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A common goal in mass spectrometry-based chemoproteomics is to directly measure the site of conjugation between the target protein and the small molecule ligand. However, these experiments are inherently challenging due to the low abundance of labeled proteins and the difficulty in identifying modification sites using standard proteomics software. Reporter tags that either generate signature fragment ions or isotopically encode target peptides can be used for the preemptive discovery of labeled peptides even in the absence of identification. We investigated the potential of BODIPY FL azide as a click chemistry enabled chemoproteomics reagent due to the presence of boron and the unique 1:4 natural abundance ratio of 10 B: 11 B. The isotopes of boron encode BODIPY-labeled peptides with a predictable pattern between the monoisotopic (M) and M+1 peaks. BODIPY-labeled peptides were identified in MS1 spectra using an R script that filters for the signature 10 B: 11 B intensity ratio and mass defect. Application of the boron detection script resulted in three times the labeled peptide coverage achieved for a BODIPY-conjugated BSA sample compared with untargeted data-dependent acquisition sequencing. Furthermore, we used the inherent HF neutral loss signature from BODIPY to assist with BODIPY-modified peptide identification. Finally, we demonstrate the application of this approach using the BODIPY-conjugated BSA sample spiked into a complex E. coli . digest. In summary, our results show that the commercially available BODIPY FL azide clicked to alkyne-labeled peptides provides a unique isotopic signature for pinpointing the site(s) of modification with the added potential for on- or off-line UV or fluorescence detection.

Selecting an adequate model to represent the mass transfer mechanisms occurring in a chromatographic process is generally complicated, which is one of the reasons why monolithic chromatography is scarcely simulated. In this study, the chromatographic separation of model proteins bovine serum albumin (BSA), β-lactoglobulin-A, and β-lactoglobulin-B on an anion exchange monolith was simulated based on experimental parameter determination, simultaneous model testing, and validation under three statistical criteria: retention time, dispersion accuracies, and Pearson correlation coefficient. Experimental characterization of morphologic, physicochemical, and kinetic parameters was performed through volume balances, pressure drop analysis, breakthrough curve analysis, and batch adsorptions. Free Gibbs energy indicated a spontaneous adsorption process for proteins and counterions. Dimensionless numbers were estimated based on height equivalent to a theoretical plate analysis, finding that pore diffusion controlled β-lactoglobulin separation, whereas adsorption/desorption kinetics was the dominant mechanism for BSA. The elution profiles were modeled using the transport dispersive model and the reactive dispersive model coupled with steric mass action (SMA) isotherms because these models allowed to consider most of the mass transport mechanisms that have been described. RDM-SMA presented the most accurate simulations at pH 6.0 and at low (250 mM) and high (400 mM) NaCl concentrations. This simulation will be used as reference to forecast the purification of these proteins from bovine whey waste and to extrapolate this methodology to other monolith-based separations using these three statistical criteria that have not been used previously for this purpose., (© 2024 Wiley‐VCH GmbH.)

As a class of point-of-care (POC) assays with visible distance readout (thermometer style), the electrophoresis titration (ET) biosensor affords high robustness, versatility, and simplicity for point-of-care quantification. However, naked-eye observation of the distance readout is unreliable in POC settings and manual processing of distance readout is time-consuming. Herein, we developed a smartphone-deployable and all-in-one machine vision for four ET biosensors (bovine serum albumin, melamine, uric acid, glutathione) to classify and quantify the samples simultaneously. To ensure accurate and rapid quantification on the smartphone, we customized the decolorization methods and edge detection operators to balance the region of interest (ROI) extraction performance and processing speed. We then established a dataset of 180 distance readout images to endow our machine vision with the ability to classify four sample types. Consequently, our machine vision demonstrated high accuracy in determining the sample type (>97.2%) and concentration (>97.3%). Moreover, expanding its applications to other targets was readily achieved by including distance readout images of other ET biosensors (e.g., hemoglobin A1c) in the dataset. Therefore, our strategy of constructing machine vision is compatible with the versatile ET biosensor technique, suggesting that the same strategy can be used for other thermometer-style POC assays., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Taylor dispersion analysis (TDA) is a rapid and precise method for determining the hydrodynamic radius (R H ) of various substances. We present a versatile TDA system with a flow-through sample injection device, two compact 3-in-1 detectors, and a high-voltage power supply. The 3D-printed detectors combine fluorimetry (FD), photometry (AD@255 nm), and contactless conductometry (C 4 D) in a single head, enabling simultaneous detection at one capillary window. Using bovine serum albumin (BSA) as a model analyte, we compare TDA with different detection methods. BSA labeled with fluorescein isothiocyanate (FITC) is analyzed in both pulse mode and capillary electrophoresis (CE) TDA. FD and AD detection yield similar R H values, except when FITC binds with small ions in the buffer. In phosphate buffer, C 4 D underestimates R H values by approximately 18 % due to BSA self-association. In Tris-based buffers, C 4 D values are 87%-96 % of AD values in pulse mode. With CE-TDA using Tris-CHES buffer, no statistical difference is found across all detections. The system is also applied to CE-TDA of various compounds, particularly charged saccharides. CE-TDA improves the accuracy of TDA results from C 4 D. We demonstrate the resolution of mixed C 4 D-TDA signals with assistance from FD and AD signals, successfully resolving gluconate peaks fully covered by another compound. The versatile system with 3-in-1 detection offers a powerful tool for TDA of mixtures and enhances sample throughput., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Automated methods for enzyme immobilization via 4-triethoxysilylbutyraldehyde (TESB) derived silicone-based coupling agents were developed. TESB and its oxidized derivative, 4-triethoxysilylbutanoic acid (TESBA), were determined to be the most effective. The resulting immobilized enzyme particles (IEPs) displayed robustness, rapid digestion, and immobilization efficiency of 51 ± 8%. Furthermore, we automated the IEP procedure, allowing for multiple enzymes, and/or coupling agents to be fabricated at once, in a fraction of the time via an Agilent Bravo. The automated trypsin TESB and TESBA IEPs were shown to rival a classical in-gel digestion method. Moreover, pepsin IEPs favored cleavage at leucine (>50%) over aromatic and methionine residues. The IEP method was then adapted for an in-situ immobilized enzyme microreactor (IMER) fabrication. We determined that TESBA could functionalize the silica capillary's inner wall while simultaneously acting as an enzyme coupler. The IMER digestion of bovine serum albumin (BSA), mirroring IEP digestion conditions, yielded a 33-40% primary sequence coverage per LC-MS/MS analysis in as little as 15 min. Overall, our findings underscore the potential of both IEP and IMER methods, paving the way for automated analysis and a reduction in enzyme waste through reuse, thereby contributing to a more cost-effective and timely study of the proteome. SIGNIFICANCE: This research introduces 4-triethoxysilylbutyraldehyde (TESB) and its derivatives as silicon-based enzyme coupling agents and an automated liquid handling method for bottom-up proteomics (BUP) while streamlining sample preparation for high-throughput processing. Additionally, immobilized enzyme particle (IEP) fabrication and digestion within the 96-well plate allows for flexibility in protocol where different enzyme-coupler combinations can be employed simultaneously. By enabling the digestion of entire microplates and reducing manual labor, the proposed method enhances reproducibility and offers a more efficient alternative to classical in-gel techniques. Furthermore, pepsin IEPs were noted to favor cleavage at leucine residues which represents an interesting finding when compared to the literature that warrants further study. The capability of immobilized enzyme microreactors (IMER) for rapid digestion (in as little as 15 min) demonstrated the system's efficiency and potential for rapid proteomic analysis. This advancement in BUP not only improves efficiency, but also opens avenues for a fully automated, mass spectrometry-integrated proteomics workflow, promising to expedite research and discoveries in complex biological studies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Dopamine (DA) is a neuromodulatory molecule that plays critical roles in many biological processes. The dysfunctions of the DA system are closely associated with several nervous system diseases. Therefore, it is urgent to establish a simple and accurate method for DA analysis. In this study, an economic and accurate DA ratiometric sensor was established using dual-emission carbon dots (DE-CDs). DE-CDs were first synthesized by the one-step solvothermal method and two separate fluorescence emission peaks at 340 and 500 nm were observed under the excitation of 310 nm. In the presence of Hg 2+ , the fluorescence signal at 340 nm was significantly quenched, while the signal at 500 nm keeps stable. Upon adding DA, the quenched signal at 340 nm was significantly recovered, whereas the signal at 500 nm remains stable. Therefore, a novel ratiometric sensor for DA analysis was established. This method shows a good linear range from 500 nM to 100 μM, and the detection limit was calculated to be 80 nM. Moreover, this established method shows excellent specificity and could be applied in real sample analysis, showing great potential for application in clinical research., (© 2024. The Author(s), under exclusive licence to The Japan Society for Analytical Chemistry.)

There is growing interest in the development of materials for enriching proteins and phosphoproteins from complex sample matrices for mass spectrometric analysis. Herein, we designed and synthesized two types of magnetic resin composites, i.e. , MTS9200@Fe 3 O 4 and FPA90CL@Fe 3 O 4 , and assessed their applications as adsorbents for enriching proteins, peptides and phosphopeptides. With the combination of Fe 3+ -IMAC interaction (MTS9200) or electrostatic attraction (FPA90CL) of resins and the adsorption of Fe 3 O 4 , the prepared composites exhibited higher capacities for adsorbing a protein (bovine serum albumin, at 195.71 and 135.03 mg g -1 for MTS9200@Fe 3 O 4 and FPA90CL@Fe 3 O 4 , respectively) than MTS9200, FPA90CL and Fe 3 O 4 . In addition, due to the contributions of the hydrophobic skeleton of resins and Fe 3 O 4 , the magnetic resin composites allowed for efficient enrichment of peptides. Moreover, through Fe 3+ -IMAC interaction or electrostatic attraction of resins and Fe-O MOAC interaction of Fe 3 O 4 with phosphate groups, phosphopeptides could also be captured. Furthermore, we employed the prepared composites for enriching proteins and phosphopeptides from human serum, where 466 and 506 proteins, and 434 and 356 phosphorylation sites, were detected from human serum after being processed with FPA90CL@Fe 3 O 4 and MTS9200@Fe 3 O 4 , respectively. Together, our work revealed the great potential of magnetic resin composites as enrichment materials for proteomics and phosphoproteomics analysis.

Current practices in synthesizing molecularly imprinted polymers face challenges-lengthy process, low-productivity, the need for expensive and sophisticated equipment, and they cannot be controlled in situ synthesis. Herein, we present a micro-reactor for in situ and continuously synthesizing trillions of molecularly imprinted polymeric nanoparticles that contain molecular fingerprints of bovine serum albumin in a short period of time (5-30 min). Initially, we performed COMSOL simulation to analyze mixing efficiency with altering flow rates, and experimentally validated the platform for synthesizing nanoparticles with sizes ranging from 52-106 nm. Molecular interactions between monomers and protein were also examined by molecular docking and dynamics simulations. Afterwards, we benchmarked the micro-reactor parameters through dispersity and concentration of molecularly imprinted polymers using principal component analysis. Sensing assets of molecularly imprinted polymers were examined on a metamaterial sensor, resulting in 81% of precision with high selectivity (4.5 times), and three cycles of consecutive use. Overall, our micro-reactor stood out for its high productivity (48-288 times improvement in assay-time and 2 times improvement in reagent volume), enabling to produce 1.4-1.5 times more MIPs at one-single step, and continuous production compared to conventional strategy., (© 2023. Springer Nature Limited.)

The digestion of proteins with proteolytic enzymes has expedited the analysis of peptide mapping. Here, we compared the digestion efficiency of soluble chymotrypsin (CT) with two immobilized CT preparations using bovine serum albumin (BSA) as the substrate. An efficient method of immobilizing chymotrypsin using formaldehyde (FA) was optimized and the conditions were applied to assess a novel immobilization reagent, triethoxysilylbutaraldehyde (TESB). Efforts to determine the best enzyme-to-substrate (E : S) ratios during digestion of denatured BSA with single-use FA-CT enzyme particles were performed by adjusting the amount of substrate used. An E : S ratio of 10 : 1 was found to be best based on the LC-MS/MS analysis data showing sequence coverage of 67%. Fabrication of immobilized enzyme microreactors (IMERs) was carried out using both (3-aminopropyl)triethoxysilane (APTES) with the idealized conditions with FA, as well as the novel procedure utilizing TESB for a proof of concept open-tubular IMER. It was found that the FA-APTES IMER had a sequence coverage of 6%, while the TESB IMER had 29% sequence coverage from MS analysis. The application of TESB in enzyme immobilization has the potential to facilitate a greater degree of enzymatic digestion with higher sequence coverage than traditional immobilization or crosslinking reagents for bottom-up proteomics.

Following health agencies warning, the use of animal origin supplements should be avoided in biological products proposed as therapy in humans. Platelet lysate and several other growth factors sources are alternatives to replace fetal calf serum, the current gold standard in clinical-grade cell culture. However, the platelet supplement's content lacks data due to different production methods. The principle behind these products relays on the lysis of platelets that release several proteins, some of which are contained in heterogeneous granules and coordinate biological functions. This study aims to analyze the composition and reproducibility of a platelet lysate produced with a standardized method, by describing several batches' protein and particle content using proteomics and dynamic light scattering. Proteomics data revealed a diversified protein content, with some related to essential cellular processes such as proliferation, morphogenesis, differentiation, biosynthesis, adhesion, and metabolism. It also detected proteins responsible for activation and binding of transforming growth factor beta, hepatocyte growth factor, and insulin-like growth factor. Total protein, biochemical, and growth factors quantitative data showed consistent and reproducible values across batches. Novel data on two major particle populations is presented, with high dispersion level at 231 ± 96 d.nm and at 30 ± 8 d.nm, possibly being an important way of protein trafficking through the cellular microenvironment. This experimental and descriptive analysis aims to support the content definition and quality criteria of a cell supplement for clinical applications., (© 2022 Wiley Periodicals LLC.)

Selective separation and enrichment of phosphoproteins are essential for understanding their important functions in almost all cellular processes. Here, taking advantage of the feature that cadmium ion (Cd 2+ ) has an overwhelming preference for phosphates, we developed a robust and simple Cd 2+ co-precipitation strategy for the selective isolation of intact phosphoproteins. After evaluating the feasibility of Cd 2+ in phosphoprotein precipitation, we compared the washing protocols for the removal of non-specific binding proteins and then used the best-performing protocol for the isolation of phosphoproteins from different complex samples. It was found that phosphoproteins can be specifically enriched from artificial protein mixtures containing α-casein, β-casein, and bovine serum albumin or plasma, in which bovine serum albumin or plasma were served as interferences with very high molar ratios. Applying this method to enrich phosphoproteins from complex cell lysates, a high specificity was confirmed by western blotting analysis with a phosphoprotein-specific kit. Finally, we successfully applied this method to the purification of caseins from drinking milk, highlighting its potential application in the studies where purified phosphoproteins were required. In a word, this Cd 2+ co-precipitation method enables universal and effective capture, enrichment, and detection of intact phosphoproteins, making it a powerful tool for the comprehensive analysis of the phosphoproteome., (© 2022 Wiley-VCH GmbH.)

Cellulose might be a promising material for surface-enhanced Raman scattering (SERS) substrates due to its wide availability, low cost, ease of fabrication, high flexibility and low optical activity. This work shows, for the first time development of the cellulose-based substrate, that owes its SERS activity to the presence of gold nanorods in its internal structure, and not only on the surface, as it is shown elsewhere, thus ensuring superior stability of the obtained material. This flexible cellulose-based substrate exhibiting plasmonic activity, provide easy and reproducible detection of different analytes via SERS technique. The substrate was prepared by introduction of gold nanorods into the cellulose fibers matrix using an eco-friendly process based on N-Methylmorpholine-N-Oxide. Au-modified cellulose fibers were used for the detection of p-Mercaptobenzoic acid and Bovine Serum Albumin by the SERS method. The obtained results show that this substrate offers large signal enhancement of 6-orders of magnitude, and high signal reproducibility with a relative standard deviation of 8.3%. Additionally, washing tests (90 °C, 20 h) showed superior stability of the as prepared plasmonic fibers, thus proving the good reusability of the substrates and the long shelf life., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Single-color reflectrometry is a sensitive and robust detection method in optical biosensor applications, for example for bioanalysis. It is based on the interference of reflected monochromatic radiation and is label free. We present a novel setup for single-color reflectometry based on the patented technology of Berner et al. from 2016. Tilting areas of micro-mirrors allow us to encode the optical reflection signal of an analyte and reference channel into a particular carrier frequency with the amplitude being proportional to the local reflection. Therefore, a single photodiode is sufficient to collect the signals from both channels simultaneously. A 180 ∘ phase shift in the tilt frequency of two calibrated micro-mirror areas leads to a superposition of the analyte and reference signal which enables an efficient reduction of the baseline offset and potential baseline offset drift. A performance test reveals that we are able to detect changes of the refractive index n down to Δn < 0.01 of saline solutions as regents. A further test validates the detection of heterogeneous binding interaction. This test compromises immobilized testosterone-bovine serum albumin on a three-dimensional layer of biopolymer as ligand and monoclonal anti-testosterone antibodies as analyte. Antibody/antigen binding induces a local growth of the biolayer and change in the refractive index, which is measured via the local change of the reflection. Reproducible measurements enable for the analysis of the binding kinetics by determining the affinity constant K A = 1.59 × 10 - 7 M - 1 . In summary, this work shows that the concept of differential Fourier spotting as novel setup for single-color reflectometry is suitable for reliable bioanalysis. Graphical Abstract., (© 2022. The Author(s).)

Freezing processes are a well-established unit operation in the biopharmaceutical industry to increase the shelf-life of protein-based drugs. While freezing reduces degradation reaction rates, it may also exert stresses such as freeze concentration. Macroscopic freeze concentration in large-scale freezing processes has been described thoroughly by examination of frozen bulk material, but the transient process leading to such freeze concentration profiles has not been monitored yet for biopharmaceutical solutions. In this study, Raman spectroscopy as a process analytical technology is demonstrated for model formulations containing monoclonal antibodies (mAbs) or bovine serum albumin (BSA) in varying concentrations of sucrose and buffer salts. Therefore, a Raman probe was immersed into a bulk volume at different heights, monitoring the freeze concentration in the liquid phase during the freezing processes. Partial least square regression models were used to quantitatively discriminate between the protein and excipients simultaneously. The freeze concentration profiles were dependend on freezing temperature and formulation with freeze concentrations up to 2.4-fold. Convection currents at the bottom of the freezing container were observed with a maximum height of 1 mm. Furthermore, freeze concentration was correlated with the sucrose concentration in a formulation. Analysis of the freeze concentration slope indicated diffusion from the bottom to the top of the container. In summary, Raman spectroscopy is a valuable tool for process validation of freeze concentration simulations and to overcome scale-dependent challenges., (© 2021 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals LLC.)

Three barbiturate squaraine dyes derived from indolenine or benzothiazole, with different barbituric acid derivatives were prepared, characterized and photophysically evaluated by standard spectroscopic methods. As expectable for squaraines, these dyes showed narrow and intense absorption and emission bands in the Vis/NIR region. The interaction of synthesized dyes with bovine and human serum albumins (BSA and HSA) was also evaluated in phosphate buffer (PB). The results revealed that upon the addition of BSA or HSA the complex dye-protein emit more fluorescence, and the emission intensity is directly proportional to the concentration of protein used (0-3.5 µM). The titration tests allowed to calculate the binding constants, in an order of magnitude of 10 4 -10 6 M, as well as the limits of detection and quantification in the nanomolar tens range. All dyes showed a good response to the interaction with both proteins, but the most pronounced envisioning their use as protein labeling was observed for the squaraine dye derived from the indolenine with a 1,3-dimethylbarbituric acid moiety. The molecular docking studies revealed the existence of a binding between the compounds and four sites on the HSA molecule, where one of these four locations is a new binding site with which this series of dye interacts., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The rational synthesis of trinuclear emissive organometallic complexes with two equivalent platinum(II) centres appended to the ancillary substituted 2,2'-bipyridyl ligand of the cyclometalated iridium(III) centre is reported here. The alkynyl-platinum moiety and cyclometalated iridium(III) centres have been separated through a non-conjugated CH 2 -O-CH 2 linkage. The emission titration with amino acids reveals that the complexes sense free amino acids. The luminescence sensing of BSA is thus attributed to the amino acid sensing ability of the complexes and confirmed by emission anisotropy and Far-UV CD spectral study. The decrease in α-helix in the CD spectra signifies the changes in the secondary structure of protein in presence of the complexes., (© 2021 Wiley-VCH GmbH.)

Cylinder-shaped NaY zeolite was used as an adsorbent for eradicating both heavy metal ions (Cu 2+ , Zn 2+ , Ni 2+ , and Co 2+ ) and proteins from the waste streams. As a pseudo-metal ion affinity adsorbent, NaY zeolite was used in the capture of heavy metal ions in the first stage. The amount (molar basis) of metal ions adsorbed onto NaY zeolite decreased in the order of Cu 2+  > Zn 2+  > Co 2+  > Ni 2+ . Bovine serum albumin (BSA) was utilized as a model of proteins used in the waste adsorption process by NaY zeolite. The adsorption capacities of NaY zeolite and Cu/NaY zeolite for BSA were 14.90 mg BSA/g zeolite and 84.61 mg BSA/g zeolite, respectively. Moreover, Cu/NaY zeolite was highly stable in the solutions made of 2 M NaCl, 500 mM imidazole or 125 mM EDTA solutions. These conditions indicated that the minimal probability of secondary contamination caused by metal ions and soluble proteins in the waste stream. This study demonstrates the potential of Cu/NaY zeolite complex as an efficient pseudo-metal chelate adsorbent that could remove metal ions and water-soluble proteins from wastewater concurrently., (Copyright © 2021 Elsevier B.V. All rights reserved.)

We report the progress on an electron-activated dissociation (EAD) device coupled to a quadrupole TOF mass spectrometer (QqTOF MS) developed in our group. This device features a new electron beam optics design allowing up to 100 times stronger electron currents in the reaction cell. The electron beam current reached the space-charge limit of 0.5 μA at near-zero electron kinetic energies. These advances enable fast and efficient dissociation of various analytes ranging from singly charged small molecules to multiply protonated proteins. Tunable electron energy provides access to different fragmentation regimes: ECD, hot ECD, and electron-impact excitation of ions from organics (EIEIO). The efficiency of the device was tested on a wide range of precursor charge states. The EAD device was installed in a QqTOF MS employing a novel trap-and-release strategy facilitating spatial mass focusing of ions at the center of the TOF accelerator. This technique increased the sensitivity 6-10 times and allows for the first time comprehensive structural lipidomics on an LC time scale. The system was evaluated for other compound classes such as intact proteins and glycopeptides. Application of hot ECD for the analysis of glycopeptides resulted in rich fragmentation with predominantly peptide backbone fragments; however, glycan fragments attributed to the ECD process were also observed. A standard small protein ubiquitin (8.6 kDa) was sequenced with 90% cleavage coverage at spectrum accumulation times of 100 ms and 98% at 800 ms. Comparable cleavage coverage for a medium-size protein (carbonic anhydrase: 29 kDa) could be achieved, albeit with longer accumulation times.

Pre-enrichment of the biological samples is a crucial step in phosphoproteomics research. At present, metal-oxide affinity chromatography (MOAC) is one of the most recognized enrichment strategy. Therefore, the design and preparation of a MOAC-based affinity material with better enrichment properties will be of great significance for the phosphoproteomics study. In this work, we obtained a novel multivariate metal-oxide microsphere (NiFe 2 O 4 @C@TiO 2 ) with a hollow and hierarchical porous structure through pyrolysis of TiO 2- modified Fe/Ni-based metal-organic frameworks (MOFs). After pyrolysis, the carbon matrix derived from the MOFs provided support and porous properties. Meanwhile, multivariate metal oxides endowed the microspheres with an excellent magnetic response property and superior enrichment performance for phosphorylated biomolecules. The unique hollow and hierarchical porous structure greatly enhanced the diffusion of phosphorylated biomolecules. Therefore, the microspheres exhibited excellent enrichment performance for phosphorylated biomolecules: a large adsorption capacity (124 μmol g -1 ), excellent selectivity (α-casein/BSA, 1:5000, m/m), perfect size-exclusion performance (α-casein digests/α-casein/BSA, 1:500:500), and extremely low detection limit (2 fmol). Furthermore, the microspheres showed excellent enrichment performance in a series of real biological samples, such as nonfat milk, serum, saliva, rat brain tissue, and plasma exosomes of patients with esophageal cancer, which further demonstrated its huge application potential in MS-based phosphoproteomics research.

Highly sensitive detection of trace amounts of substances is crucial for broad applications in healthcare, environmental monitoring, antiterrorism, etc., where cost effectiveness and portability are often demanded. Here, an ultrasensitive sensor is reported that can detect an angstrom-thick layer of adsorbed molecules through image acquisition and processing. The sensor features a centimeter-scale plasmonic metasurface with spatially varying geometry, where the light scattering is dependent on both the adsorbed substances and spatial locations. When illuminated with narrowband light (such as from a light emitting diode), the intensity pattern recorded on the metasurface changes with the surface-adsorbed molecules, enabling label-free, sensitive, and spectrometer-free molecular detection. The centimeter-scale size of the sensing area interfaces well with consumer-level imaging sensors on mobile devices without the need for microscopic optics and offers a high signal-to-noise ratio by leveraging the multimillion pixels for noise reduction. It is experimentally demonstrated that a single layer of Al 2 O 3 molecules deposited on the sensor, with a thickness of approximately one angstrom, can be detected by analyzing the images taken of the sensing chip. Furthermore, by integrating the sensor into a microfluidic setup, quantitative detection of BSA/anti-BSA immune complex formation events is demonstrated, which agrees well with the Langmuir isotherm model., (© 2021 Wiley-VCH GmbH.)

The sensitivity and reproducibility of the lateral flow assay can be influenced by multiple factors, such as the size of gold nanoparticles (GNPs) employed. Here, we evaluated the analytical performance of single-sized and mixed-sized GNPs using a simple lateral flow assay (LFA) platform. This platform was used as a model assay to diagnose albumin levels and demonstrate the analytical performance of single-sized and mixed-sized GNPs in LFA tests. Two sizes of GNPs@anti-bovine serum albumin (BSA) conjugate proteins were mixed at different ratios. The unique optical properties of the GNPs induced a distinguishing color-shedding effect on the single- and mixed-sized GNPs@anti-BSA conjugates interacting with the target analyte BSA spotted on the test line. The use of mixed-sized GNPs@anti-BSA conjugates enhanced signal relative to the 20 nm GNPs, and provided superior stability compared with solely employing the large GNPs (50 nm). The proposed platform in this study could provide an efficient BSA detection mechanism that can be utilized as a model biomarker for confronting chronic kidney disease.

Whey represents a valuable protein source for human nutrition. Whey composition varies with respect to process characteristics during milk processing. For efficient exploitation of this dairy side stream, reliable analytical methods are essential. The aim of this study was to develop and validate an RP-HPLC-DAD method for the simultaneous quantification of the minor (lactoferrin, lactoperoxidase, bovine serum albumin) and major (α-lactalbumin, β-lactoglobulin) whey proteins. Seven RP-columns were compared and the composition of the mobile phase was optimized to achieve baseline separation. In validation experiments the limits of detection (LOD < 8 mg/L) and quantification (LOQ < 24 mg/L) were determined. Validity was proofed by precision (>96%), accuracy (95% - 103%) and recovery (96% - 102%) measurements. Peak homogeneity was confirmed by SDS-PAGE. The individual working ranges were adjusted to the estimated protein concentrations in whey, allowing direct analysis without sample preparation at a method runtime of 23 min., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The adoption of process analytical technologies by the biopharmaceutical industry can reduce the cost of therapeutic drugs and facilitate investigation of new bioprocesses. Control of critical process parameters to retain critical product quality attributes within strict bounds is important for ensuring a consistently high product quality, but developing the sophisticated analytical technologies required has proven to be a major challenge. Here, we demonstrate a new optical technique for continuous monitoring of protein species as they are eluted from a chromatographic column, even when they fully co-elute with other protein species, without making any assumption about or peak-fitting to the elution profile. To achieve this, we designed and constructed a time-resolved intrinsic fluorescence lifetime chromatograph, and established an analytical framework for deconvolving and quantifying distinct but co-eluting protein species in real time. This proof-of-concept technology has potentially useful applications as a process analytical technology and more generally as an analytical technique for label-free quantification of proteins in mixtures., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: J.E.H., S.A., G.A., and P.A.D. are co-authors on a patent application which could increase in value if the methods and ideas described in this paper find widespread application., (Copyright © 2021. Published by Elsevier B.V.)

In this study, a key issue to be addressed is the safe disposal of hybridoma instability. Hybridoma technology was used to produce anti-O. viverrini monoclonal antibody. Previous studies have shown that antibody production via antibody phage display can sustain the hybridoma technique. This paper presents the utility of antibody phage display technology for producing the phage displayed KKU505 Fab fragment and using experiments in concomitant with molecular simulation for characterization. The phage displayed KKU505 Fab fragment and characterization were successfully carried out. The KKU505 hybridoma cell line producing anti-O. viverrini antibody predicted to bind to myosin was used to synthesize cDNA so as to amplify the heavy chain and the light chain sequences. The KKU505 displayed phage was constructed and characterized by a molecular modeling in which the KKU505 Fab fragment and -O. viverrini myosin head were docked computationally and it is assumed that the Fab fragment was specific to -O. viverrini on the basis of mass spectrometry and Western blot. This complex interaction was confirmed by molecular simulation. Furthermore, the KKU505 displayed phage was validated using indirect enzyme-linked immunosorbent assays (ELISA) and immunohistochemistry. It is worthy to note that ELISA and immunohistochemistry results confirmed that the Fab fragment was specific to the -O. viverrini antigen. Results indicated that the approach presented herein can generate anti-O. viverrini antibody via the phage display technology. This study integrates the use of phage display technology together with molecular simulation for further development of monoclonal antibody production. Furthermore, the presented work has profound implications for antibody production, particularly by solving the problem of hybridoma stability issues., Competing Interests: The authors have declared that no competing interests exist.

In this study, a catalytic polydopamine-remodeling gold nanoparticle sensitized with an antinucleolin AS1411 probe (pAu nanoprobe) is synthesized, where the surface of the gold nanoparticles (AuNPs) is modified with a spontaneous self-polymerization of a polydopamine coating that imparts the probe functionalization ability and antispecific protein binding while the intrinsic catalytic property of the AuNPs is preserved. The functionalized AS1411 probe exerts specific recognition with nucleolin protein that is found to be overexpressed on the surface of breast cancer cells (MDA-MB-231). Scanning electron microscopy (SEM) confirms that the specific binding of the pAu nanoprobe occurs at the cancer cell surface. Taking advantage of the catalytic ability of the pAu nanoprobe in reducing blue-colored methylene blue (MB) to colorless leuco-MB, a colorimetric biosensing platform is established based on the accessible catalytic active sites on the pAu nanoprobe toward MB. The specific binding inhibits the pAu nanoprobe from efficiently catalyzing the reduction of MB, resulting in a " turn-off " catalytic biosensing platform. The catalytic conversion of MB is inversely proportional to the concentration of the nucleolin protein and the cancer cells, yielding a detection limit of 15 pM of the nucleolin protein and two cancer cells. The presence of five orders of magnitude higher concentration of bovine serum albumin hardly affects the catalytic ability of the pAu nanoprobe, that is, 88% catalytic ability is still preserved, which validates the specificity of the proposed pAu nanoprobe. In particular, a distinct color contrast creates a significant signal-to-noise ratio so as to enable single-cell level detection of two cancer cells by naked-eye judgment. Moreover, the undiluted, real human serum samples spiked with the cancer cells were examined with an impressive recovery of 94 ± 0.3%, which holds great promise in cancer cell screening.

An optical and dielectric biosensor based on a liquid crystal (LC)-photopolymer composite was established in this study for the detection and quantitation of bovine serum albumin (BSA). When the nematic LC E7 was doped with 4-wt.% NOA65, a photo-curable prepolymer, and photopolymerized by UV irradiation at 20 mW/cm 2 for 300 s, the limit of detection determined by image analysis of the LC optical texture and dielectric spectroscopic measurements was 3400 and 88 pg/mL for BSA, respectively, which were lower than those detected with E7 alone (10 μg/mL BSA). The photopolymerized NOA65, but not the prepolymer prior to UV exposure, contributed to the enhanced optical signal, and UV irradiation of pristine E7 in the absence of NOA65 had no effect on the optical texture. The effective tilt angle θ , calculated from the real-part dielectric constant ε ', decreased with increasing BSA concentration, providing strong evidence for the correlation of photopolymerized NOA65 to the intensified disruption in the vertically oriented LC molecules to enhance the optical and dielectric signals of BSA. The optical and dielectric anisotropy of LCs and the photo-curable dopant facilitate novel quantitative and signal amplification approaches to potential development of LC-based biosensors.

As a nonspecific phosphomonoesterase, alkaline phosphatase (ALP) plays a pivotal role in tissue mineralization and osteogenesis which is an important biomarker for the clinical diagnosis of bone and hepatobiliary diseases. Herein, we described a novel electrochemical method that used aminoferrocene (AFC) as an electroactive probe for the ALP activity detection. In the condition with imidazole and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), the AFC probe could be directly labeled on single-stranded DNA (ssDNA) by one-step conjugation. Specifically, thiolated ssDNA at 3'-terminals was modified to the electrode surface through Au-S bond. In the condition without ALP, AFC could be labeled on ssDNA by conjugating with phosphate groups. In the presence of ALP, phosphate groups were catalyzed to be removed from the 5'-terminal of ssDNA. The AFC probe cannot be labeled on ssDNA. Thus, the electrochemical detection of ALP activity was achieved. Under optimal conditions, the strategy presented a good linear relationship between current intensity and ALP concentration in the range of 20 to 100 mU/mL with the limit of detection (LOD) of 1.48 mU/mL. More importantly, the approach rendered high selectivity and satisfactory applicability for ALP activity detection. In addition, this method has merits of ease of operation, low cost, and environmental friendliness. Thus, this strategy presents great potential for ALP activity detection in practical applications. An easy, sensitive and reliable strategy was developed for the detection of alkaline phosphatase activity via electrochemical "Signal off".

Vibrational circular dichroism (VCD) spectroscopy has emerged as a powerful platform to quantify chirality, a vital biological property that performs a pivotal role in the metabolism of life organisms. With a photoelastic modulator (PEM) integrated into an infrared spectrometer, the differential response of a sample to the direction of circularly polarized light can be used to infer conformation handedness. However, these optical components inherently exhibit chromatic behavior and are typically optimized at discrete spectral frequencies. Advancements of discrete frequency infrared (DFIR) spectroscopic microscopes in spectral image quality and data throughput are promising for use toward analytical VCD measurements. Utilizing the PEM advantages incorporated into a custom-built QCL microscope, we demonstrate a point scanning VCD instrument capable of acquiring spectra rapidly across all fingerprint region wavelengths in transmission configuration. Moreover, for the first time, we also demonstrate the VCD imaging performance of our instrument for site-specific chirality mapping of biological tissue samples. This study offers some insight into future possibilities of examining small, localized changes in tissue that have major implications for systemic diseases and their progression, while also laying the groundwork for additional modeling and validation in advancing the capability of VCD spectroscopy and imaging.

Optical tweezers have emerged as a powerful tool for the non-invasive trapping and manipulation of colloidal particles and biological cells 1,2 . However, the diffraction limit precludes the low-power trapping of nanometre-scale objects. Substantially increasing the laser power can provide enough trapping potential depth to trap nanoscale objects. Unfortunately, the substantial optical intensity required causes photo-toxicity and thermal stress in the trapped biological specimens 3 . Low-power near-field nano-optical tweezers comprising plasmonic nanoantennas and photonic crystal cavities have been explored for stable nanoscale object trapping 4-13 . However, the demonstrated approaches still require that the object is trapped at the high-light-intensity region. We report a new kind of optically controlled nanotweezers, called opto-thermo-electrohydrodynamic tweezers, that enable the trapping and dynamic manipulation of nanometre-scale objects at locations that are several micrometres away from the high-intensity laser focus. At the trapping locations, the nanoscale objects experience both negligible photothermal heating and light intensity. Opto-thermo-electrohydrodynamic tweezers employ a finite array of plasmonic nanoholes illuminated with light and an applied a.c. electric field to create the spatially varying electrohydrodynamic potential that can rapidly trap sub-10 nm biomolecules at femtomolar concentrations on demand. This non-invasive optical nanotweezing approach is expected to open new opportunities in nanoscience and life science by offering an unprecedented level of control of nano-sized objects, including photo-sensitive biological molecules.

Molecular detection in complex mixtures is of great importance in biomedical diagnosis, food safety, and environmental monitoring. Although surface-enhanced Raman scattering serves as one of the most promising detection methods, metal surfaces are prone to contamination, making the direct detection of small molecules in mixtures elusive. Metal nanoparticle-loaded hydrogels have been used for the exclusion of large adhesive molecules and direct detection of small molecules. Here, we design microgels containing highly concentrated gold nanoparticles through the simultaneous formation of hydrogel and gold nanoparticles in emulsion droplets. Monodisperse water-in-oil droplets are microfluidically prepared to contain a gold precursor, hydrogel precursor, and photoinitiator. Upon ultraviolet irradiation, a hydrogel is gradually formed in the drop by photocross-linking at which gold nanoparticles are synthesized and grown by photo and thermal reduction. The in situ synthesis provides the uniform distribution of gold nanoparticles at very high concentrations without aggregation, which is otherwise very difficult to achieve. Using the microgels, small molecules in albumin solutions can be detected by Raman measurement with high signal sensitivity and reproducibility in the absence of interruption from albumin. As a proof of concept, we demonstrate the direct detection of pyocyanin, a biomarker for Pseudomonas infection spiked in unpurified saliva.

Protein denaturation involves a change in the protein structure with the loss of activity, which proceeds via various intermediates. The possible intermediate structures account largely for understanding the process of unfolding. Hence, considerable attention is required to characterize partially unfolded protein states and to gain more insight into the information about the sequence and steps involved in protein folding mechanisms. In this report, a stepwise unfolding of bovine serum albumin (BSA) with guanidine hydrochloride (GuHCl) has been investigated using Raman spectroscopy in the amide I and III regions. Two-dimensional (2D) correlation analysis has been applied to reveal information on the sequential order and the dynamic properties of interaction during the unfolding process. Raman spectral signatures in the amide I region revealed that there is no significant change in secondary structures up to 2 M concentration of GuHCl. However, 2D correlation analysis further supports the observation by inferring the strengthening of secondary structure at the expense of tertiary structure. At a higher concentration of GuHCl (2-4 M), there is an accumulation of random and β-sheet structures that is mediated by small connecting segments of helices. It further accelerates the unfolding of helices and a complete collapse of structure. These analyses establish the ability of Raman spectroscopy to estimate the ensemble of secondary structures present in proteins. The results reveal the mechanistic details of unfolding, characterizing structure of intermediates even at high concentrations, and understanding the evolution of various secondary structures with respect to each other during unfolding. Such observations can be helpful in understanding the factors affecting the shape and size of proteins during folding/unfolding.

Magnetic levitation, which is a magnetic phenomenon of levitating particles suspended in a paramagnetic liquid under a nonuniform magnetic field, is a powerful tool for determining densities and magnetic properties of micro- and nanoparticles. The levitation height of particles in the magnetic field depends on the magnetic susceptibility and density difference between the object and the surrounding liquid. Here, we developed a magnetic susceptibility-based protein detection scheme in a low-cost and miniaturized magnetic levitation setup consisting of two opposing magnets to create a gradient of a magnetic field, a glass capillary channel to retain the sample, and two side mirrors to monitor inside the channel. The method includes the use of polymeric microspheres as mobile assay surfaces and magnetic nanoparticles as labels. The assay was realized by capturing the target protein to the polymer microspheres. Then, magnetic nanoparticles were attached onto the resulting microsphere-protein complex, creating a significant difference in the magnetic properties of polymer microspheres compared to those without protein. The change in the magnetic properties caused a change in the levitation height of the microspheres. The levitation heights and their distribution were then correlated to the amount of target proteins. The method enabled a detection limit of ∼110 fg/mL biotinylated bovine serum albumin in serum. With the sandwich immunoassay developed for mouse immunoglobulin G, detection limits of 1.5 ng/mL and >10 ng/mL were achieved in buffer and serum, respectively. This approach sensed the minute changes in the volume magnetic susceptibility of the microspheres with a resolution of 4.2 × 10 -8 per 1 μm levitation height change.

High levels of performance and stability have been demonstrated for conjugated polymer thin-film transistors in recent years, making them promising materials for flexible electronic circuits and displays. For sensing applications, however, most research efforts have been focusing on electrochemical sensing devices. Here we demonstrate a highly stable biosensing platform using polymer transistors based on the dual-gate mechanism. In this architecture a sensing signal is transduced and amplified by the capacitive coupling between a low- k bottom dielectric and a high- k ionic elastomer top dielectric that is in contact with an analyte solution. The new design exhibits a high signal amplification, high stability under bias stress in various aqueous environments, and low signal drift. Our platform, furthermore, while responding expectedly to charged analytes such as the protein bovine serum albumin, is insensitive to changes of salt concentration of the analyte solution. These features make this platform a potentially suitable tool for a variety of biosensing applications.

A three-dimensional structured porous graphene oxide-polyethylenimine bead (pGP) is synthesized for immobilizing gold nanoparticles and modifying glutathione molecules (denoted as pGP/AuG). The pGP/AuG has open pore structure, honeycomb-like channels, and excellent hydrophilicity. By taking advantages of the porous structure, abundant binding sites, and multivalent interactions between glycopeptides and both glutathione molecules and free amino groups, the pGP/AuG is adopted to the selective enrichment of N-linked glycopeptides with low limit of detection (2 fmol), high enrichment selectivity (1:500), binding capacity (333.3 mg/g), recovery yield (91.3 ± 2.1%), and repeatability (< 6.0% RSD) using matrix-assisted laser desorption/ionization time of flight mass spectrometry detection method. Furthermore, the practical applicability of pGP/AuG is evaluated, in which 209 N-glycosylated peptides corresponding to 128 N-glycosylated proteins are identified from 1 μL human serum in three independent analysis procedures, suggesting the great potential for application in glycoproteome fields.Graphical abstract Schematic presentation of preparation for porous graphene oxide-based hydrophilic beads (pGP/AuG) with honeycomb-like microstructure. The pGP/AuG was successfully used for enriching and identifying glycopeptides from actual biological sample.

Protein N -glycosylation plays a crucial role in the folding, transportation, and localization of proteins, and participates in many important biological processes such as receptor activation and signal transduction. An increasing number of studies have shown that abnormal protein glycosylation is closely related to various diseases. Therefore, N -glycosylated proteins are potential candidates for become new biomarkers or drug targets. The current research strategy for N -glycosylated proteins is to first digest the protease into peptides and then identify them by mass spectrometry. In sample preparation processing, enrichment and separation of N -glycopeptides have become the vital step for glycoproteomic analysis. However, because of their low abundance and poor ionization, mass spectrometric identification of N -glycopeptides in complex samples remains a challenging task. Therefore, the development of a selective enrichment strategy for N -glycopeptides from complex biological samples is necessary. In this work, new functional nanomaterials were prepared through a simple, convenient, and efficient two-step "Au-S" reaction. In short, the "Au-S" bond was first used to load nano-gold wires on the surface of molybdenum disulfide (MoS 2 ), and then to connect these wires to 4-mercaptophenylboronic acid (4-MPB). The resulting nanomaterial named MoS 2 /Au/4-MPB was successfully prepared by serial functionalization of ultra-thin two-dimensional MoS 2 , nano-gold wires and 4-MPB for the efficient enrichment of N -glycopeptides. The layered structure of molybdenum disulfide nanomaterials can provide numerous modifiable sites for the reaction, allowing for convenient modification of the nano-gold wires. The functional group 4-MPB has high affinity for N -glycopeptides, and it can selectively enrich them in biological samples. In order to evaluate the N -glycopeptide enrichment performance of MoS 2 /Au/4-MPB, standard proteins human immunoglobulin G (IgG) and bovine serum albumin (BSA) trypsin digests were used. Thus, facilitated interactions with N -glycopeptides for boric acid-based retention could be expected. Low femtomolar detection sensitivity, 1:1000 enrichment selectivity, and 100 μg/mg loading capacity were achieved for N -glycopeptide enrichment. In the application of this material to biological samples, exosomes were chosen as the research objects. On the one hand, exosomes serve as a new class of carriers for intercellular communication and drug delivery. Almost all types of cells can secrete exosomes. On the other hand, urine is another potential source for liquid biopsy samples in addition to blood. Obtaining urine samples is a less invasive process, and urine samples are easier to handle and save as compared to blood samples. Exosomes can also be detected in urine. Cell-specific components such as proteins, lipids, and nucleic acids can provide diverse information, which is of great significance in disease diagnosis and treatment. However, there are only a few studies on urinary exosomes N -glycoprotein group. In this research, N -glycoproteins were successfully identified in urine exosomes. In three replicate experiments, 536, 515 and 487 N -glycosylated peptides were identified from 279, 270 and 279 N -glycoproteins, respectively. After de-duplication of the three sets of data, a total of 768 N -glycopeptides corresponding to 377 N -glycoproteins were identified. This result indicates that the novel nanocomposite has good enrichment selectivity and sensitivity for N -glycopeptides enrichment in complex biological samples, and this method provides a new method for glycoproteomics research.

Identification of peptides and proteins is a common task in mass spectrometry-based proteomics but often fails to deliver a comprehensive list of identifications. Downstream analysis, quantitative or qualitative, depends on the outcome of this process. Despite continuous improvement of computational methods, a large fraction of the screened peptides and/or proteins remains unidentified. We introduce here pacMASS, a method that de novo predicts the elemental composition of peptides and small proteins based on a single accurate mass, ie, the observed monoisotopic or average mass. This novel approach returns in a fast and memory efficient manner a limited number of elemental compositions per queried peptide or protein., (© 2019 John Wiley & Sons, Ltd.)

With recent advances in LC-MS systems, current MS-based proteomics has an increasing need for automated, high-throughput sample preparation with neglectable sample loss. In this study, we developed a microfluidic system for fully automated proteomics sample preparation. All of the required proteomics sample preparation steps for both protein digestion and peptide fractionation are fully integrated into a disposable plastic chip device (named AutoProteome Chip). The AutoProteome Chip packed with mixed-mode ion exchange beads and C18 membrane in tandem could be fabricated with very low cost and high stability in organic reagents. Benefiting from its low backpressure, the AutoProteome Chip could be precisely driven by gas pressure, which could be easily multiplexed. As low as 2 ng of standard protein BSA could be trapped into the AutoProteome chip and processed within 2 h. Fully automated processing of 10 μg of protein extracts of HEK 293T cells achieved more than 97% of digestion efficiency with missed cleavage less than 2 and comparable performance with conventional approaches. More than 4700 proteins could be readily identified within 80 min of LC-MS analysis with good label-free quantification performance (Pearson correlation coefficient >0.99). Furthermore, deep proteome profiling by integrated high-pH RP fractionation in the same AutoProteome Chip resulted in more than 7500 proteins being identified from only 20 μg of protein extracts of HEK 293T cells and comparable reprodicibility as single-shot analysis. The AutoProteome Chip system provided a valuable prototype for developing a fully automated proteome analysis workflow and for proteomic applications with high demand for processing throughput, reproducibility, and sensitivity.

C-reactive protein (CRP) is a clinical biomarker for inflammatory diseases. In this work, we present a simple and fast colorimetric method for CRP detection that employs citrate-capped gold nanoparticles (AuNPs) and a CRP-binding aptamer as sensing elements. The aptamer consisted in a guanine rich single-stranded DNA (ssDNA) that adsorbs onto the surface of the AuNPs. In the presence of the CRP, the ssDNA releases from the AuNPs surface to interact preferentially with the protein to form guanine-quadruplexes. The exposure of the unprotected AuNPs to buffer salts leads to aggregation and subsequent color change from red-wine to blue-purple that was readily seen by the naked eye. The AuNPs aggregation was monitored using UV-Vis spectroscopy and the CRP concentration in the samples could be correlated with the aggregation ratio (A 670nm /A 520nm ). A linear sensing range of 0.889-20.7 μg/mL was found. The detection limit (LOD) was 1.2 μg/mL which is comparable to the typical clinical cutoff concentration in high-sensitivity CRP assays (1 μg/mL) and lower than the detection limit of nephelometric methods used in clinical practice. This method can provide a fast (5 min analysis time), simple, and sensitive way for CRP detection, with negligible interference of bovine serum albumin (BSA) up to concentrations of 100 nM., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Extracellular vesicles (EVs) are lipid bilayer-bounded particles that are actively synthesized and released by cells. The main components of EVs are lipids, proteins, and nucleic acids and their composition is characteristic to their type and origin, and it reveals the physiological and pathological conditions of the parent cells. The concentration and protein composition of EVs closely relate to their functions; therefore, total protein determination can assist in EV-based diagnostics and disease prognosis. Here, we present a simple, reagent-free method based on attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy to quantify the protein content of EV samples without any further sample preparation. After calibration with bovine serum albumin, the protein concentration of red blood cell-derived EVs (REVs) were investigated by ATR-FTIR spectroscopy. The integrated area of the amide I band was calculated from the IR spectra of REVs, which was proportional to the protein quantity in the sample' regardless of its secondary structure. A spike test and a dilution test were performed to determine the ability to use ATR-FTIR spectroscopy for protein quantification in EV samples, which resulted in linearity with R 2 values as high as 0.992 over the concentration range of 0.08 to 1 mg/mL. Additionally, multivariate calibration with the partial least squares (PLS) regression method was carried out on the bovine serum albumin and EV spectra. R 2 values were 0.94 for the calibration and 0.91 for the validation set. The results indicate that ATR-FTIR measurements provide a reliable method for reagent-free protein quantification of EVs. Graphical abstract.

A high-performance molecularly imprinted sensing platform inspired by natural recognition mechanisms was fabricated to detect protein by employing a linear electro-polymerizable molecularly imprinted polymer as macromonomer. This was achieved via the combination of a biosensor fabrication with a self-assembly imprinting technique without the use of chemical labels. An amphipathic electroactive copolymer was designed as macro-monomer to maintain structural integrity of the protein template via self-assembly, resulting in generation of a 3D construction around the protein molecule to form imprinted sites. Electro-polymerization was utilized not only to anchor imprinted sites but also to enhance electron transfer. The adaptable sensing platform was based on a strengthened recognition reaction between the MIP layer and template protein after the generation of an electroactive network. Bovine serum albumin (BSA) and trypsin were used as model proteins to investigate the method's generality, which gave broad detection ranges of 10 -14 -10 -5  mg mL -1 for BSA and 10 -13 -10 -8  mg mL -1 for trypsin. These results indicate that the proposed fabrication offers an effective and versatile strategy for protein recognition., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Isobaric peptide termini labeling (IPTL) is an attractive protein quantification method because it provides more accurate and reliable quantification information than traditional isobaric labeling methods (e.g., TMT and iTRAQ) by making use of the entire fragment-ion series instead of only a single reporter ion. The multiplexing capacity of published IPTL implementations is, however, limited to three. Here, we present a selective maleylation-directed isobaric peptide termini labeling (SMD-IPTL) approach for quantitative proteomics of LysC protein digestion. SMD-IPTL extends the multiplexing capacity to 4-plex with the potential for higher levels of multiplexing using commercially available 13 C/ 15 N labeled amino acids. SMD-IPTL is achieved in a one-pot reaction in three consecutive steps: (1) selective maleylation at the N-terminus; (2) labeling at the ε-NH 2 group of the C-terminal Lys with isotopically labeled acetyl-alanine; (3) thiol Michael addition of an isotopically labeled acetyl-cysteine at the maleylated N-terminus. The isobarically labeled peptides are fragmented into sets of b- and y-ion clusters upon LC-MS/MS, which convey not only sequence information but also quantitative information for every labeling channel and avoid the issue of ratio distortion observed with reporter-ion-based approaches. We demonstrate the SMD-IPTL approach with a 4-plex labeled sample of bovine serum albumin (BSA) and yeast lysates mixed at different ratios. With the use of SMD-IPTL for labeling and a narrow precursor isolation window of 0.8 Th with an offset of -0.2 Th, accurate ratios were measured across a 10-fold mixing range of BSA in a background of yeast proteome. With the yeast proteins mixed at ratios of 1:5:1:5, BSA was detected at ratios of 0.94:2.46:4.70:9.92 when spiked at 1:2:5:10 ratios with an average standard deviation of peptide ratios of 0.34.

With its ability to continuously separate and collect charged analytes, free-flow electrophoresis (FFE) has become a useful tool for the purification and real-time analysis of biological mixtures. This work presents a new free-flow counterflow gradient focusing (FF-CGF) mechanism that uses a novel velocity gradient to counterbalance electrophoretic migration. This counterflow gradient is created by simply introducing fluid flow through the sidewalls of the FFE chamber. The theoretical foundation and device design for FF-CGF are provided in this work, followed by implementation and validation, with a detailed discussion on future opportunities and challenges. Initial results show promise, with the potential to improve FFE resolution and offer versatility. Compared to existing focusing techniques, such as free-flow isotachophoresis and isoelectric focusing, no complex buffer compositions are required.

Flavonoids interfere with colorimetric protein assays in a concentration- and structure-dependent manner. Degree (≥3) and position (C 3 ) of -OH substitution was associated with intensified interference (p < 0.05). Significant overestimation of protein (~3-5 folds) could occur at higher flavonoid concentrations (>5 μM) and is particularly evident at lower protein concentrations (25-250 μg/ml). Since, healthy human urinary protein (<200 μg/ml) and flavonoids urinary excretion (0.5-2 μg/ml) levels fall in this range, overestimation of protein concentration with flavonoids consumption in diet, including natural supplements, remains relevant issue for diagnostic and research labs. Protein precipitation by acetone to remove interfering flavonoid successfully resolves the problem., Competing Interests: Declaration of competing interest Authors have no conflict of interest to declare., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The aflatoxin (AF) albumin adduct is often used as a biomarker for aflatoxin exposure in humans. An ELISA method previously used for aflatoxin serum albumin in human blood was used to analyse bovine serum samples (n = 22) collected from dairy cattle during an aflatoxin mitigation study in Kenya. Albumin adduct data were compared with aflatoxin M 1 (AFM 1 ) levels in corresponding milk samples from these cows. The concentration ranged from < LOD to 487.9 pg/mL for AFM 1 and < LOD and 96.3 pg/mg for aflatoxin-albumin. This study indicates that aflatoxin-albumin adducts could be used as a measure of chronic aflatoxin exposure in dairy cattle.

Ultrasensitive detection of nonlabelled bovine serum albumin is performed in micro/nanofluidic chips using a photothermal optical phase shift (POPS) detection system. Currently, micro- and nanofluidics allow the analysis of various single cells, and their targets of interest are shifting from nucleic acids to proteins. Previously, our group developed photothermal detection techniques for the sensitive detection of nonfluorescent molecules. For example, we developed a thermal lens microscope (TLM) with ultrahigh sensitivity at the single-molecule level and a POPS detector that is applicable to nanochannels smaller than the wavelength of light. The POPS detector also realized the detection of nonlabelled proteins in nanochannels, although its detection sensitivity is less than that of the TLM in microchannels due to insufficient background light reduction. To overcome this problem, we developed a new POPS detector using relay optics for further reduction of the background light. In addition, heat transfer from the sample solution to the nanochannel wall was thoroughly investigated to achieve ultrahigh sensitivity. The limit of detection (LOD) obtained with the new POPS detector is 30 molecules in 1.0 fL. Considering this LOD, the performance of the new POPS detector is comparable with that of the TLM. Owing to the applicability of the POPS detector for sensitive detection even in nanochannels or single-μm channels, which cannot be realized with the TLM, combinations of the POPS detector and separation techniques employing unique nanochannel properties will contribute to advances in single-cell proteomics in the future.

Determining the concentration of protein samples generally is accomplished either by measuring the UV absorbance at 280 nm or by reacting the protein quantitatively with dyes and/or metal ions (Bradford, Lowry, or BCA assays). For purified proteins, UV absorbance remains the most popular method because it is fast, convenient, and reproducible; it does not consume the protein; and it requires no additional reagents, standards, or incubations. No method of protein concentration determination is perfect because each is subject to a different set of constraints such as interference of buffer components and contaminating proteins in direct UV determination ( A 280 ) or reactivity of individual proteins and buffer components with the detecting reagents in colorimetric assays. In cases in which protein concentration is critical (e.g., determination of catalytic rate constants for an enzyme), it may be advisable to compare the results of several assays., (© 2020 Cold Spring Harbor Laboratory Press.)

Protein analysis by desorption electrospray ionization mass spectrometry (DESI-MS) is limited and often accompanied by a mass-dependent loss in sensitivity as protein molecular weight increases. Previously, incomplete dissolution was identified as a potential contributing factor to this limitation for larger proteins. Here, we developed a unique two-step configuration in which a prewetting solvent is applied to the sample surface proximal to DESI analysis by a wetting quill to increase dissolution time and the detection of larger proteins. After optimizing the system with a mixture of proteins containing cytochrome c, myoglobin, and chymotripsinogen, we demonstrate the ability of delayed desorption to improve the analysis of larger proteins such as bovine serum albumin. Albumin and other serum proteins, including even larger ones, were also detected directly from diluted goat serum. An additional feature of this technique is the ability to deliver multiple solvents with potential synergistic or cooperative effects. For example, when using acetonitrile solutions of formic acid and ammonium bicarbonate as the prewetting and DESI spray solvent, respectively, the intensity of chymotrypsinogen improved dramatically compared to controls but less so for smaller proteins such as myoglobin and cytochrome c. Adduct removal was also observed for all proteins. These early results demonstrate the ability of this two-step technique for the use of multiple additives and increased dissolution times compared to standard DESI-MS experiments.

Protein modification by chemical reagents has played an essential role in the treatment of human diseases. However, the reagents currently used are limited to the covalent modification of cysteine and lysine residues. It is thus desirable to develop novel methods that can covalently modify other residues. Despite the fact that the carboxyl residues are crucial for maintaining the protein function, few selective labeling reactions are currently available. Here, we describe a novel reactive probe, 3-phenyl-2 H -azirine, that enables chemoselective modification of carboxyl groups in proteins under both in vitro and in situ conditions with excellent efficiency. Furthermore, proteome-wide profiling of reactive carboxyl residues was performed with a quantitative chemoproteomic platform.

The Bradford assay is a quick and fairly sensitive method for measuring the concentrations of proteins. It is based on the shift in absorbance maximum of Coomassie Brilliant Blue G-250 dye from 465 to 595 nm following binding to denatured proteins in solution., (© 2020 Cold Spring Harbor Laboratory Press.)

We developed an electrolyte pre-deposition-based saliva pattern modulation method to detect ovulation with high accuracy and reliability. Ovulation tests using human saliva have advantages in terms of the earlier ovulation detection and more convenient sample collection procedure; however, accuracy is low, which is a critical limitation given that the concentrations of salivary constituents can vary depending on the health status of the tested individual and subjective user judgement of the test result. In this study, we quantitatively analyzed saliva patterns according to the concentrations of electrolytes and proteins in the ovulation test and found that changes in the saliva pattern during the ovulatory period can be controlled by sodium chloride (NaCl) pre-deposition, which directly affects the accuracy of ovulation detection. The 100 nmol NaCl pre-deposition condition proved optimal, being two-fold more sensitive to changes in saliva pattern versus the non-pre-deposition condition (accuracy of ovulation detection = 66.6% and 33.3%, respectively). Although accuracy remained insufficient for actual applications compared to the urine-based ovulation detection method, we expect that the electrolyte pre-deposition method will greatly contribute to enhancing the performance of saliva-based ovulation detection tests, toward a commercially satisfactory level of accuracy.