite, through in situ attenuated total reflection Fourier-transform infrared (ATR-FTIR), P K-edge X-ray absorption near-edge structure (XANES) spectroscopy, and surface complexation modeling. Phosphate synergistically cosorbed with Cd on goethite predominantly through P-bridged ternary complexes (≡Fe-P-Cd) and electrostatic interactions at wide pH conditions. Likewise, P and Cd exhibited synergistic cosorption on birnessite by forming P-bridged ternary complexes (≡Mn-P-Cd) and weak competitive sorption at the layer edge sites. As pH and Cd loading increased, the surface P species transitioned from a binary complex to a ternary complex and/or Cd3(PO4)2 precipitate for both goethite and birnessite. Compared to that in solution at pH 8, the formation of Cd3(PO4)2 was inhibited by the presence of goethite and birnessite, ascribed to the specific adsorption of P and Cd, more pronounced in birnessite due to the stronger sorption of Cd at its vacant sites. The discovered cosorption mechanisms of P and Cd have important implications for understanding and predicting their mobility and availability in Cd-contaminated settings.

The Effect of Dihydromyricetin (DMY) on the Mechanism of Soy Protein Isolate/Inulin/Dihydromyricetin Interaction: Structural, Interfacial, and Functional Properties.

The combination of proteins with polysaccharides and polyphenols is expected to improve their physicochemical and functional properties. In this study, a novel plant-based antioxidant emulsifier was formed by soybean protein isolate (SPI), inulin (INU), and dihydromyricetin (DMY). Based on the binary system of SPI/INU, we focused on exploring the effect of the DMY concentration (0.5 mg/mL~2.5 mg/mL) on the formation and properties of the ternary complex. The structure, interaction mechanism, and interfacial and functional properties of the ternary complex were investigated. The results indicate that compared to the SPI/INU binary complex, the SPI/INU/DMY ternary complex had a significant decrease in particle size (~100 nm) and a slight decrease in absolute zeta potential. The SPI/INU binary complex with DMY mainly interacted by hydrogen bonding and hydrophobic interactions. Due to the incorporation of DMY, the structure of SI was denser and more flexible. The ternary complex exhibited an ideal three-phase contact angle and demonstrated better foaming and antioxidant ability. Additionally, compared to SPI/INU, the ternary complex had a significant improvement in EAI. These results provide a strategy for polyphenols to modify the structure, interfacial properties, and functions of protein/polysaccharide complexes. This provides a potential reference for the preparation of more ternary complexes with excellent emulsifying and antioxidant properties for application in emulsions.

An analysis of acid-labile subunit (ALS) levels in children with short stature born with normal weight.

The acid-labile subunit (ALS) is a protein best known for its function in stabilising the insulin like growth factor-1/2-insulin-like growth factor-1 binding protein 3/5 (IGF-1/2-IGFBP3/5) binary complex by creating the ternary complex and in consequence regulating the biological activity of IGF-1. The aim of the study was to assess ALS concentrations in a chosen population of children with short stature taking into account their clinical diagnosis. A total of 109 prepubertal children were involved in the study - 85 children in the study group and 24 in controls. In all the children IGF-1, IGFBP3, and ALS were measured. The study group was divided according to diagnosis into groups: growth hormone deficiency (GHD), constitutional delay of growth and puberty (CDGP), idiopathic short stature (ISS), and familial short stature (FSS). In the control group the ALS concentration ranged from 4.81 to 13.66 μg/mL. In the whole study group the ALS concentration ranged from 2.73 to 15.81 μg/mL. The difference between both groups was statistically significant (p < 0.0001, R = 0.39). A strong, statistically significant correlation between ALS levels and age was observed, but only in the study group (p < 0.0001, r = 0.59). The ALS standard deviation score (SDS) was not significantly different between the control and CDGP children (p = 0.0644). The ALS concentration was significantly lower in children with short stature. There was, however, no difference between the subgroups of the study group. There was no significant difference in ALS SDS between the control group and children with constitutional delay of growth and development. The usefulness of ALS in routine short stature diagnostics is uncertain, but it might play a role in the diagnosis of children with ISS and CDGP in the future.

Speciation studies of binary and ternary complexes formed with fe(II) ion 2,6-pyridinedicarboxylic acid and several amino acids

The chemical speciation of ternary systems involving Fe(II), 2,6-pyridinedicarboxylic acid (H2Dipi), and various amino acids (glycine, β-alanine, α-alanine, leucine, methionine, lysine, phenylalanine, threonine, serine, proline, valine, and histidine) was investigated in this work. Potentiometric titrations were conducted for each system in aqueous solution at 25 °C using 1 mol dm−3 NaCl solution as the ionic medium. The resulting data were analyzed using the least-squares program LETAGROP. Additionally, the binary system of Fe(II)-2,6-pyridinedicarboxylic acid in aqueous solution at 25 °C with NaCl 1.0 mol dm−3 as the ionic medium was also examined. Species distribution diagrams were generated, and the relative stability parameter Δlog K was calculated.

Dyeing behaviour of iron(III)-gallic acid complexes on wool as function of pH-dependent iron(III)-complex stoichiometry

Iron complexes with polyphenolics e.g. gallotannins and flavonoids have been used since ancient times for coloration of textiles, painting and preparation of inks. The composition of iron gall inks on historical artefacts has been investigated with modern instrumental methods. However the dyeing behaviour of iron-complexes with polyphenols still bases on empirical knowledge. In this study Fe(III)-complexes with gallic acid were used as model system to elucidate their pH dependent sorption and dyeing behaviour on wool. At dyebath pH 3 high sorption of Fe(III)-gallic acid complexes was observed, however darker dyeings were obtained at dyebath pH 4. Model calculations for the complex species present in the dyebath indicated the presence of the binary complex [Fe(III)(GA2−)]+ as main species at pH 3, while at pH 4 the ternary species [Fe(III)(GA2−)2]- prevails. The dye sorption bases on Van-der-Waals forces, while ionic interactions between Fe(III)-complex and keratin are of minor relevance. The elaborated theoretical model also explains the influence of dyebath pH in dyeing with gallotannins, polyphenols and flavonoids in presence of iron salts and thus is of particular relevance for improved utilisation of these natural colorants.

Structures of a DNA Polymerase Caught while Incorporating Responsive Dual-Functional Nucleotide Probes.

Functionalizing nucleic acids using DNA polymerases is essential in biophysical and biotechnology applications. This study focuses on understanding how DNA polymerases recognize and incorporate nucleotides with diverse chemical modifications, aiming to develop advanced nucleotide probes. We present the crystal structures of ternary complexes of Thermus aquaticus DNA polymerase (KlenTaq) with C5-heterocycle-modified environment-sensitive 2'-deoxyuridine-5'-triphosphate (dUTP) probes. These nucleotides include SedUTP, BFdUTP and FBFdUTP, which bear selenophene, benzofuran and fluorobenzofuran, respectively, at the C5 position of uracil, and exhibit high conformational sensitivity. SedUTP and FBFdUTP serve as dual-app probes, combining a fluorophore with X-ray anomalous scattering Se or 19F NMR labels. Our study reveals that the size of the heterocycle influences how DNA polymerase families A and B incorporate these modified nucleotides during single nucleotide incorporation and primer extension reactions. Remarkably, FBFdUTP's responsiveness enabled real-time monitoring of the binary complex formation and polymerase activity through fluorescence and 19F NMR. Comparative analysis of incorporation profiles, fluorescence, 19F NMR data, and crystal structures of ternary complexes highlights the enzyme's plasticity. Key insights are provided into the role of gatekeeper amino acids (Arg660 and Arg587) in accommodating and processing these modified substrates, offering a structural basis for next-generation nucleotide probe development.

Structures of a DNA Polymerase Caught while Incorporating Responsive Dual‐Functional Nucleotide Probes

Functionalizing nucleic acids using DNA polymerases is essential in biophysical and biotechnology applications. This study focuses on understanding how DNA polymerases recognize and incorporate nucleotides with diverse chemical modifications, aiming to develop advanced nucleotide probes. We present the crystal structures of ternary complexes of Thermus aquaticus DNA polymerase (KlenTaq) with C5‐heterocycle‐modified environment‐sensitive 2′‐deoxyuridine‐5′‐triphosphate (dUTP) probes. These nucleotides include SedUTP, BFdUTP and FBFdUTP, which bear selenophene, benzofuran and fluorobenzofuran, respectively, at the C5 position of uracil, and exhibit high conformational sensitivity. SedUTP and FBFdUTP serve as dual‐app probes, combining a fluorophore with X‐ray anomalous scattering Se or 19F NMR labels. Our study reveals that the size of the heterocycle influences how DNA polymerase families A and B incorporate these modified nucleotides during single nucleotide incorporation and primer extension reactions. Remarkably, FBFdUTP's responsiveness enabled real‐time monitoring of the binary complex formation and polymerase activity through fluorescence and 19F NMR. Comparative analysis of incorporation profiles, fluorescence, 19F NMR data, and crystal structures of ternary complexes highlights the enzyme's plasticity. Key insights are provided into the role of gatekeeper amino acids (Arg660 and Arg587) in accommodating and processing these modified substrates, offering a structural basis for next‐generation nucleotide probe development.

Ternary π-π Stacking Complexes by Allosteric Regulation in Multilayer Nanographenes.

Construction of π-π stacking supramolecular complexes with more than two different components is challenging due to the weak and directionless nature of dispersion interactions. Here, we report ternary complexes of a ditopic nanographene tetraimide (1), α-substituted phthalocyanine (Pc), and polyaromatic hydrocarbons (PAHs) in solution and the crystalline state via allosteric regulation. Binding of one Pc gives rise to significant distortion and conformational changes in 1 that in turn lead to the inhibition of the second binding of Pc. The conformational changes associated with first binding allowed an allosteric binding of a third component (PAHs) to form ternary complexes in solution. 1H NMR titration revealed moderately high thermodynamic stability for the ternary complexes in CDCl3. Competition between allosterically regulated ternary complexes ([Pc·1·PAH]) and 1:2 stoichiometric binary complexes of 1 with PAHs ([PAH·1·PAH]) was elucidated. Further, the selective formation of ternary complexes in solution led to the generation of ternary cocrystals from a 1:1:1 mixture of three components in solution. Our work shows that large π-conjugated nanographenes designed with allosteric recognition sites allow the construction of multilayer ternary complexes in solution and the solid state even with dispersive π-π interactions.

Reverse hierarchical DED assembly in the cFLIP-procaspase-8 and cFLIP-procaspase-8-FADD complexes

cFLIP, a master anti-apoptotic regulator, targets the FADD-induced DED complexes of procaspase-8 in death receptor and ripoptosome signaling pathways. Several tumor cells maintain relatively high levels of cFLIP in achieving their immortality. However, understanding the three-dimensional regulatory mechanism initiated or mediated by elevated levels of cFLIP has been limited by the absence of the atomic coordinates for cFLIP-induced DED complexes. Here we report the crystal plus cryo-EM structures to uncover an unconventional mechanism where cFLIP and procaspase-8 autonomously form a binary tandem DED complex, independent of FADD. This complex gains the ability to recruit FADD, thereby allosterically modulating cFLIP assembly and partially activating caspase-8 for RIPK1 cleavage. Our structure-guided mutagenesis experiments provide critical insights into these regulatory mechanisms, elucidating the resistance to apoptosis and necroptosis in achieving immortality. Finally, this research offers a unified model for the intricate bidirectional hierarchy-based processes using multiprotein helical assembly to govern cell fate decisions.

Modulating the Amine-CO2 Interaction Strength: Toward Efficient Carbon Capture.

This study contributes to a comprehensive understanding of the interactions between CO2 and amines at the molecular level by exploring the rotational spectra of binary complexes between CO2 and eight different amines through pulsed-jet Fourier transform microwave spectroscopy and quantum chemical calculations. The findings reveal a consistent pattern in which CO2 is bonded to the amino group, primarily through a C···N tetrel bond, while being supported by C-H···O/C hydrogen bonds. Notably, the binding energies increase from primary through tertiary amines and with increasing chain length of the alkyl groups. These groups are found to enhance the electron density at the amino group significantly, thereby facilitating the formation of stronger C···N tetrel bonds. The insights provided into how the interaction strength is modulated by the geometries of amines are deemed essential for the design of more effective CO2 adsorption materials, thus advancing carbon capture technologies.

Design of a Cereblon construct for crystallographic and biophysical studies of protein degraders

The ubiquitin E3 ligase cereblon (CRBN) is the target of therapeutic drugs thalidomide and lenalidomide and is recruited by most targeted protein degraders (PROTACs and molecular glues) in clinical development. Biophysical and structural investigation of CRBN has been limited by current constructs that either require co-expression with the adaptor DDB1 or inadequately represent full-length protein, with high-resolution structures of degrader ternary complexes remaining rare. We present the design of CRBNmidi, a construct that readily expresses from E. coli with high yields as soluble, stable protein without DDB1. We benchmark CRBNmidi for wild-type functionality through a suite of biophysical techniques and solve high-resolution co-crystal structures of its binary and ternary complexes with degraders. We qualify CRBNmidi as an enabling tool to accelerate structure-based discovery of the next generation of CRBN based therapeutics.

Pre-fusion AAA+ remodeling of target-SNARE protein complexes enables synaptic transmission.

Membrane fusion is driven by SNARE complex formation across cellular contexts, including vesicle fusion during synaptic transmission. Multiple proteins organize trans-SNARE complex assembly and priming, leading to fusion. One target membrane SNARE, syntaxin, forms nanodomains at the active zone, and another, SNAP-25, enters non-fusogenic complexes with it. Here, we show that the AAA+ protein NSF (N-ethylmaleimide sensitive factor) and SNAP (soluble NSF attachment protein) must act prior to fusion. We show that syntaxin clusters are conserved, that NSF colocalizes with them, and characterize SNARE populations within and near these clusters using cryo-EM. Supercomplexes of NSF, α-SNAP, and either a syntaxin tetramer or two binary complexes of syntaxin-SNAP-25 reveal atomic details of SNARE processing and show how sequential ATP hydrolysis drives disassembly. These results suggest a functional role for syntaxin clusters as reservoirs and a corresponding role for NSF in syntaxin liberation and SNARE protein quality control preceding fusion.

Increased rainfall frequency enhances dry seasonal soil mineral-associated organic carbon in a subtropical forest

Climate models predict longer and more severe droughts, and alterations in the frequency and intensity of rainfall events. However, how changing precipitation patterns affect soil organic carbon (SOC), particulate organic carbon (POC), and mineral-associated organic carbon (MAOC) remains unclear. Here, we conducted a three-year rainfall manipulation experiment with ambient rainfall as the control, removal of half the total rainfall amount with unaltered frequency (DRA), and increased rainfall frequency with the total amounts unchanged (IRF) in a subtropical forest. The results showed that the rainfall treatments did not change SOC content or fractions during the wet season. In the dry season, DRA significantly increased topsoil POC but did not change SOC or MAOC. IRF significantly increased POC and MAOC levels. The increased MAOC was associated with the newly formed binary complexes of Fe3+, Al3+, and Ca2+, and the adsorption/precipitation of reduced short-range-ordered Fe oxides, likely derived from the enhanced reductive dissolution in the IRF treatment. Our results suggest that changes in rainfall frequency affect organo-mineral interactions more profoundly relative to the rainfall amount and that these effects are season-dependent. Therefore, moisture-sensitive geochemical processes play an essential role in SOC stabilization in subtropical forests.

Deciphering Pathways and Thermodynamics of Protein Assembly Using Native Mass Spectrometry.

Protein oligomerization regulates many critical physiological processes, and its dysregulation can contribute to dysfunction and diseases. Elucidating the assembly pathways and quantifying their underlying thermodynamic and kinetic parameters are crucial for a comprehensive understanding of biological processes and for advancing therapeutics targeting abnormal protein oligomerization. Established binding assays, with limited mass precision, often rely on simplified models for data interpretation. In contrast, high-resolution native mass spectrometry (nMS) can directly determine the stoichiometry of biomolecular complexes in vitro. However, quantification is hindered by the fact that the relative abundances of gas-phase ions generally do not reflect solution concentrations due to nonuniform response factors. Recently, slow mixing mode (SLOMO)-nMS, which can quantify the relative response factors of interacting species, has been demonstrated to reliably measure the affinity (Kd) of binary biomolecular complexes. Here, we introduce an extended form of SLOMO-nMS that enables simultaneous quantification of the thermodynamics in multistep association reactions. Application of this method to homo-oligomerization of concanavalin A and insulin confirmed the reliability of the assay and uncovered details about the assembly processes that had previously resisted elucidation. Results acquired using SLOMO-nMS implemented with charge detection shed new light on the binding of recombinant human angiotensin-converting enzyme 2 and the SARS-CoV-2 spike protein. Importantly, new assembly pathways were uncovered, and the affinities of these interactions, which regulate host cell infection, were quantified. Together, these findings highlight the tremendous potential of SLOMO-nMS to accelerate the characterization of protein assembly pathways and thermodynamics and, in so doing, enhance fundamental biological understanding and facilitate therapeutic development. https://orcid.org/0000-0002-3389-7112.

Rapid separation of bile acid isomers via ion mobility mass spectrometry by complexing with spiramycin.

Bile acid (BA) is one of the main active components of bile and has multiple isomers, the structure or content of its isomers often changes due to diseases and other health problems; thus, the accurate detection of BA isomers is very important. In this study, two groups of BA isomers of glycine-conjugated BAs and taurine-conjugated BAs were simultaneously separated and quantitatively analyzed by ion mobility mass spectrometry (IM-MS). Especially, baseline mobility separation between the isomers was achieved by the formation of binary complexes via simple interaction with spiramycin (SPM), for which a separation resolution (Rp-p) of 1.96was reached. Moreover, BA isomers were quantitatively analyzed, and the limit of detection (LOD) of absolute quantification for TCDCA/TUDCA and GUDCA/GCDCA/GHDCA was 0.514 and 0.611ng∙mL-1, respectively; the LODs for molar ratio ranges of relative quantification for TCDCA/TUDCA, GUDCA/GHDCA, and GCDCA/GHDCA were 1:18-30:1, 1:18-21:1, and 1:19-21:1, respectively. Additionally, BA isomers analyzed in pig bile powder and bear bile powder were measured, which were in good consistency with those labeled, revealing the differences in BA composition and content between the two powders. Finally, BA detection and recovery analyses were performed on serum samples, with a recovery rate of ≥73.69%, RSD of ≤6.8%, and SR (standard deviation of recoveries, the degree of difference between measured values and average recovery) of ≤1.27. Due to the simple, rapid, and lack of need for complex sample preparation and chromatographic separation, the proposed method can be an effective method for BA detection in practical samples.