Phosphoryl Research Articles

Role of extracellular polymeric substances in the immobilization of hexavalent chromium by Shewanella putrefaciens CN32 unsaturated biofilms

Microbial remediation provides a promising avenue for the management and restoration of heavy metal-contaminated soils. Microorganisms in soils usually exist within unsaturated biofilms, however, their response to heavy metals is still limited compared to saturated biofilms. This work investigated the Cr(VI) immobilization by Shewanella putrefaciens CN32 unsaturated biofilms, and explored the underlying mechanisms of Cr(VI) complexation. Results reveal a dose-dependent toxicity of Cr(VI) to the growth of the unsaturated biofilms. During the early growth stage, the Cr(VI) addition stimulated more extracellular polymeric substances (EPS) production. In the meantime, the EPS were demonstrated to be the primary components for Cr(VI) immobilization, which accounted for more than 60% of the total adsorbed Cr(VI). The Fourier transform infrared spectra and X-ray photoelectron spectra corroborated that the binding sites for immobilizing Cr(VI) were hydroxyl, carboxyl, phosphoryl and amino functional groups of the proteins and polysaccharides in EPS. However, for the starved unsaturated biofilms, EPS were depleted and the EPS-bound Cr(VI) were released, which caused approximately 60% of the adsorbed Cr(VI) onto cell components and further aggravated the Cr(VI) stress to cells. This work extends our understanding about the Cr(VI) immobilization by unsaturated biofilms, and provides useful information for remediation of heavy metal-contaminated soils.

Mechanism of Triphosphate Hydrolysis by Human MAT2A at 1.07 Å Resolution.

Human methionine adenosyltransferase MAT2A provides S-adenosyl-l-methionine (AdoMet) for methyl-transfer reactions. Epigenetic methylations influence expression patterns in development and in cancer. Transition-state analysis and kinetic studies have described the mechanism of AdoMet and triphosphate formation at the catalytic site. Hydrolysis of triphosphate to pyrophosphate and phosphate by MAT2A is required for product release and proceeds through a second chemical transition state. Crystal structures of MAT2A with analogues of AdoMet and pyrophosphate were obtained in the presence of Mg2+, Al3+, and F-. MgF3- is trapped as a PO3- mimic in a structure with malonate filling the pyrophosphate site. NMR demonstrates that MgF3- and AlF30 are bound by MAT2A as mimics of the departing phosphoryl group. Crystallographic analysis reveals a planar MgF3- acting to mimic a phosphoryl (PO3-) leaving group. The modeled transition state with PO3- has the phosphorus atom sandwiched symmetrically and equidistant (approximately 2 Å) between a pyrophosphate oxygen and the water nucleophile. A catalytic site arginine directs the nucleophilic water to the phosphoryl leaving group. The catalytic geometry of the transition-state reconstruction predicts a loose transition state with characteristics of symmetric nucleophilic displacement.

The Relationship between Enzyme Conformational Change, Proton Transfer, and Phosphoryl Transfer in β-Phosphoglucomutase

Molecular details for the timing and role of proton transfer in phosphoryl transfer reactions are poorly understood. Here, we have combined QM models, experimental NMR measurements, and X-ray structures to establish that the transition of an archetypal phosphoryl transfer enzyme, βPGM, from a very closed near-attack conformation to a fully closed transition state analogue (TSA) conformation triggers both partial proton transfer from the general acid–base residue to the leaving group oxygen and partial dissociation of the transferring phosphoryl group from the leaving group oxygen. Proton transfer continues but is not completed throughout the reaction path of the phosphoryl transfer with the enzyme in the TSA conformation. Moreover, using interacting quantum atoms (IQA) and relative energy gradient (REG) analysis approaches, we observed that the change in the position of the proton and the corresponding increased electrostatic repulsion between the proton and the phosphorus atom provide a stimulus for phosphoryl transfer in tandem with a reduction in the negative charge density on the leaving group oxygen atom. The agreement between solution-phase 19F NMR measurements and equivalent QM models of βPGMWT and βPGMD10N TSA complexes confirms the protonation state of G6P in the two variants, validating the employed QM models. Furthermore, QM model predictions of an AlF4 distortion in response to the proton position are confirmed using high resolution X-ray crystal structures, not only providing additional validation to the QM models but also further establishing metal fluorides as highly sensitive experimental predictors of active-site charge density distributions.

Crystal structure of brigatinib Form A (Alunbrig®), C29H39ClN7O2P

The crystal structure of brigatinib Form A has been solved and refined using synchrotron X-ray powder diffraction data and optimized using density functional theory techniques. Brigatinib Form A crystallizes in space group P-1 (#2) with a = 9.59616(20), b = 10.9351(3), c = 14.9913(6) Å, α = 76.1210(13), β = 79.9082(11), γ = 74.0802(6)°, V = 1458.497(15) Å3, and Z = 2. Structure solution was complicated by the lowest cost factor solution having an unreasonable conformation of the dimethylphosphoryl group. The second-best structure yielded a better refinement. The crystal structure is characterized by alternating layers of aliphatic and aromatic portions of the molecules along the b-axis. Strong N–H⋯N hydrogen bonds link the molecules into pairs, with a graph set R2,2(8). There is a strong intramolecular N–H⋯O hydrogen bond to the phosphoryl group, which determines the orientation of this group. The powder pattern has been submitted to ICDD® for inclusion in the Powder Diffraction File™ (PDF®).

Synthesis, IR spectroscopic and structural studies of erbium and nickel complexes with N,N'-tetraethyl-N''-trifluoroacetylphosphoroustriamide

A synthesis procedure was developed and a new carbacylamidophosphate type ligand N,N'-tetraethyl-N''-trifluoroacetylphosphoroustriamide (CF3C(O)NHP(O)(NC2H5)2, HL) that contains C(O)NP(O) chelating fragment was isolated in the crystalline state. A mononuclear erbium complex [Er(HL)3(NO3)3] and a tetranuclear nickel complex [Ni4L4(OCH3)4(CH3OH)4] were isolated in the crystalline state. The suggestion about the type and coordination mode of the ligand in complexes was made based on IR spectroscopic investigations: deprotonated (acido-) form in bidentate manner in nickel complex and neutral form in monodentate manner in erbium complex. According to X-ray structural studies, different coordination modes of the ligand in complexes were determined: bidentate chelate manner via the oxygen atoms of the phosphoryl and carbonyl groups of the ligand with the formation of six-membered chelate cycles in case of nickel complex and monodentate manner via the oxygen atom of the phosphoryl group of the ligand in case of erbium complex, the coordination polyhedron of which was interpreted as a distorted three-handed trigonal prism.

Identification of novel benzothiopyranones with ester and amide motifs derived from active metabolite as promising leads against Mycobacterium tuberculosis.

We reported three distinct series of novel benzothiopyranones, derived from an active metabolite (M-1) of anti-TB agent 6b. These small molecules were evaluated for their biological activities against a range of Mycobacterium tuberculosis (M.tuberculosis) strains. Preliminary druggability evaluation demonstrated that M-1 showed good aqueous solubility and hepatocyte stability. Benzothiopyranones with acyl, sulfonyl and phosphoryl groups exhibited potent invitro inhibitory activity against M.tuberculosis H37Rv and low cytotoxicity. In particular, compound 3d, containing a benzoate fragment, displayed marked metabolic stability and potent invitro activity against drug-resistant tuberculosis clinical strains. Further druggability evaluation based on the identified compounds 3d, 4e and 5b is ongoing for the discovery of promising anti-TB agents.

The protein–protein interaction network of the Escherichia coli EIIANtr regulatory protein reveals a role in cell motility and metabolic control

The nitrogen-related PTSNtr system, present in many Proteobacteria including Escherichia coli, acts as a phosphorelay cascade composed of the EINtr, NPr and EIIANtr proteins. Phosphotransfer initiates with phosphoenolpyruvate-dependent EINtr autophosphorylation, the phosphoryl group is then transferred to NPr and finally to a conserved histidine residue on EIIANtr. The reporter metabolites l-glutamine and 2-oxoglutarate reciprocally regulate EINtr autophosphorylation (Lee et al., 2013) and consequently the phosphorylation status of the PTSNtr components is controlled by the availability of nitrogen and carbon. The final phosphate acceptor, EIIANtr, regulates a range of cellular process by acting as the central hub of a complex protein–protein interaction network. Contact between EIIANtr and its target proteins is usually regulated by the EIIANtr phosphorylation status. In this study we performed ligand fishing assays coupled to label-free quantitative proteomics to examine the protein–protein interaction network of E. coli EIIANtr and a phosphomimic variant of the protein. The ligand fishing data, along with phenotypic analysis, indicated that EIIANtr interacts with proteins related to chemotaxis and thereby regulates cell motility. Important metabolic enzymes were also identified as potential EIIANtr binding partners.

Phosphorylated protein modification analysis on normal liver and Exo-celiac liver of Glyptosternum maculatum.

This study aimed to reveal the biological function and molecular mechanism of phosphorylated proteins in the normal liver (NG) and Exo-celiac liver (WG) of Glyptosternum maculatum and potential plateau-adaption mechanisms of G. maculatum. A multivariate analysis was performed on proteomic quantitative data (label-free group) and phosphorylated proteome data (phosphorylation group) to reveal protein characteristics. The differentially expressed proteins (DEPs) between NG and WG in the two groups were analysed. Enrichment analysis of these DEPs was performed prior to the protein-protein interaction (PPI) analysis. Finally, an integrated interaction network was constructed to reveal the biological mechanism of the DEP-mediated signal transduction process. The NG and WG samples in the phosphorylation group were well distinguished compared to the label-free group. A total of 49 and 313 DEPs were identified in the label-free and phosphorylation groups, respectively. These DEPs, including LIM and calponin homology domains-containing protein 1 (LIMCH1) and DEAD(Asp-Glu-Ala-Asp)-Box Helicase 51 (DDX51), were mainly assembled in functions such as cell adhesion. Two PPI networks were constructed using DEPs in the two groups. Finally, an integrated interaction network was constructed using co-DEP Ferredoxin 1 (FDX1) and associated pathways, including RNA transport. LIMCH1 and DDX51 might play important roles in the organogenesis of normal liver and Exo-celiac liver in G. maculatum via the cell adhesion function. Moreover, FXD1 might be associated with the plateau-adaption mechanisms of G. maculatum via participation in the RNA transport pathway.

Diazophosphonates: Effective Surrogates for Diazoalkanes in Pyrazole Synthesis.

Diazophosphonates, readily prepared from α-ketophosphonates by oxidation of the corresponding hydrazones in batch or in flow, are useful partners in 1,3-dipolar cycloaddition reactions to alkynes to give N-H pyrazoles, including the first intramolecular examples of such a process. The phosphoryl group imbues a number of desirable properties into the diazo 1,3-dipole. The electron-withdrawing nature of the phosphoryl stabilizes the diazo compound making it easier to handle, whilst the ability of the phosphoryl group to migrate readily in a [1,5]-sigmatropic rearrangement enables its transfer from C to N to aromatize the initial cycloadduct, and hence its facile removal from the final pyrazole product. Overall, the diazophosphonate acts as a surrogate for the much less stable diazoalkane in cycloadditions, with the phosphoryl group playing a vital, but traceless, role. The cycloaddition proceeds more readily with alkynes bearing electron-withdrawing groups, and is regiospecific with asymmetrical alkynes. The potential of diazophosphonates for use in bioorthogonal cycloadditions is demonstrated by their facile addition to strained alkynes.

Exploring the Mechanism of Shikimate Kinase through Quantum Mechanical and Molecular Mechanical (QM/MM) Methods

The chemical step of Shikimate Kinase Helicobacter pylori, involving the transfer of a phosphoryl group, has been studied by using quantum mechanical and molecular mechanical (QM/MM) methods. Understanding the mechanism of this chemical step, present in bacteria and other microorganisms but absent in humans, can lead to the development of novel drugs for the treatment of common diseases caused by those pathogenic organisms. Different mechanisms including associative, dissociative, and concerted have been proposed up to now but there is not a consensus on the type of pathway that the reaction follows. Herein, we found that the mechanism has features from the associative and concerted types. An analysis of the free energy landscape of the chemical step reveals that the reaction is a two-step process without a well-defined intermediate state.

Multigram Synthesis of Difluoromethylene Phosphonic and Phosphinic Amides and Phosphine Oxides via Formal [2,3]-Sigmatropic Allyl Phosphite–Allylphosphonate Rearrangement

AbstractWe describe a method for the preparation of CF2-P(V) building blocks and monomers for biological and materials chemistry applications in multigram quantities based on a formal [2,3]-sigmatropic phospha-Wittig rearrangement of readily available fluoroallyl bis(amido)phosphites, amido(aryl)phosphonites, and diarylphosphinites. The proposed intramolecular phosphorylation approach complements the currently prevailing phosphoryldifluoromethylation methods by providing a straightforward access to difluoromethylene phosphonate analogues bearing dialkylamino and/or aryl substituents at the phosphoryl group. An important advantage of the developed method is that it does not rely on ozone-depleting HCF2Cl and CF2Br2 necessary for the preparation of phosphoryldifluoromethylating reagents. 2,3,3-Trifluoroallyloxy P(III) derivatives substituted with two dialkylamino and/or aryl groups underwent a facile [2,3]-rearrangement to give difluoromethylene phosphonic and phosphinic amides and phosphine oxides on up to a 0.15 mol (30 g) scale. The reaction was extended to P(III) derivatives of 1-substituted 2,3,3-trifluoro- and 3,3-difluoroallylic alcohols readily available from carbonyl compounds and, respectively, 1,1,1,2-tetrafluoroethane and O-protected 2,2,2-trifluoroethanols. Products derived from O-(tetrahydropyran-2-yl)-2,2,2-trifluoroethanol were synthesized and deprotected in a one-pot multistep protocol to give phosphonic and phosphinic amide and phosphine oxide analogues of α,α-difluoro-β-ketophosphonates on a multigram scale.

Adenylate Kinase-Catalyzed Reaction of AMP in Pieces: Enzyme Activation for Phosphoryl Transfer to Phosphite Dianion.

The binding of adenosine 5'-triphosphate (ATP) and adenosine 5'-monophosphate (AMP) to adenylate kinase (AdK) drives closure of lids over the substrate adenosyl groups. We test the hypothesis that this conformational change activates AdK for catalysis. The rate constants for Homo sapiens adenylate kinase 1 (HsAdK1)-catalyzed phosphoryl group transfer to AMP, kcat/Km = 7.0 × 106 M-1 s-1, and phosphite dianion, (kHPi)obs ≤1 × 10-4 M-1 s-1, show that the binding energy of the adenosyl group effects a ≥7.0 × 1010-fold rate acceleration of phosphoryl transfer from ATP. The third-order rate constant of kcat/KHPiKEA = 260 M-2 s-1 for 1-(β-d-erythrofuranosyl)adenine (EA)-activated phosphoryl transfer to phosphite dianion was determined, and the isohypophosphate reaction product characterized by 31P NMR. The results demonstrate the following: (i) a ≥14.7 kcal/mol stabilization of the transition state for phosphoryl transfer by the adenosyl group of AMP and a ≥2.6 × 106-fold rate acceleration from the EA-driven conformational change and (ii) the recovery of ≥8.7 kcal/mol of this transition state stabilization for EA-activated phosphoryl transfer from ATP to phosphite.

Laboratory studies on the effect of adsorbed microbial extracellular polymeric substances on the acidity of selected variable‐charge soils

AbstractExtracellular polymeric substances (EPS) secreted by rhizobacteria are ubiquitous in the environment and can enhance the negative charge of variable‐charge soils. Soil negative charge is directly related to pH buffering capacity (pHBC); it is essential to examine the effect of EPS on soil acidity and pHBC. We investigated the effect of adsorbed EPS on the acidification of five variable‐charge soils in China, which were subjected to different land use: an Oxisol and four Ultisols. Spectroscopic analysis revealed that EPS was specifically adsorbed onto soil surfaces through iron–oxygen–phosphorus/aluminum–oxygen–phosphorus (Fe‐O‐P/Al‐O‐P) bonds formed between phosphoryl groups of EPS and soil Al/Fe oxides. During the formation of Fe‐O‐P/Al‐O‐P bonds, there was ligand exchange between the EPS phosphoryl groups and the surface hydroxyl groups of Fe/Al oxides of the soils. The released OH– played an important role in soil acidity amelioration by the adsorbed EPS through neutralizing H+ and increasing soil suspension pH. After acidification, the pH of EPS‐treated soils was higher than that of the controls. This was attributed to a combined effect of enhanced soil pHBC, and protonation of organic anions (e.g., COO– to COOH) of EPS to decrease the activity of protons. The adsorbed EPS ameliorated prior soil acidity, inhibited new soil acidification, and promoted hydrolysis and re‐adsorption of Al. This was evident as the proportion of organically bound Al and adsorbed hydroxyl Al increased, whereas that of exchangeable Al decreased after EPS adsorption. Therefore, this study can serve as a reference for the molecular design of cost‐effective and environmentally friendly amendments for acid soils.

Conformational Analysis of N-Alkyl-N-[2-(diphenylphosphoryl)ethyl]amides of Diphenylphosphorylacetic Acid: Dipole Moments, IR Spectroscopy, DFT Study.

Experimental and theoretical conformational analysis of N-methyl-N-[2-(diphenylphosphoryl)ethyl]diphenylphosphorylacetamide, N-butyl-N-[2-(diphenylphosphoryl)ethyl]diphenylphosphorylacetamide, and N-octyl-N-[2-(diphenylphosphoryl)ethyl]diphenylphosphorylacetamide was carried out by the methods of dipole moments, IR spectroscopy, and Density Functional Theory (DFT) B3PW91/6-311++G(df,p) calculations. In solution, these N,N-dialkyl substituted bisphosphorylated acetamides exist as a conformational equilibrium of several forms divided into two groups—with Z- or E-configuration of the carbonyl group and alkyl substituent, and syn or anti arrangement of the phosphoryl-containing fragments relative to the amide plane. The substituents at the phosphorus atoms have eclipsed cis- or staggered gauche-orientation relative to the P=O groups, and cis orientation of the substituents is due to the presence of intramolecular H-contacts P=O...H−Cphenyl or p,π conjugation between the phosphoryl group and the phenyl ring. Preferred conformers of acetamides molecules are additionally stabilized by various intramolecular hydrogen contacts with the participation of oxygen atoms of the P=O or C=O groups and hydrogen atoms of the methylene and ethylene bridges, alkyl substituents, and phenyl rings. However, steric factors, such as a flat amide fragment, the bulky phenyl groups, and the configuration of alkyl bridges, make a significant contribution to the realization of preferred conformers.

It takes two to tango: Unraveling a new post-translational modification involved in SnRK2.6 activation

Protein kinases are a conserved superfamily of proteins that play an essential role in signal transduction by attaching a phosphoryl group to serine, threonine, or tyrosine residues of a protein, hence converting them to phosphoproteins. This process, which can be reversed by protein phosphatases, regulates vital cellular processes in plants, including metabolism, proliferation, and death. It is also key to trigger response mechanisms to drought, increased salinity, and other abiotic stresses (Zhu, 2016).

Mass Spectrometry Analysis of SARS-CoV-2 Nucleocapsid Protein Reveals Camouflaging Glycans and Unique Post-Translational Modifications.

The devastating coronavirus disease 2019 (COVID-19) pandemic has prompted worldwide efforts to study structural biological traits of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its viral components. Compared to the Spike protein, which is the primary target for currently available vaccines or antibodies, knowledge about other virion structural components is incomplete. Using high-resolution mass spectrometry, we report a comprehensive post-translational modification (PTM) analysis of nucleocapsid phosphoprotein (NCP), the most abundant structural component of the SARS-CoV-2 virion. In addition to phosphoryl groups, we show that the SARS-CoV-2 NCP is decorated with a variety of PTMs, including N-glycans and ubiquitin. Based on newly identified PTMs, refined protein structural models of SARS-CoV-2 NCP were proposed and potential immune recognition epitopes of NCP were aligned with PTMs. These data can facilitate the design of novel vaccines or therapeutics targeting NCP, as valuable alternatives to the current vaccination and treatment paradigm that is under threat of the ever-mutating SARS-CoV-2 Spike protein.

Effects of Weak Nonspecific Interactions with ATP on Proteins.

Adenosine triphosphate (ATP) is an immensely well-studied metabolite serving multiple key biochemical roles as the major chemical energy currency in living systems, a building block of ribonucleic acids, and a phosphoryl group donor in kinase-mediated signaling. Intriguingly, ATP has been recently proposed to act as a hydrotrope that inhibits aggregation of amyloidogenic proteins; however, the underlying mechanism and the general physicochemical effect that coexistence with ATP exerts on proteins remain unclear. By combining NMR spectroscopy and MD simulations, here we observed weak but unambiguously measurable and concentration-dependent noncovalent interactions between ATP and various proteins. The interactions were most pronounced for an intrinsically disordered protein (α-synuclein) and for residues in flexible regions (e.g., loops or termini) of two representative folded proteins (ubiquitin and the dimeric ubiquitin-binding domain of p62). As shown by solution NMR, a consequence of the ATP-protein interaction was altered hydration of solvent-exposed residues in the protein. The observation that ATP interacted with all three proteins suggests that ATP is a general nonspecific binder of proteins. Several complementary biophysical methods further confirmed that, at physiological concentrations of ∼5-10 mM, ATP starts to form oligomeric states via magnesium-chelating and chelation-independent mechanisms, in agreement with previous studies. Although the observed ATP-protein interaction was relatively weak overall, the high ratio of ATP (monomeric free ATP, mono- and divalent ion-bound ATP, oligomeric and chelated ATP) to proteins in cells suggests that most proteins are likely to encounter transient interactions with ATP (and chemically similar metabolites) that confer metabolite-mediated protein surface protection.

Structures of full-length VanR from Streptomyces coelicolor in both the inactive and activated states.

Vancomycin has historically been used as a last-resort treatment for serious bacterial infections. However, vancomycin resistance has become widespread in certain pathogens, presenting a serious threat to public health. Resistance to vancomycin is conferred by a suite of resistance genes, the expression of which is controlled by the VanR-VanS two-component system. VanR is the response regulator in this system; in the presence of vancomycin, VanR accepts a phosphoryl group from VanS, thereby activating VanR as a transcription factor and inducing expression of the resistance genes. This paper presents the X-ray crystal structures of full-length VanR from Streptomyces coelicolor in both the inactive and activated states at resolutions of 2.3 and 2.0 Å, respectively. Comparison of the two structures illustrates that phosphorylation of VanR is accompanied by a disorder-to-order transition of helix 4, which lies within the receiver domain of the protein. This transition generates an interface that promotes dimerization of the receiver domain; dimerization in solution was verified using analytical ultracentrifugation. The inactive conformation of the protein does not appear intrinsically unable to bind DNA; rather, it is proposed that in the activated form DNA binding is enhanced by an avidity effect contributed by the receiver-domain dimerization.

Phosphoryl Prodrugs: Characteristics to Improve Drug Development.

Phosphoryl prodrugs are key compounds in drug development. Biologically active phosphoryl compounds often have negative charges on the phosphoryl group, and as a result, frequently have poor pharmacokinetic (PK) profiles. The use of lipophilic moieties bonded to the phosphorus (or attached oxygen atoms) masks the negative charge of the phosphoryl group, cleavage releasing the active molecule. The use of prodrugs to improve the PK of active parent molecules is an essential step in drug development. This review highlights promising trends in terminal elimination half-life, Cmax, clearance, oral bioavailability, and cLogP in phosphoryl prodrugs. We focus on specific prodrug families: esters, amidates, and ProTides. We conclude that moderating lipophilicity is a key part of prodrug success. This type of evaluation is important for drug development, regardless of clinical application. It is our hope that this analysis, and future ones like it, will play a significant role in prodrug evolution.

Simultaneous 3-Nitrophenylhydrazine Derivatization Strategy of Carbonyl, Carboxyl and Phosphoryl Submetabolome for LC-MS/MS-Based Targeted Metabolomics with Improved Sensitivity and Coverage.

Metabolomics is a powerful and essential technology for profiling metabolic phenotypes and exploring metabolic reprogramming, which enables the identification of biomarkers and provides mechanistic insights into physiology and disease. However, its applications are still limited by the technical challenges particularly in its detection sensitivity for the analysis of biological samples with limited amount, necessitating the development of highly sensitive approaches. Here, we developed a highly sensitive liquid chromatography tandem mass spectrometry method based on a 3-nitrophenylhydrazine (3-NPH) derivatization strategy that simultaneously targets carbonyl, carboxyl, and phosphoryl groups for targeted metabolomic analysis (HSDccp-TM) in biological samples. By testing 130 endogenous metabolites including organic acids, amino acids, carbohydrates, nucleotides, carnitines, and vitamins, we showed that the derivatization strategy resulted in significantly improved detection sensitivity and chromatographic separation capability. Metabolic profiling of merely 60 oocytes and 5000 hematopoietic stem cells primarily isolated from mice demonstrated that this method enabled routine metabolomic analysis in trace amounts of biospecimens. Moreover, the derivatization strategy bypassed the tediousness of inferring the MS fragmentation patterns and simplified the complexity of monitoring ion pairs of metabolites, which greatly facilitated the metabolic flux analysis (MFA) for glycolysis, the tricarboxylic acid (TCA) cycle, and pentose phosphate pathway (PPP) in cell cultures. In summary, the novel 3-NPH derivatization-based method with high sensitivity, good chromatographic separation, and broad coverage showed great potential in promoting metabolomics and MFA, especially in trace amounts of biospecimens.