Proteomic and phosphoproteomic profiling of urinary small extracellular vesicles in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most prevalent form of primary liver cancer and a major cause of cancer-related mortality worldwide. Small extracellular vesicles (sEVs) are heterogeneous populations of membrane-structured vesicles that can be found in many biological fluids and are currently considered as a potential source of disease-associated biomarkers for diagnosis. The purpose of this study was to define the proteomic and phosphoproteomic landscape of urinary sEVs in patients with HCC. Mass spectrometry-based methods were used to detect the global proteome and phosphoproteome profiles of sEVs isolated by differential ultracentrifugation. Label-free quantitation analysis showed that 348 differentially expressed proteins (DEPs) and 548 differentially expressed phosphoproteins (DEPPs) were identified in the HCC group. Among them, multiple phosphoproteins related to HCC, including HSP90AA1, IQGAP1, MTOR, and PRKCA, were shown to be upregulated in the HCC group. Pathway enrichment analysis indicated that the upregulated DEPPs participate in the regulation of autophagy, proteoglycans in cancer, and the MAPK/mTOR/Rap1 signaling pathway. Furthermore, kinase-substrate enrichment analysis revealed activation of MTOR, AKT1, MAP2Ks, and MAPKs family kinases in HCC-derived sEVs, indicating that dysregulation of the MAPK and mTOR signaling pathways may be the primary sEV-mediated molecular mechanisms involved in the development and progression of HCC. This study demonstrated that urinary sEVs are enriched in proteomic and phosphoproteomic signatures that could be further explored for their potential use in early HCC diagnostic and therapeutic applications.

[Preparation of magnetic carbon nitride composite toward phosphopeptide enrichment].

Protein phosphorylation plays an important role in cellular signaling and disease development. Advances in mass spectrometry-based proteomics have enabled qualitative and quantitative phosphorylation studies as well as in-depth biological explorations for biomarker discovery and signaling pathway analysis. However, the dynamic changes that occur during phosphorylation and the low abundance of target analytes render direct analysis difficult because mass spectral detection offers no selectivity, unlike immunoassays such as Western blot and enzyme-linked immunosorbent assay (ELISA). The present study aimed to solve one of the key problems in the specific and efficient isolation of phosphorylated peptides. A method based on a magnetic carbon nitride composite coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was developed for the enrichment and analysis of phosphopeptides with low abundance in complex samples. Magnetic carbon nitride composite was synthesized and characterized by electron microscopy, infrared spectroscopy, and X-ray diffractometry. The composite showed a well-distributed two-dimensional layered structure and functional groups with excellent paramagnetic performance. Two classical phosphoproteins, namely, α- and ß-caseins, were selected as model phosphorylated samples to assess the performance of the proposed enrichment technique. The magnetic carbon nitride composite exhibited high selectivity and sensitivity for phosphopeptide enrichment. The limit of detection was determined by MALDI-TOF-MS analysis to be 0.1 fmol. The selectivity of the method was investigated using the digest mixtures of α-casein, ß-casein, and bovine serum albumin (BSA) with different mass ratios (1∶1∶1000, 1∶1∶2000, and 1∶1∶5000). Direct analysis of the samples revealed the dominance of spectral signals from the abundant peptides in BSA. After enrichment with the magnetic carbon nitride composite, the high concentration of background proteins was washed away and only the signals of the phosphopeptides were captured. The signals from the casein proteins were clearly observed with little background noise, indicating the high selectivity of the composite material. The robustness of the method was tested by assessing the reusability of the same batch of magnetic carbon nitride materials over 20 cycles of enrichment. The composite showed nearly the same enrichment ability even after several cycles of reuse, demonstrating its potential applicability for a large number of clinical samples. Finally, the method was applied to the analysis of phosphopeptides from several commonly used phosphoprotein-containing samples, including skimmed milk digest, human serum, and human saliva; these samples are significant in the analysis of food quality, disease biomarkers, and liquid biopsies for cancer. Without enrichment, no phosphopeptide was detected because of the high abundance of nonphosphopeptide materials dominating the spectral signals obtained. After pretreatment with the developed magnetic carbon nitride composite, most of the phosphosites were identified with high selectivity and sensitivity via MALDI-TOF-MS. These results revealed the practicality of the developed approach for clinical applications. In addition, our method may potentially be employed for phosphoproteomics with real complex biological samples.

High-throughput proteomic analysis of extracellular vesicles from saliva by chemical probe-based array.

Extracellular vesicles (EVs) are cell-released, nucleus-free particles with a double-membrane structure that effectively prevents degradation of internal components by a variety of salivary enzymes. Saliva is an easily accessible biofluid that contains a wealth of valuable information for disease diagnosis and monitoring and especially reflect respiratory and digestive tract diseases. However, the lack of efficient and high-throughput methods for proteomic analysis of salivary biomarkers poses a significant challenge. Herein, we designed a salivary EV amphiphile-dendrimer supramolecular probe (SEASP) array which enables efficient enrichment and in situ detection of EVs protein biomarkers. Detergent Tween-20 washing of SEASP arrays removes high abundance of heteroproteins from saliva well. This array shows good analytical performance in the linear range of 10 µL-150 µL (LOD = 0.4 µg protein, or 10 µL saliva), exhibiting a good recovery (80.0 %). Compared to ultracentrifugation (UC), this procedure provides simple and convenient access to high-purity EVs (1.3 × 109 particles per mg protein) with good physiological status and structure. Coupling with mass spectrometry based proteomic analysis, differentially expressed proteins as selected asthma biomarkers have been screened. Then, we validated the proteomics primary screening results through clinical samples (100 µL each) using the SEASP array. Utilizing the dual antibody fluorescence technology, SEASP enables the simultaneous high-throughput detection of two proteins. Therefore, the EVs marker protein CD81 could be used as an internal standard to normalize the number of EVs, which was stably expressed in EVs. Proteomics and array results suggested that HNRNPU (P = 4.9 * 10-6) and MUC5B (P = 4.7 * 10-11) are promising protein biomarkers for infantile asthma. HNRNPU and MUC5B may be associated with disease onset and subtypes. The SEASP arrays provide a significant advancement in the field of salivary biomarker. The array enables high-throughput in situ protein detection for highly viscous and complex biological samples. It provides a rapid, low-cost, highly specific screening procedure and experimental basis for early disease screening and diagnosis in the field of liquid biopsy.

Bilateral efforts to improve SERS detection efficiency of exosomes by Au/Na7PMo11O39 Combined with Phospholipid Epitope Imprinting.

Detection of cancer-related exosomes in body fluids has become a revolutionary strategy for early cancer diagnosis and prognosis prediction. We have developed a two-step targeting detection method, termed PS-MIPs-NELISA SERS, for rapid and highly sensitive exosomes detection. In the first step, a phospholipid polar site imprinting strategy was employed using magnetic PS-MIPs (phospholipids-molecularly imprinted polymers) to selectively isolate and enrich all exosomes from urine samples. In the second step, a nanozyme-linked immunosorbent assay (NELISA) technique was utilized. We constructed Au/Na7PMo11O39 nanoparticles (NPs) with both surface-enhanced Raman scattering (SERS) property and peroxidase catalytic activity, followed by the immobilization of CD9 antibodies on the surface of Au/Na7PMo11O39 NPs. The Au/Na7PMo11O39-CD9 antibody complexes were then used to recognize CD9 proteins on the surface of exosomes enriched by magnetic PS-MIPs. Lastly, the high sensitivity detection of exosomes was achieved indirectly via the SERS activity and peroxidase-like activity of Au/Na7PMo11O39 NPs. The quantity of exosomes in urine samples from pancreatic cancer patients obtained by the PS-MIPs-NELISA SERS technique showed a linear relationship with the SERS intensity in the range of 6.21 × 107-2.81 × 108 particles/mL, with a limit of detection (LOD) of 5.82 × 107 particles/mL. The SERS signal intensity of exosomes in urine samples from pancreatic cancer patients was higher than that of healthy volunteers. This bidirectional MIPs-NELISA-SERS approach enables noninvasive, highly sensitive, and rapid detection of cancer, facilitating the monitoring of disease progression during treatment and opening up a new avenue for rapid early cancer screening.

Oriented docking of the template for improved imprinting efficiency toward peptide with modifications.

Molecular imprinting polymers (MIPs) are synthetic receptors as biomimetic materials for various applications ranging from sensing to separation and catalysis. However, currently existing MIPs are stuck to some of the issues including the longer preparation steps and poor performance. In this report, a facile and one-pot strategy by integrating the in-situ growth of magnetic nanoparticles and reversed phase microemulsion oriented molecularly imprinting strategy to develop magnetic molecular imprinted nanocomposites was proposed. Through self-assembling of the template, it brought up highly ordered and uniform arrangement of the imprinting structure, which offered faster adsorption kinetic as adsorption equilibrium was achived within 15 min, higher adsorption capacity (Qmax = 48.78 ± 1.54 µmol/g) and high affinity (Kd = 127.63 ± 9.66 µM) toward paradigm molecule-adenosine monophosphate (AMP) compared to the conventional bulk imprinting. The developed MIPs offered better affinity and superior specificity which allowed the specific enrichment toward targeted phosphorylated peptides from complex samples containing 100-fold more abundant interfering peptides. Interestingly, different types of MIPs can be developed which could targetly enrich the specific phosphorylated peptides for mass spectrometry analysis by simply switching the templates, and this strategy also successfully achieved imprinting of macromolecular peptides. Collectively, the approach showed broad applicability to target specific enrichment from metabolites to phosphorylated peptides and providing an alternative choice for selective recognition and analysis from complex biological systems.

Investigation of rare earth-based magnetic nanocomposites for specific enrichment of exosomes from human plasma.

Exosomes, also known as small extracellular vesicles, are widely present in a variety of body fluids (e.g., blood, urine, and saliva). Exosomes are becoming an alternative promising source of diagnostic markers for disease rich in cargo of metabolites, proteins, and nucleic acids. However, due to the low abundance and structure similarity with protein complex, the efficient isolation of exosomes is one of the most important issues for biomedical applications. With a higher order of f-orbitals in rare earth element, it will have strong adsorption toward the phosphate group on the surface of the phospholipid bilayer of exosomes. In this study, we systematically investigated the ability of various rare earths interacting with phosphate-containing molecules and plasma exosomes. One of the best binding europium was selected and used to synthesize core-shell magnetic nanomaterials (Fe3O4@SiO2@Eu2O3) for the enrichment of exosomes from human plasma. The developed nanomaterials exhibited higher enrichment capacity, less time consumption and more convenient handling compared to commonly used ultracentrifugation method. The nanomaterials were applied to separate exosomes from the plasma of patients with hepatocellular carcinoma and healthy controls for metabolomics study with high-resolution mass spectrometry, where 70 differentially expressed metabolites were identified, involving amino acid and lipid metabolic pathway. We anticipated the rare earth-based materials to be an alternative approach on exosome isolation for disease diagnosis or postoperative clinical monitoring.

Proteomic Discovery and Array-Based Validation of Biomarkers from Urinary Exosome by Supramolecular Probe.

Exosomes are nanoscale, membrane-enclosed vesicles with contents similar to their parent cells, which are rich in potential biomarkers. Urine, as a noninvasive sampling body fluid, has the advantages of being simple to collect, stable in protein, diverse and not regulated by homeostatic mechanisms of the body, making it a favorable target for studying tumor biomarkers. In this report, the urinary exosomal proteome was analyzed and high-throughput downstream validation was performed using a supramolecular probe-based capture and in situ detection. The technology demonstrated the efficient enrichment of exosomes with a high concentration (5.5 × 1010 particles/mL) and a high purity (2.607 × 1010 particles/mg) of exosomes from urine samples. Proteomic analysis of urine samples from patients with hepatocellular carcinoma and healthy individuals combined with proteomic screening techniques revealed that 68 proteins were up-regulated in patients with hepatocellular carcinoma. As a proof-of-principle study, three of these differentially expressed proteins, including OLFM4, HDGF and GDF15, were validated using the supramolecular probe-based array (48 samples per batch). These findings demonstrate the great potential of this approach toward a liquid biopsy for the discovery and validation of biomarkers from urinary exosomes, and it can be extended to various biological samples with lower content of exosomes.

Mesoporous molecularly imprinted nanoparticles with peptide mimics for the detection of phenolic compounds.

Immobilized enzymes outperform free enzymes in many properties and are widely used in environmental monitoring, engineering applications, food and medical fields. Based on the developed immobilization techniques, the search for immobilization with wider applicability, lower cost and more stable enzyme properties is of significant importance. In this study, we reported a molecular imprinting strategy for immobilizing peptide mimics of DhHP-6 on mesoporous materials. The DhHP-6 molecularly imprinted polymer (MIP) showed much higher adsorption capacity than raw mesoporous silica toward DhHP-6. The DhHP-6 peptide mimics was immobilized on the surface of mesoporous silica for the fast detection of phenolic compounds, a widely spread pollutant with highly toxic and difficult in degradation. Immobilized enzyme of DhHP-6-MIP exhibited higher peroxidase activity, better stability, and recyclability than free peptide. Notably, DhHP-6-MIP showed excellent linearity for the detection of the two phenols with detection limits of 0.28 µM and 0.25 µM, respectively. In combination with the spectral analysis and PCA method, DhHP-6-MIP provided better discrimination between the six phenolic compounds (phenol, catechol, resorcinol, hydroquinone, 2-chlorophenol, 2, 4-dichlorophenol). Our study showed that immobilization of peptide mimics by the molecular imprinting strategy using mesoporous silica as carriers was a simple and effective approach. The DhHP-6-MIP has great potentiality for the monitoring and degradation of environmental pollutants.

Epitope Imprinting of Phospholipids by Oriented Assembly at the Oil/Water Interface for the Selective Recognition of Plasma Membranes.

Phospholipids, as fundamental building blocks of the cell membrane, play important roles for molecule transportation, cell recognition, etc. However, due to the structural diversity and amphipathic nature, there are few methods for the specific recognition of lipids as compared to other biomolecules such as proteins and glycans. Herein, we developed a molecular imprinting strategy for controllable imprinting toward the polar head of phospholipid exposed on the surface of cellular membranes for recognition. Phosphatidylserine, as unique lipid on the outer membrane leaflet of exosome and also hallmark for cell apoptosis, was imprinted with the developed method. The phosphatidylserine imprinted materials showed high efficiency and specific targeting capability not only for apoptotic cell imaging but also for the isolation of exosomes. Collectively, the synthesized molecularly imprinted materials have great potential for selective plasma membrane recognition for targeted drug delivery and biomarker discovery.

Glycoproteomic Analysis of Urinary Extracellular Vesicles for Biomarkers of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) accounts for the most common form of primary liver cancer cases and constitutes a major health problem worldwide. The diagnosis of HCC is still challenging due to the low sensitivity and specificity of the serum α-fetoprotein (AFP) diagnostic method. Extracellular vesicles (EVs) are heterogeneous populations of phospholipid bilayer-enclosed vesicles that can be found in many biological fluids, and have great potential as circulating biomarkers for biomarker discovery and disease diagnosis. Protein glycosylation plays crucial roles in many biological processes and aberrant glycosylation is a hallmark of cancer. Herein, we performed a comprehensive glycoproteomic profiling of urinary EVs at the intact N-glycopeptide level to screen potential biomarkers for the diagnosis of HCC. With the control of the spectrum-level false discovery rate ≤1%, 756 intact N-glycopeptides with 154 N-glycosites, 158 peptide backbones, and 107 N-glycoproteins were identified. Out of 756 intact N-glycopeptides, 344 differentially expressed intact N-glycopeptides (DEGPs) were identified, corresponding to 308 upregulated and 36 downregulated N-glycopeptides, respectively. Compared to normal control (NC), the glycoproteins LG3BP, PIGR and KNG1 are upregulated in HCC-derived EVs, while ASPP2 is downregulated. The findings demonstrated that specific site-specific glycoforms in these glycoproteins from urinary EVs could be potential and efficient non-invasive candidate biomarkers for HCC diagnosis.

Supramolecular Exosome Array for Efficient Capture and In Situ Detection of Protein Biomarkers.

Exosomes are an emerging source for disease biomarker discovery due to the high stability of proteins protected by phospholipid bilayers. However, liquid biopsy with exosomes remains challenging due to the extreme complexity of biological samples. Herein, we introduced an amphiphile-dendrimer supramolecular probe (ADSP) for the efficient capture and high-throughput analysis of exosomes, enabling the array-based assay for marker proteins. Amphiphilic amphotericin B was functionalized onto highly branched globular dendrimers, which can then insert into the exosome membrane efficiently, forming a supramolecular complex through multivalent interactions between the probe and the bilayer of exosomes. The ADSP can be easily coated onto magnetic beads or the nitrocellulose membrane, facilitating the capture of exosomes from a minimum amount of clinical samples. The captured exosomes can be detected with target protein antibodies via Western blotting or in a high-throughput array-based dot blotting format. This new strategy exhibited excellent extraction capability from trace body fluids with superior sensitivity (less than 1 µL plasma), good quantitation ability (R2 > 0.99), and high throughput (96 samples in one batch) using clinical plasma samples. The combination of proteomics and ADSP will provide a platform for the discovery and validation of protein biomarkers for cancer diagnosis and prognosis.

Improved performance of proteomic characterization for Panax ginseng by strong cation exchange extraction and liquid chromatography-mass spectrometry analysis.

Panax ginseng is a precious and ancient medicinal plant. The completion of its genome sequencing has laid the foundation for the study of proteome and peptidome. However, the high abundance of secondary metabolites in ginseng reduces the identification efficiency of proteins and peptides in mass spectrometry. In this report, strong cation exchange pretreatment was carried out to eliminate the interference of impurities. Based on the charge separation of proteolytic peptides and metabolites, the sensitivity of mass spectrometry detection was greatly improved. After pretreatment, 2322 and 2685 proteins were identified from the root and stem leaf extract. Further, the ginseng peptidome was analyzed based on this optimized strategy, where 970 and 653 endogenous peptides were identified from root and stem leaf extract, respectively. Functional analysis of proteins and endogenous peptides provided valuable information on the biological activities, metabolic processes, and ginsenoside biosynthesis pathways of ginseng.

SERS Resolving of the Significance of Acetate on the Enhanced Catalytic Activity of Nanozymes.

Understanding the structure-activity correlation and reaction mechanism of the catalytic process in an acetic acid-sodium acetate (HAc-NaAc) buffer environment is crucial for the design of efficient nanozymes. Here, we first reported a lattice restructuration of Au-LaNiO3-δ nanofibers (NFs) after acidification with the HAc-NaAc buffer to show a significantly enhanced oxidase-like property. Surface-enhanced Raman spectroscopy (SERS) and density functional theory (DFT) calculation confirm the direct evidence for the formation of specific enhanced intermediate O-O species after acidification, indicating that the insertion of the carboxyl group in the A-Au/LaNiO3-δ NFs plays crucial roles in both producing vacancies in HAc-NaAc solution from its dissociation during the catalytic process and the protection of the vacancies, which can be directly interacted with oxygen in the environment to produce O-O species, realizing the enhanced oxidation of substrate molecules. The insertion of the carboxyl group increased the oxidase-like catalytic activity by 2.38 times and the SERS activity by 5.27 times. This strategy offers a way to construct an efficient nanozyme-linked immunosorbent assay system for the diagnosis of cancer through the highly sensitive SERS identification of exosomes.

Highly effective identification of drug targets at the proteome level by pH-dependent protein precipitation.

Fully understanding the target spaces of drugs is essential for investigating the mechanism of drug action and side effects, as well as for drug discovery and repurposing. In this study, we present an energetics-based approach, termed pH-dependent protein precipitation (pHDPP), to probe the ligand-induced protein stability shift for proteome-wide drug target identification. We demonstrate that pHDPP works for a diverse array of ligands, including a folate derivative, an ATP analog, a CDK inhibitor and an immunosuppressant, enabling highly specific identification of target proteins from total cell lysates. This approach is compared to thermal and solvent-induced denaturation approaches with a pan-kinase inhibitor as the model drug, demonstrating its high sensitivity and high complementarity to other approaches. Dihydroartemisinin (DHA), a dominant derivative of artemisinin to treat malaria, is known to have an extraordinary effect on the treatment of various cancers. However, the anti-tumor mechanisms remain unknown. pHDPP was applied to reveal the target space of DHA and 45 potential target proteins were identified. Pathway analysis indicated that these target proteins were mainly involved in metabolism and apoptosis pathways. Two cancer-related target proteins, ALDH7A1 and HMGB1, were validated by structural simulation and AI-based target prediction methods. And they were further validated to have strong affinity to DHA by using cellular thermal shift assay (CETSA). In summary, pHDPP is a powerful tool to construct the target protein space to reveal the mechanism of drug action and would have broad application in drug discovery studies.

A Tyrosine Phosphoproteome Analysis Approach Enabled by Selective Dephosphorylation with Protein Tyrosine Phosphatase.

Protein tyrosine phosphorylation (pTyr) plays a prominent role in signal transduction and regulation in all eukaryotic cells. In conventional immunoaffinity purification (IP) methods, phosphotyrosine peptides are isolated from the digest of cellular protein extracts with a phosphotyrosine-specific antibody and are identified by tandem mass spectrometry. However, low sensitivity, poor reproducibility, and high cost are universal concerns for IP approaches. In this study, we presented an antibody-free approach to identify phosphotyrosine peptides by using protein tyrosine phosphatase (PTP). It was found that most of the PTPs including PTP1B, TCPTP, and SHP1 can efficiently and selectively dephosphorylate phosphotyrosine peptides. We then designed a workflow by combining two Ti4+-IMAC-based phosphopeptide enrichment steps with PTP-catalyzed dephosphorylation for tyrosine phosphoproteomics analysis. This workflow was first validated by selective detection of phosphotyrosine peptides from semicomplex samples and then applied to analyze the tyrosine phosphoproteome of Jurkat T cells. Around 1000 putative former phosphotyrosine peptides were identified from less than 500 µg of cell lysate. The tyrosine phosphosites on the majority of these peptides could be unambiguously determined for over 70% of them possessing only one tyrosine residue. It was also found that the tyrosine sites identified by this method were highly complementary to those identified by the SH2 superbinder-based method. Therefore, the combination of Ti4+-IMAC enrichment with PTP dephosphorylation provides an alternative and cost-effective approach for tyrosine phosphoproteomics analysis.

Molecular imprinting of glycoproteins: From preparation to cancer theranostics.

Glycoprotein imprinted polymers have rapidly grown as excellent receptors for cancer targeting, diagnostics, inhibition, and nanomedicines as they specifically target glycans and glycosites overexpressed in various tumors. Compared to natural antibodies, they are easy to synthesize, stable, and cost-efficient. Currently, no study specifically discusses glycoproteins imprinting strategies for cancer theranostics. In this review, firstly we explored various factors involved in designing and synthesis of glycoprotein imprinted materials, including, the characteristics and choice of monomers for imprinting, types of templates and their interactions involved, and the imprinting methods. Secondly, the integration of these MIPs with different probes that have been applied for in vitro and in vivo targeting for cancer diagnostics including biosensing and bioimaging, and image-guided therapeutic applications as nanomedicines. These Glycoprotein imprinted polymers have been found to specifically target the glycoprotein biomarkers and glycosylated cell receptors overexpressed in different cancers and have been reported as excellent diagnostic tools. As nanomedicines, they have been potentially employed in various modes of cancer therapy such as targeted drug delivery, photodynamic therapy, photothermal therapy, and nanoMIPs themselves as therapeutics for locally killing tumor cells. Although the research is still in its early stages and no real-world clinical trials on humans have been conducted, nanoMIPs have a promising future in this field. We believe these findings will pave the way for MIPs in advanced diagnostics, antibody treatment, and immunotherapy as future nanomedicine for real-world cancer theranostics.

Probing the methotrexate-protein interactions by proteomics and thermostability assay for drug resistance study.

Screening of drug targets is critical to understand the mechanism of action of the drug. The aim of this study is to screen the drug-resistant target proteins of the anticancer drug methotrexate (MTX) by using chemical proteomics and to further study the interaction between MTX and its target protein in vitro and in vivo according to the principle of the increasing thermal stability of the target protein after binding with the drug molecule. The results showed that 21 drug resistance related proteins of MTX including phosphoglycerate kinase 1 (PGK1) were detected by quantitative proteomics. The expression of PGK1 increased with the prolongation of incubation time of MTX, indicating PGK1 protein is affected by MTX time dependently in cells. Further the results of the study on the interaction between MTX and PGK1 in vitro and in vivo using cellular thermal shift assay (CETSA) showed that the level of PGK1 in MTX-treated groups was higher than that in the control group under the stimulation of higher temperature conditions, indicating that PGK1 has direct interactions with MTX. The present study provided the data and theoretical support for the study of the resistant target proteins of MTX and a novel point for the extension application of MTX.

A Simplified Thermal Proteome Profiling Approach to Screen Protein Targets of a Ligand.

Thermal proteome profiling is a powerful energetic-based chemical proteomics method to reveal the ligand-protein interaction. However, the costly multiplexed isotopic labeling reagent, mainly Multiplexed isobaric tandem mass tag (TMT), and the long mass spectrometric time limits the wide application of this method. Here a simple and cost-effective strategy by using dimethyl labeling technique instead of TMT labeling is reported to quantify proteins and by using the peptides derived from the same protein to determine significantly changed proteins in one LC-MS run. This method is validated by identifying the known targets of methotrexate and geldanamycin. In addition, several potential off-targets involved in detoxification of reactive oxygen species pathway are also discovered for geldanamycin. This method is further applied to map the interactome of adenosine triphosphate (ATP) in the 293T cell lysate by using ATP analogue, adenylyl imidodiphosphate (AMP-PNP), as the ligand. As a result, a total of 123 AMP-PNP-sensitive proteins are found, of which 59 proteins are stabilized by AMP-PNP. Approximately 53% and 20% of these stabilized candidate protein targets are known as ATP and RNA binding proteins. Overall, above results demonstrated that this approach could be a valuable platform for the unbiased target proteins identification with reduced reagent cost and mass spectrometric time.

Analysis of therapeutic monoclonal antibody glycoforms by mass spectrometry for pharmacokinetics study.

Monoclonal antibodies (mAbs), are one of the most important protein drugs have attracted increasing attention. However, the pharmacokinetics of mAbs has not been fully investigated due to the complexity of protein drugs. Traditonal immuno-based approaches can not recognize the proteoforms of mAbs because of the long development cycles, prohibitive cost, and interactions between different proteins. Therefore, reliable qualitative and quantitative analysis of the proteoforms of mAbs in biological samples is of crucial importance. Herein, a novel method was developed for absolute quantitation of mAbs and their glycoforms in complex biological samples such as serum and tissues. With the combination of HILIC enrichment and parallel reaction monitoring by high resolution mass spectrometry, most of the glycoforms can be accurately quantified at the fmol level through the use of the model mAb of bevacizumab. More importantly, the structural confirmation can be achieved simultaneously without the need for additional experiments. This strategy can be readily applied to the pharmacokinetic study of glycosylation modification and biomarker discovery for clinical applications.

Gomisin A is a Novel Isoform-Specific Probe for the Selective Sensing of Human Cytochrome P450 3A4 in Liver Microsomes and Living Cells.

Nearly half of prescription medicines are metabolized by human cytochrome P450 (CYP) 3A. CYP3A4 and 3A5 are two major isoforms of human CYP3A and share most of the substrate spectrum. A very limited previous study distinguished the activity of CYP3A4 and CYP3A5, identifying the challenge in predicting CYP3A-mediated drug clearance and drug-drug interaction. In the present study, we introduced gomisin A (GA) with a dibenzocyclooctadiene skeleton as a novel selective probe of CYP3A4. The major metabolite of GA was fully characterized as 8-hydroxylated GA by LC-MS and NMR. CYP3A4 was assigned as the predominant isozyme involved in GA 8-hydroxylation by reaction phenotyping assays, chemical inhibition assays, and correlation studies. GA 8-hydroxylation in both recombinant human CYP3A4 and human liver microsomes followed classic Michaelis-Menten kinetics. The intrinsic clearance values indicated that CYP3A4 contributed 12.8-fold more than CYP3A5 to GA 8-hydroxylation. Molecular docking studies indicated different hydrogen bonds and π-π interactions between CYP3A4 and CYP3A5, which might result in the different catalytic activity for GA 8-hydroxylation. Furthermore, GA exhibited a stronger inhibitory activity towards CYP3A4 than CYP3A5, which further suggested a preferred selectivity of CYP3A4 for the transformation of GA. More importantly, GA has been successfully applied to selectively monitor the modulation of CYP3A4 activities by the inducer rifampin in hepG2 cells, which is consistent with the level change of CYP3A4 mRNA expression. In summary, our results suggested that GA could be used as a novel probe for the selective sensing of CYP3A4 in tissue and cell preparations.