Biotin-streptavidin Interaction Research Articles

Click reaction-assisted construction of antifouling immunosensors for electrochemical detection of cancer biomarkers in human serum

Herein, an electrochemical immunosensor capable of detecting targets in human serum with ultralow fouling and high sensitivity was successfully constructed through a click reaction. One designed biotinylated peptide was associated with the other peptide functionalized with biotin and azide groups at its two terminals, to form a branched antifouling peptide through the streptavidin-biotin affinity interaction, and it was attached to the electrode to construct an antifouling interface. Then, the exposed azide group on the branched peptide was used to link the antibody modified with 5′-dibenzocyclooctyne (DBCO), and the immunosensor was thus constructed with the aid of the click reaction between the azide group and the DBCO. The developed antifouling immunosensor demonstrated a wide linear response range for carcinoembryonic antigen (CEA) and a low limit of detection of 40 fg mL−1, which is much superior to the sensing performance of the immunosensor prepared through the normal EDC/NHS method. The immunosensor was capable of assaying CEA in undiluted serum samples with satisfying accuracy when compared with a clinically approved method. This work offered an effective strategy to develop various biosensors through the conjugation of antifouling peptides with different antibodies.

Adenovirus Fibers as Ultra-Stable Vehicles for Intracellular Nanoparticle and Protein Delivery.

Protein-based carriers are promising vehicles for the intracellular delivery of therapeutics. In this study, we designed and studied adenovirus protein fiber constructs with potential applications as carriers for the delivery of protein and nanoparticle cargoes. We used as a basic structural framework the fibrous shaft segment of the adenovirus fiber protein comprising of residues 61–392, connected to the fibritin foldon trimerization motif at the C-terminal end. A fourteen-amino-acid biotinylation sequence was inserted immediately after the N-terminal, His-tagged end of the construct in order to enable the attachment of a biotin moiety in vivo. We report herein that this His-tag biotinylated construct folds into thermally and protease-stable fibrous nanorods that can be internalized into cells and are not cytotoxic. Moreover, they can bind to proteins and nanoparticles through the biotin–streptavidin interaction and mediate their delivery to cells. We demonstrate that streptavidin-conjugated gold nanoparticles can be transported into NIH3T3 fibroblast and HeLa cancer cell lines. Furthermore, two streptavidin-conjugated model proteins, alkaline phosphatase and horseradish peroxidase can be delivered into the cell cytoplasm in their enzymatically active form. This work is aimed at establishing the proof-of-principle for the rational engineering of diverse functionalities onto the initial protein structural framework and the use of adenovirus fiber-based proteins as nanorods for the delivery of nanoparticles and model proteins. These constructs could constitute a stepping stone for the development of multifunctional and modular fibrous nanorod platforms that can be tailored to applications at the sequence level.

Paper-Based Progesterone Sensor Using an Allosteric Transcription Factor.

Progesterone monitoring is an essential component of in vitro fertilization treatments and reproductive management of dairy cows. Gold-standard biosensors for progesterone monitoring rely on antibodies, which are expensive and difficult to procure. We have developed an alternative transcription factor-based sensor that is superior to conventional progesterone biosensors. Here, we incorporate this transcription factor-based progesterone sensor into an affordable, portable paperfluidic format to facilitate widespread implementation of progesterone monitoring at the point of care. Oligonucleotides labeled with a fluorescent dye are immobilized onto nitrocellulose via a biotin–streptavidin interaction. In the absence of progesterone, these oligonucleotides form a complex with a transcription factor that is fluorescently labeled with tdTomato. In the presence of progesterone, the fluorescent transcription factor unbinds from the immobilized DNA, resulting in a decrease in tdTomato fluorescence. The limit of detection of our system is 27 nm, which is a clinically relevant level of progesterone. We demonstrate that transcription factor-based sensors can be incorporated into paperfluidic devices, thereby making them accessible to a broader population due to the portability and affordability of paper-based devices.

Immobilization of β-Galactosidase by Encapsulation of Enzyme-Conjugated Polymer Nanoparticles Inside Hydrogel Microparticles.

Increasing the shelf life of enzymes and making them reusable is a prominent topic in biotechnology. The encapsulation inside hydrogel microparticles (HMPs) can enhance the enzyme’s stability by preserving its native conformation and facilitating continuous biocatalytic processes and enzyme recovery. In this study, we present a method to immobilize β-galactosidase by, first, conjugating the enzyme onto the surface of polymer nanoparticles, and then encapsulating these enzyme-conjugated nanoparticles (ENPs) inside HMPs using microfluidic device paired with UV-LEDs. Polymer nanoparticles act as anchors for enzyme molecules, potentially preventing their leaching through the hydrogel network especially during swelling. The affinity binding (through streptavidin-biotin interaction) was used as an immobilization technique of β-galactosidase on the surface of polymer nanoparticles. The hydrogel microparticles of roughly 400 μm in size (swollen state) containing unbound enzyme and ENPs were produced. The effects of encapsulation and storage in different conditions were evaluated. It was discovered that the encapsulation in acrylamide (AcAm) microparticles caused an almost complete loss of enzymatic activity. Encapsulation in poly(ethylene glycol) (PEG)-diacrylate microparticles, on the other hand, showed a residual activity of 15–25%, presumably due to a protective effect of PEG during polymerization. One of the major factors that affected the enzyme activity was presence of photoinitiator exposed to UV-irradiation. Storage studies were carried out at room temperature, in the fridge and in the freezer throughout 1, 7 and 28 days. The polymer nanoparticles showcased excellent immobilization properties and preserved the activity of the conjugated enzyme at room temperature (115% residual activity after 28 days), while a slight decrease was observed for the unbound enzyme (94% after 28 days). Similar trends were observed for encapsulated ENPs and unbound enzyme. Nevertheless, storage at −26°C resulted in an almost complete loss of enzymatic activity for all samples.

A ratiometric fluorescent nanoprobe for signal amplification monitoring of intracellular telomerase activity.

Telomerase is considered a valuable diagnostic and prognostic cancer biomarker. Accurate and reliable detection of telomerase activity is of great value in clinical diagnosis, screening of inhibitors, and therapeutics. Here, we developed a novel amplified fluorescence resonance energy transfer (FRET) nanoprobe for highly sensitive and reliable monitoring of intracellular telomerase activity. The nanoprobe (QDSA@DNA) was composed of a streptavidin-modified quantum dot (QDSA) which was functionalized with a telomerase primer sequence (TP) and Cy5-tagged signal switching sequence (SS) through biotin-streptavidin interaction. When the nanoprobe was assembled, the Cy5 was in close proximity to the QDSA, resulting in high FRET efficiency from the QDSA to Cy5. In the presence of telomerase, the TP could be extended to produce telomeric repeat units, which was complementary to the loop of SS. Thus, the SS could hybridize with elongated sequences to form a rigid double-stranded structure, which forced the Cy5 away from the surface of the QDSA, causing low FRET efficiency. Furthermore, due to the production of multiple repeat units by telomerase, multiple hairpin structures could be opened, yielding significant fluorescence ratio (FQDsa/FCy5) enhancement for sensing of telomerase activity. In this way, the combination of a FRET and target-assisted strategy in a nanoprobe improved the detection accuracy and amplified the detection signal, respectively. The QDSA@DNA nanoprobe also showed high selectivity, excellent nuclease stability, and good biocompatibility. More importantly, this nanoprobe was found to be an excellent platform for efficient monitoring of intracellular telomerase activity, providing a potential platform in tumor diagnosis and screening of telomerase-related inhibitors.

A labeling strategy with effective preservation of fluorophores for expansion single-molecule localization microscopy (Ex-SMLM).

Expansion microscopy (ExM) significantly improves the resolution of conventional diffraction-limited optical microscopy by using physically expanding biological samples. Combining ExM with single-molecule localization microscopy (SMLM) could further enhance the resolving power of SMLM, which is typically in the order of 20-30 nm. However, to make this combination successful, we need to solve three key issues related to sample preparation, including mainly hydrogel shrinking in an ionic photoswitching buffer, fluorescence photobleaching due to a free-radical reaction and reduced labelling efficiency from protease digestion. Re-embedding polyacrylamide gel or using an improved photoswitching buffer with a low ionic strength is able to minimize or even solve the hydrogel shrinking problem, while the development of post-expansion labelling approaches avoids fluorescence bleaching. However, the preservation of protein epitopes (which determines the labelling efficiency) remains to be challenging. In this paper, we propose to tackle this challenge by introducing the highly selective and stable biotin-streptavidin interaction into the post-expansion labelling strategy. After upgrading the popular immunolabelling linkage scheme from Epitope-Primary antibody-Secondary antibody-Fluorophores to Epitope-Primary antibody-Secondary antibody-Biotin-Streptavidin-Fluorophores, we were able to label protein epitopes with biotin, which was stable during the expansion process, and thus avoid the troublesome problem in preserving protein epitopes or antibodies. We demonstrate that combining Ex-SMLM with the new post-expansion linkage scheme enables new possibilities in resolving the detailed arrangement of Nup133 proteins in the nuclear pore complex, which helps researchers to observe a clearer structure. This study provides new opportunities for studying the ultrastructural details of subcellular organelles or even biomacromolecules, using the conventional SMLM system.

Nanowired dual-electrodes surface to monitor cerebral ischemia by current-volt measurements.

The level of clotting protein 'factor IX' (FIX) is highly associated with cerebral ischemia, and this research work has developed a sensitive detection of FIX on dielectrode sensor by current-volt measurement. Sensing area was grown with zinc oxide nanowire to attach more probe for FIX interaction. Aptamer was utilized as the detection probe and attached on the sensing electrode surface through amine-aldehyde chemical linkage. In addition, biotin-streptavidin interaction was utilized to attach the higher number aptamers on the electrode surface connected with dual-probe station. FIX detection limit was found as 10 fM in the phosphate buffer saline spiked samples and 1:320 dilution of human serum. The linear ranges were as 10 fM to 100pM and 1:320 to 1:80, respectively. With a good determination co-efficient [y = 2.6813x-3.8467; R 2 = 0.9479] this biosensing strategy helps to quantify FIX and monitor the condition of cerebral ischemia.

Abstract 9749: High-Avidity Binding Drives Nucleation of Amyloidogenic Transthyretin Monomer in Cardiac Amyloidosis

Amyloidosis involves stepwise growth of fibrils assembled from soluble precursor proteins. Mutations of plasma carrier protein transthyretin (TTR) are associated with hereditary transthyretin amyloidosis (ATTR). TTR naturally folds into a stable tetramer for its function, whereas conditions and mutations that foster aberrant monomer formation facilitate TTR oligomeric aggregation and subsequent fibril extension. We investigated the dynamics of early assembly of oligomers by wild-type TTR, in comparison to its TTR V30M and TTR V122I disease mutants. We selected buffer conditions for monitoring time-dependent redistribution among monomer, dimer, tetramer, and oligomer contents in the presence and absence of multimeric TTR seeds. The seeds were artificially constructed recombinant multimers that contained 20-40 TTR subunits via engineered biotin-streptavidin interactions. As expected, these multimer seeds rapidly nucleated TTR monomers and dimers into larger complexes, while having no effect on tetramers. In vivo , streptavidin-induced multimers formed ATTR-like deposits in the heart and the kidney following i.v. injection in mice. While all three variants prominently deposited in the kidney glomerulus, only TTR V30M resulted in extensive deposition in the heart. The cardiac TTR deposits varied in size and shape and were localized to the intermyofibrillar space along the capillaries. These results are consistent with the notion of monomeric TTR to engage high-avidity interactions with tissue amyloids. Our multimeric induction approach therefore provides a new model for studying the initiation of TTR deposition in the heart.

Short- and long-term effect of colorectal cancer targeting peptides conjugated to gold nanoparticles in rats' liver and colon after single exposure.

Peptides play important roles in the diagnosis, prognostic predictors, and treatment of various kinds of cancer. Peptides (p.C, p.L and p.14), derived from the phage display peptide libraries, specifically binds to colorectal cancer (CRC) cells in vitro. To allow tumor specificity and selectivity for in vivo diagnosis of CRC, biotinylated p.C, p.L and p.14 were conjugated to AuNPs (14nm) via the biotin-streptavidin interaction. Male Wistar rats were intravenously injected with a single dose (100µg/kg body weight) of AuNPs (citrate-AuNPs, PEG-AuNPs, p.C-PEG-, p.L-PEG- and p.14-PEG-AuNPs). Animals were monitored for behavioral changes, and sacrificed either 14days or 84days post-injection. Biochemical changes, oxidative stress, and histology of the liver and colon were assessed. No significant changes were noted in the rats injected with all the AuNPs, except p.L-PEG-AuNPs that caused significant toxicity (p < 0.05) 14days post-exposure when compared to control group, as evidenced by increased relative liver weight, increased malondialdehyde levels and histological changes in the liver. These changes, however, returned to normalcy 84days post-injection. It can be concluded, based on these findings, that p.L induced a transient toxicity in rats after a single intravenous injection, and can therefore be considered non-toxic long-term after a single exposure.

Near-Infrared Fluorescence Immunoassay Using Streptavidin-Conjugated Oxygen-Doped Carbon Nanotubes

Single-walled carbon nanotubes (CNTs) have been introduced to near-infrared (NIR) immunoassay systems for recent years. NIR lights have low absorption and scattering of biological matter and avoid autofluorescence from blood. Compared with other NIR molecules and materials, CNTs have several advantages such as strong emissions, large Stokes-shifts and high photo-stabilities as NIR labels. Previously, our research group reported that immunoprecipitation (IP) was successfully conducted between IgG-conjugated CNTs and protein G magnetic beads. Although the system can ideally measure target analytes at concentrations as low as 600 pmol/L, the practical reaction efficiency was insufficient, around ~18% [1] .To improve the IP reaction yield, we here made the streptavidin-conjugated oxygen-doped CNTs (o-CNT-SA) that can selectively bind with biotin molecules. The o-CNTs [3] were coated by streptavidin which combined with phospholipid polyethylene glycol (SA-PEG). While IgG forms a 1:1 complex with an antigen, SA can bind biotin molecules at four sites. The strength of streptavidin-biotin interaction is larger than antigen-antibody interaction, therefore it expects that the reaction efficiency become larger.Some amounts of phospholipid polyethylene glycol (PL-PEG) were added to stabilize the dispersion state of o-CNT-SA. For assessing the IP reactivity, the fluorescent intensity ratio between the eluted solution and the original o-CNT-SA dispersion was calculated (Fig. 1(a)). Figure 1(b) shows the relationship of the reaction efficiency and SA-PEG/PL-PEG concentrations. The contour lines indicate the reaction efficiency. At around ~50 µg/mL of SA-PEG concentration, the reaction efficiency shows the maximum. On the other hand, the reaction efficiency is better when the PL-PEG concentration is low while o-CNTs aggregate occurs at the PL-PEG concentration of ~144 µg/mL. Figure 1(c) shows the NIR fluorescence spectra observed from the eluted and the original o-CNT-SA solutions. By adjusting the concentrations of o-CNTs, SA-PEG and PL-PEG, the IP reaction yield increased up to 79.3 %. In this condition, molecular number ratio of o-CNT and SA-PEGs was estimated to be 1 : 11 – 46. Comparing the length of o-CNT, 500 nm, with the diameter of streptavidin, 8.2 nm, it expects that SA-PEGs adsorb on almost all surface of o-CNT. In the poster, we will show the detailed experimental procedures and discuss the proper structure of the o-CNT-SA NIR label based on the obtained results.[1] Y. Iizumi et al., ACS Appl. Mater. Interfaces, 5, 7665 (2013).[2] Y. Iizumi et al., Sci. Rep., 8, 6272 (2018). Figure 1

Nets of biotin-derived gold nanoparticles as a label for the C-reactive protein immunoassay.

This study presents a promising approach for the one-pot generation of the biotin-derived gold nanoparticles (GNPs@biotin). We report a direct method for the reduction of tetrachloroauric acid with biotin and generation of the labels due to nets formed via biotin-streptavidin interactions. The synthesized GNPs@biotin have a characteristic plasmon maximum, excellent colloidal stability, and streptavidin coupling efficiency. The size of the GNPs@biotin:streptavidin nets and the efficiency of interaction with specific antibodies can be easily customized by the component concentrations and time of their interaction. Moreover, the proposed labels require no additional reagents or manipulations for the synthesis, separation, or purification. The developed labels were applied for the detection of the model antigen of C-reactive protein (CRP) as a major inflammation biomarker. The assembling labels demonstrated a competitive advantage limit of CRP detection (LOD) of 1.2ng/mL and a limit of quantification (LOQ) of 3.9ng/mL in human plasma comparable to classical immunoassays. Moreover, the proposed approach is universal and can be potentially applied for the quantitative determination of other biomarkers in a variety of immunoassays in a combination with specific biotinylated antibodies.

Development of a multi-layering protein grafting process on miniaturized monolithic columns for weak affinity nano liquid chromatography application purposes

Affinity chromatography is a powerful technique to identify and quantify weak ligand-protein interactions (Kd in the range of mM to 0.1µM). In some fields such as Fragment Based Drug Discovery, the detection of very weak affinities (mM) is of utmost importance since weak ligands can be good starting points for the conception of high affinity ligands. However, the identification of such weak ligands can be hampered by the limited bulk density of active target grafted onto the support. At the same time, downscaling the chromatographic column is of utmost interest when scarce and/or expensive proteins are targeted. In this context, we herein present a novel approach of protein immobilization to improve the bulk density of active protein grafted onto organic capillary monolithic columns. The proposed approach is based on the streptavidin-biotin interaction and consists of successive grafting steps of biotinylated target protein onto streptavidin layers through a multi-layering process. Concanavalin A was used as model protein. The study focuses on the optimization of the grafting conditions to maximize the amount of active protein during the multi-layering process and highlights the impact of the biotinylation ratio of the protein. It is demonstrated that a 3-layer grafting process allows to improve the bulk density of active sites by a 2-fold factor compared to a single layer. This improvement in protein density allows to increase the affinity range of this technique to the millimolar range.

DNAzyme-Based Biosensors: Immobilization Strategies, Applications, and Future Prospective.

Since their discovery almost three decades ago, DNAzymes have been used extensively in biosensing. Depending on the type of DNAzyme being used, these functional oligonucleotides can act as molecular recognition elements within biosensors, offering high specificity to their target analyte, or as reporters capable of transducing a detectable signal. Several parameters need to be considered when designing a DNAzyme-based biosensor. In particular, given that many of these biosensors immobilize DNAzymes onto a sensing surface, selecting an appropriate immobilization strategy is vital. Suboptimal immobilization can result in both DNAzyme detachment and poor accessibility toward the target, leading to low sensing accuracy and sensitivity. Various approaches have been employed for DNAzyme immobilization within biosensors, ranging from amine and thiol-based covalent attachment to non-covalent strategies involving biotin-streptavidin interactions, DNA hybridization, electrostatic interactions, and physical entrapment. While the properties of each strategy inform its applicability within a proposed sensor, the selection of an appropriate strategy is largely dependent on the desired application. This is especially true given the diverse use of DNAzyme-based biosensors for the detection of pathogens, metal ions, and clinical biomarkers. In an effort to make the development of such sensors easier to navigate, this paper provides a comprehensive review of existing immobilization strategies, with a focus on their respective advantages, drawbacks, and optimal conditions for use. Next, common applications of existing DNAzyme-based biosensors are discussed. Last, emerging and future trends in the development of DNAzyme-based biosensors are discussed, and gaps in existing research worthy of exploration are identified.

Design and Evaluation of Multiplex One-Step Reverse Transcription PCR-Dipstick Chromatography Method for the Analysis of Seven Respiratory Pathogens.

Influenza A, influenza B, severe acute respiratory syndrome coronavirus 2, adenovirus, respiratory syncytial virus, Mycoplasma pneumoniae, and Chlamydophila pneumoniae are common pathogens that can cause severe pneumonia and other symptoms, resulting in acute lower respiratory tract infections. The objective of this study was to design and evaluate a sensitive and specific multiplex one-step reverse transcription PCR (RT-PCR)–dipstick chromatography method for simultaneous rapid detection of these seven pathogens. Streptavidin-coated blue latex particles were used to read out a positive signal. Based on the DNA–DNA hybridization of oligonucleotide sequences (Tag) for forward primer with the complementary oligonucleotide sequence (cTag) on the dipstick and biotin–streptavidin interactions, PCR products were able to be illuminated visually on the dipstick. The specificity and the limit of detection (LOD) were also evaluated. Moreover, the clinical performance of this method was compared with Sanger sequencing for 896 samples. No cross reaction with other pathogens was found, confirming the high specificity of this method. The LOD was 10 copies/µL for each of the tested pathogens, and the whole procedure took less than 40 min. Using 896 samples, the sensitivity and specificity were shown to be no lower than 94.5%. The positive predictive value was higher than 82.1%, and the negative predictive value was higher than 99.5%. The kappa value between the PCR–dipstick chromatography method and Sanger sequencing ranged from 0.869 to 0.940. In summary, our one-step RT-PCR–dipstick chromatography method is a sensitive and specific tool for rapidly detecting multiplex respiratory pathogens.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00284-021-02621-7.

Magnetic hyperthermia mediated by Escherichia coli for targeted cancer therapy

Heterogeneous cancers are usually recalcitrant to the conventional treatment method involving chemotherapy and radiotherapy. This issue was addressed by development of magnetic hyperthermia therapy (MHT) based on Escherichia coli. To be functional, E. coli was constructed to display the bivalent anti-HER2 affibody (denoted ZH2) on cell surface. The ZH2-displayed strain was further equipped with the biotin-binding motif (AP) which was anchored on the inner leaflet of cell membrane. After permeabilization, the engineered E. coli was loaded with streptavidin-linked magnetic nanoparticles (SiMNPs). The biotinylated AP tethers it to SiMNPs through the biotin-streptavidin interaction. As a result, SiMNPs-loaded E. coli was selectively internalized by HER2-positive cancer cells. Following the application of alternating magnetic field, the viability of cancer cells decreased to 20%. The administration in vivo also significantly caused tumor regression in mice xenografted with human ovarian cancer cells. Overall, this study proposes a new strategy of targeted cancer therapy based on the E. coli-mediated MHT.

Construction of a Dye-Sensitized and Gold Plasmon-Enhanced Cathodic Photoelectrochemical Biosensor for Methyltransferase Activity Assay.

DNA methyltransferases may function as important biomarkers of cancers and genetic diseases. Herein, we develop a dye-sensitized and gold plasmon-enhanced cathodic photoelectrochemical (PEC) biosensor on the basis of p-type covalent organic polymers (COPs) for the signal-on measurement of M.SssI methyltransferase (M.SssI MTase). The cathodic PEC biosensor is constructed by the in situ growth of p-type COP films onto a glass coated with indium tin oxide and the subsequent assembly of biotin- and HS-labeled double-stranded DNA (dsDNA) probes onto the COP film via biotin-streptavidin interaction. The dsDNA probe contains the recognition sequence of M.SssI MTase. The COP thin films possess a porous ultrathin nanosheet structure with abundant active sites, facilitating the generation of a high photocurrent compared with the hydrothermally synthesized ones. The presence of DNA methyltransferases can prevent the digestion of restriction endonuclease HpaII, consequently inducing the introduction of gold nanoparticles (AuNPs) to the dsDNA probes via the S-Au bond and the intercalation of rhodamine B (RhB) into the DNA grooves to produce a high photocurrent due to the dye-photosensitized enhancement and AuNP-mediated surface plasmon resonance. However, in the absence of M.SssI MTase, HpaII digests the dsDNA probes, and neither AuNPs nor RhB can be introduced onto the electrode surface, leading to a low photocurrent. This cathodic PEC biosensor possesses high sensitivity and good selectivity, and it can screen the inhibitors and detect M.SssI MTase in serum as well.

An in situ activity assay for lysyl oxidases

The lysyl oxidase family of enzymes (LOXs) catalyze oxidative deamination of lysine side chains on collagen and elastin to initialize cross-linking that is essential for the formation of the extracellular matrix (ECM). Elevated expression of LOXs is highly associated with diverse disease processes. To date, the inability to detect total LOX catalytic function in situ has limited the ability to fully elucidate the role of LOXs in pathobiological mechanisms. Using LOXL2 as a representative member of the LOX family, we developed an in situ activity assay by utilizing the strong reaction between hydrazide and aldehyde to label the LOX-catalyzed allysine (-CHO) residues with biotin-hydrazide. The biotinylated ECM proteins are then labeled via biotin-streptavidin interaction and detected by fluorescence microscopy. This assay detects the total LOX activity in situ for both overexpressed and endogenous LOXs in cells and tissue samples and can be used for studies of LOXs as therapeutic targets.

Enhancing Comprehensive Analysis of Newly Synthesized Proteins Based on Cleavable Bioorthogonal Tagging.

Protein synthesis and degradation responding to environmental cues is critical for understanding the mechanisms involved. Chemical proteomics introducing bioorthogonal tagging into proteins and isolation by biotin affinity purification is applicable for enrichment of newly synthesized proteins (NSPs). Current enrichment methods based on biotin-streptavidin interaction lack efficiency to release enriched NSPs under mild conditions. Here we designed a novel method for enriching newly synthesized peptides by click chemistry followed by release of enriched peptides via tryptic digestion based on cleavable bioorthogonal tagging (CBOT). CBOT-modified peptides can further enhance identification in mass spectrometry analysis and provide a confirmation by small mass shift. Our method achieved an improvement in specificity (97.1%) and sensitivity for NSPs in cell lysate, corresponding to profiling at a depth of 4335 NSPs from 2 mg of starting materials in a single LC-MS/MS run. In addition, the CBOT strategy can quantify NSPs when coupling a pair of isotope-labeled azidohomoalanine (AHA/hAHA) with decent reproducibility. Furthermore, we applied it to analyze newly synthesized proteomes in the autophagy process after 6 h rapamycin stimulation in cells, 2910 NSPs were quantified, and 337 NSPs among them were significantly up- and down-regulated. We envision CBOT as an effective and alternative approach for bioorthogonal chemical proteomics to study stimuli-sensitive subsets.

Single-cell profiling of D-2-hydroxyglutarate using surface-immobilized resazurin analogs

D-2-hydroxyglutarate (D2HG) is over-produced as an oncometabolite due to mutations in isocitrate dehydrogenases (IDHs). Accumulation of D2HG can cause the dysfunction of many enzymes and genome-wide epigenetic alterations, which can promote oncogenesis. Quantification of D2HG at single-cell resolution can help understand the phenotypic signatures of IDH-mutant cancers and identify effective therapeutics. In this study, we developed an analytical method to detect D2HG levels in single cancer cells by adapting cascade enzymatic reactions on a resazurin-based fluorescence reporter. The resazurin probe was immobilized to the sensing surface via biotin-streptavidin interaction. This surface chemistry was rationally optimized to translate the D2HG levels to sensitive fluorescence readouts efficiently. This D2HG assay demonstrated good selectivity and high sensitivity toward D2HG, and it was compatible with the previously developed single-cell barcode chip (SCBC) technology. Using the SCBC platform, we performed simultaneous single-cell profiling of D2HG, glucose uptake, and critical oncogenic signaling proteins in single IDH-mutant glioma cells. The results unveiled the complex interplays between metabolic and oncogenic signaling and led to the identification of effective combination targeted therapy against these IDH-mutant glioma cells.

Abstract #1038067: Clinical Hypothyroidism Masked Biochemically due to Biotin Supplements???

Biotin( Vitamin B7) is widely available as an over the counter supplement for skin, hair and nail health. Many immunoassays use the biotin-streptavidin interaction as an immobilizing system, in which biotin can be readily incorporated into a hormone. Ingestion of high doses of biotin has been reported to cause abnormal results in thyroid function tests by this assay interference. Hereby we describe the case of a patient with clinical hypothyroidism but biochemically hyperthyroid due to biotin supplementation.