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Porous organic cages (POCs) are an emerging class of porous materials that have aroused considerable research interest because of their unique characteristics, including good solubility and a well-defined intrinsic cavity. However, there have so far been no reports of chiral POCs as chiral stationary phases (CSPs) for enantioseparation by high-performance liquid chromatography (HPLC). Herein, we report the first immobilization of a chiral POC, NC1-R, on thiol-functionalized silica using a mild thiol-ene click reaction to prepare novel CSPs for HPLC. Two CSPs (CSP-1 and CSP-2) with different spacers have been prepared. CSP-1, with a cationic imidazolium spacer, exhibited excellent enantioselectivity for the resolution of various racemates. Twenty-three and 12 racemic compounds or chiral drugs were well enantioseparated on the CSP-1-packed column under normal-phase and reversed-phase conditions, respectively, including alcohols, diols, esters, ethers, ketones, epoxides, organic acids, and amines. In contrast, chiral resolution using CSP-2 (without a cationic imidazolium spacer)-packed column B was inferior to that of column A, demonstrating the important role of the cationic imidazolium spacer for chiral separation. The chiral separation capability of column A was also compared with that of two most popular commercial chiral columns, Chiralpak AD-H and Chiralcel OD-H, which exhibits good chiral recognition complementarity with the two commercial chiral columns. In addition, five positional isomers dinitrobenzene, nitroaniline, chloroaniline, bromoaniline, and iodoaniline were also well separated on column A. The effects of temperature, mobile phase composition, and injected analyte mass for separation on column A were investigated. Column A also showed good stability and reproducibility after repeated injections. This work demonstrates that chiral POCs are promising chiral materials for HPLC enantioseparation.

Large cohorts of samples from multiple batches are usually required for global metabolomic studies to characterize the metabolic state of human disease. As such, it is critical to eliminate systematic variation and truly reveal the biologically associated alterations. In this study, we proposed a reference material-based approach (Ref-M) for data correction by liquid chromatography-mass spectrometry and represented by an analysis of multibatch human serum samples. The reference material was generated by mixing serum from healthy donors and distributed to each extraction batch of subject samples. Pooled quality control samples and isotopic internal standards were then applied in each acquisition batch for data quality control. Finally, each metabolite in subject samples was normalized by its counterpart in the reference serum. We demonstrated that Ref-M significantly enhanced the numbers of efficient features and effectively eliminated the batch variation of 522 serum samples of healthy individuals, benign pulmonary nodules, and lung cancer patients. Twenty differential metabolites were identified to distinguish lung cancer from healthy controls in the training set. The discriminant model was validated in an independent data set with an area under the receiver operating characteristics (ROC) curve (AUC) of 0.853. Another 40 serum samples further tested with Ref-M were achieved an AUC of 0.843 by the established model. Our results showed that the reference material-based approach presents the potential to improve the data comparability and precision for biomarker discovery in large-scale metabolomic studies.

N-linked protein glycosylation is a key regulator in various biological functions. Previous studies have shown that aberrant glycosylation is associated with many diseases. Therefore, it is essential to elucidate protein modifications of glycosylation by quantitatively profiling intact N-linked glycopeptides. Data independent acquisition (DIA) mass spectrometry (MS) is a cost-effective, flexible, and high throughput method for global proteomics. However, substantial challenges are still present in the quantitative analysis of intact glycopeptides with high accuracy at high throughput. In this study, we have established a novel integrated platform for the DIA analysis of intact glycopeptides isolated from complex samples. The established analysis platform utilizes a well-designed DIA-MS method for raw data collection, a spectral library constructed specifically for intact glycopeptide quantification providing accurate results by the inclusion of Y ions for quantification, and filtering of quantified intact glycopeptides with low-quality MS2 spectra automatically using a set of criteria. Intact glycopeptides isolated from human serum were used to evaluate the performance of the integrated platform. By utilizing 100 isolation windows for DIA data acquisition, a well-constructed human serum spectral library containing 1123 non-redundant intact glycopeptides with Y ions, and the automated data inspection, 620 intact glycopeptides were quantified with high confidence from DIA-MS. In summary, our integrated platform can serve as a reliable quantitative tool for characterizing intact glycopeptides isolated from complex biological samples to assist our understanding of biological functions of N-linked glycosylation.

Recent advances in mass spectrometry (MS)-based proteomics allow the measurement of turnover rates of thousands of proteins using dynamic labeling methods, such as pulse stable isotope labeling by amino acids in cell culture (pSILAC). However, when applying the pSILAC strategy to multicellular animals (e.g., mice), the labeling process is significantly delayed by native amino acids recycled from protein degradation in vivo, raising a challenge of defining accurate protein turnover rates. Here, we report JUMPt, a software package using a novel ordinary differential equation (ODE)-based mathematical model to determine reliable rates of protein degradation. The uniqueness of JUMPt is to consider amino acid recycling and fit the kinetics of the labeling amino acid (e.g., Lys) and whole proteome simultaneously to derive half-lives of individual proteins. Multiple settings in the software are designed to enable simple to comprehensive data inputs for precise analysis of half-lives with flexibility. We examined the software by studying the turnover of thousands of proteins in the pSILAC brain and liver tissues. The results were largely consistent with the proteome turnover measurements from previous studies. The long-lived proteins are enriched in the integral membrane, myelin sheath, and mitochondrion in the brain. In summary, the ODE-based JUMPt software is an effective proteomics tool for analyzing large-scale protein turnover, and the software is publicly available on GitHub (https://github.com/JUMPSuite/JUMPt) to the research community.

Small extracellular vesicles (sEVs), often referred to as exosomes, are potential biomarkers for noninvasive cancer diagnosis. However, because of their phenotype heterogeneity, precise detection of tumor-derived sEVs is a great challenge. Herein, a dual-aptamer-assisted AND logic gate was fabricated for sensitive electrochemical detection of tumor-derived sEVs based on a cyclic enzymatic signal amplification strategy. Four different tumor-derived sEVs were used to verify the feasibility of the AND logic gate, and CCRF-CEM sEVs were successfully detected by this assay. The electrochemical assay shows a good linear response from 4 × 103 to 8 × 107 particles/μL, with a detection limit of 920 particles/μL, for CCRF-CEM sEVs, indicating potential application in accurate cancer diagnostics.

The ratiometric detection of cysteine (Cys) and homocysteine (Hcy) is very challenging because of their highly similar chemical structures and properties. By introducing the phenylethynyl group into a coumarin dye as the sensing group, the ratiometric fluorescent probe CP was developed to selectively and rapidly discriminate between Cys and Hcy. With a single-wavelength excitation, the presence of Cys or Hcy induced the probe to produce distinct ratiometric fluorescence changes: from red (λmaxem = 608 nm) to blue (λmaxem = 485 nm) toward Cys and from red to mixed red/blue toward Hcy. Moreover, the probe was capable of visualizing and discriminating between intracellular Cys and Hcy in HeLa cells and zebrafish by exhibiting different ratiometric fluorescence signals.

Homocysteine (Hcy) is one of the important biomarkers of clinical diagnosis, which is closely related to the occurrence and development of many diseases. Current analysis methods have difficulties in detecting Hcy in cells and living organisms. As a powerful technique, fluorescence methods combined the laser confocal imaging technology can achieve real-time visual tracking in cells and in vivo. Herein, we establish a conjugated copolymer-based fluorescence nanosensor (DPA-PFNP-Cu(II)) using the connected 2,7-dibromofluorene and 4,7-bis (2-bromothiophen-5-yl)-2-1-3-benzothiadiazole as the main chain. The competitive coordination between Hcy and Cu(II) allows the fluorescence of the polymer off to on. Finally, the nanosensor is applied for in situ imaging of Hcy levels in the kidney and liver of diabetic mice and is found that Hcy levels were positively correlated with the degree of diabetes. Notably, the depth of tissue penetration of the nanosensor enables Hcy detection of the liver and kidney through in vivo imaging without damage. Two-photon imaging and in vivo imaging achieve consistent results, which correct each other, improving the accuracy of the test result. The present works provide a new imaging technique for studying the occurrence and development of diabetes and screening of new drugs for treatment at the living level.

MicroRNAs (miRNAs) in cancer cell-derived exosomes are important cancer biomarkers. Herein, a sensitive hybridization chain reaction (HCR) electrochemical assay was fabricated for the detection of exosomal microRNA-122 (miR-122). The hairpin DNA (hpDNA) probes were first immobilized on the surface of a gold electrode. In the presence of miR-122, the hairpin structure of the hpDNA could be opened and triggered the HCR through the cross-opening and hybridization of two helper DNA hairpins. Long nicked double helixes generated from HCR are used to capture more RuHex and increase the signal of differential pulse voltammetry (DPV). In this assay, the density of the hpDNA probes on the surface of the gold electrode was precisely controlled by the simultaneous immobilization of hpDNA and short 12 nucleotides single-stranded DNA (S-12), providing a very high amplification efficiency. More importantly, the false positive signal could be reduced or completely eliminated by applying exonuclease I (Exo I) before the introduction of target miR-122. Under optimal conditions, the assay offers very high sensitivity with an attomolar level detection limit, a linear range with 9 orders of magnitude, and specificity in single mismatch discrimination. This sensitive electrochemical assay could successfully evaluate the miR-122 concentration in different cancer-derived exosomes, indicating its potential use in cancer diagnostics.

Methodologies that facilitate high-throughput proteomic analysis are a key step toward moving proteome investigations into clinical translation. Data independent acquisition (DIA) has potential as a high-throughput analytical method due to the reduced time needed for sample analysis, as well as its highly quantitative accuracy. However, a limiting feature of DIA methods is the sensitivity of detection of low abundant proteins and depth of coverage, which other mass spectrometry approaches address by two-dimensional fractionation (2D) to reduce sample complexity during data acquisition. In this study, we developed a 2D-DIA method intended for rapid- and deeper-proteome analysis compared to conventional 1D-DIA analysis. First, we characterized 96 individual fractions obtained from the protein standard, NCI-7, using a data-dependent approach (DDA), identifying a total of 151,366 unique peptides from 11,273 protein groups. We observed that the majority of the proteins can be identified from just a few selected fractions. By performing an optimization analysis, we identified six fractions with high peptide number and uniqueness that can account for 80% of the proteins identified in the entire experiment. These selected fractions were combined into a single sample which was then subjected to DIA (referred to as 2D-DIA) quantitative analysis. Furthermore, improved DIA quantification was achieved using a hybrid spectral library, obtained by combining peptides identified from DDA data with peptides identified directly from the DIA runs with the help of DIA-Umpire. The optimized 2D-DIA method allowed for improved identification and quantification of low abundant proteins compared to conventional unfractionated DIA analysis (1D-DIA). We then applied the 2D-DIA method to profile the proteomes of two breast cancer patient-derived xenograft (PDX) models, quantifying 6,217 and 6,167 unique proteins in basal- and luminal- tumors, respectively. Overall, this study demonstrates the potential of high-throughput quantitative proteomics using a novel 2D-DIA method.

Cells perform various functions by proteins via protein complexes. Characterization of protein complexes is critical to understanding their biological and clinical significances and has been one of the major efforts of functional proteomics. To date, most protein complexes are characterized by in vitro system from protein extracts after cells or tissues are lyzed, and it has been challenging to determine which of these protein complexes are formed in intact cells. Herein, we report an approach to preserve protein complexes using in vivo cross-linking followed by size exclusion chromatography and data independent acquisition mass spectrometry. This approach enables the characterization of in vivo protein complexes from cells or tissues, which allows the determination of protein complexes in clinical research. More importantly, the described approach can identify protein complexes that are not detected by in vitro system, which provide unique protein function information.

For better investigating the complicated relationships between H

The ratiometric detection of cysteine (Cys) and homocysteine (Hcy) is very challenging because of their highly similar chemical structures and properties. By introducing the phenylethynyl group into a coumarin dye as the sensing group, the ratiometric fluorescent probe

We describe a mass spectrometry (MS) analytical platform resulting from the novel integration of acoustic droplet ejection (ADE) technology, an open-port interface (OPI), and electrospray ionization (ESI)-MS that creates a transformative system enabling high-speed sampling and label-free analysis. The ADE technology delivers nanoliter droplets in a touchless manner with high speed, precision, and accuracy. Subsequent sample dilution within the OPI, in concert with the capabilities of modern ESI-MS, eliminates the laborious sample preparation and method development required in current approaches. This platform is applied to a variety of experiments, including high-throughput (HT) pharmacology screening, label-free in situ enzyme kinetics, in vitro absorption, distribution, metabolism, elimination, pharmacokinetic and biomarker analysis, and HT parallel medicinal chemistry.

Here, we fabricated a novel photoelectrochemical (PEC) aptasensor based on Br,N-codoped TiO2/CdS quantum dots (QDs) sensitization structure with excellent energy level arrangement for supersensitiv...

Biothiols are essential reactive sulfur species (RSS), which play crucial roles in various critical physiological activities. To clarify their complex correlations in signal transduction and metabolism pathways, methods to distinguish H2S, cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) at the same time are highly needed. Herein, we deliberately integrate 7-diethylcoumarin and resorufin through an ether bond to develop RC for simultaneous differentiation of Cys, Hcy, GSH, and H2S for the first time. RC displays distinct fluorescence colors in response to H2S, Cys, Hcy, and GSH as red, blue-red, blue-green-red, and green-red, respectively, both in aqueous media and living cells. This work not only reported a robust molecule, RC, to disentangle the intricate interplay networks among different biothiols in biosystems but also demonstrated a strategy for the design of multisignal fluorescent probes for the simultaneous sensing of diverse RSS species as well as other biological species.

Homocysteine (Hcy) is one of the important biomarkers of clinical diagnosis, which is closely related to the occurrence and development of many diseases. Current analysis methods have difficulties in detecting Hcy in cells and living organisms. As a powerful technique, fluorescence methods combined the laser confocal imaging technology can achieve real-time visual tracking in cells and

Tn-antigen (Tn), a single N-acetylgalactosamine (GalNAc) monosaccharide attached to protein Ser/Thr residues, is found on most cancer yet rarely detected in adult normal tissues as reported in previous studies, featuring it as one of the most distinctive signatures of cancer. Although it is important in cancer, Tn-antigen modified glycoproteins are not entirely clear owing to the lack of a suitable method. Knowing the Tn-glycosylated proteins and glycosylation sites are essential to the diagnosis and therapeutics of cancer associated with the expression of Tn. Here, we introduce a method named EXoO-Tn for large-scale mapping of Tn-glycosylated proteins and glycosylation sites. EXoO-Tn utilizes solid-phase immobilization of proteolytic peptides of proteins, modifies Tn-antigens by glycosyltransferase C1GalT1 with isotopically-labeled UDP-Gal((13)C(6)) to tag and convert Tn-antigen to Gal((13)C(6))-Tn, which gives rise to a unique glycan mass. The exquisite Gal((13)C(6)) modified Tn-antigens are then recognized by a human-gut-bacterial enzyme, OpeRATOR, and released at the N-termini of the Gal((13)C(6))-Tn-occupied Ser/Thr residues from immobilized peptides to yield site-containing glycopeptides. The effectiveness of EXoO-Tn was benchmarked by analyzing Jurkat cells, where 947 Tn-glycosylation sites from 480 glycoproteins were mapped. The EXoO-Tn was further applied for the analysis of pancreatic cancer sera, where Tn-glycoproteins were identified. Given the significance of Tn-antigen in cancer, EXoO-Tn is anticipated to have a broad utility in clinical investigations.

Here, a photoelectrochemical (PEC) biosensor was established by a cosensitization strategy with cascade energy level arrangement for ultrasensitive detection of prostate-specific antigen (PSA). The proposed cosensitization strategy was based on the well-matched energy level arrangement of four kinds of organic photoactive materials, in which poly{4,8-bis[5-(2-ethylhexyl)thiophen-2-yl]benzo[1,2-b:4,5-b′]dithiophene-2,6-diyl-alt-3-fluoro-2-[(2-ethylhexyl)-carbonyl]thieno[3,4-b]thiophene-4,6-diyl} (PTB7-Th) was used as the photoactive material and perylenetetracarboxyl diimide (PDI), fullerene (nano-C60), and polyaniline (PANI) were employed as the sensitizers. The resulting PTB7-Th/PDI/nano-C60/PANI cascade cosensitization structure with narrow energy level gradient (

Aberrant glycosylation has been shown to associate with disease progression, and with glycoproteins representing the major protein component of biological fluids this makes them attractive targets for disease monitoring. Leveraging glycoproteomic analysis via mass spectrometry (MS) could provide the insight into the altered glycosylation patterns that relate to disease progression. However, investigation of large sample cohorts requires rapid, efficient, and highly reproducible sample preparation. To address the limitation, we developed a high-throughput method for characterizing glycans, glycosites, and intact glycopeptides (IGPs) derived from N-linked glycoproteins. We combined disparate peptide enrichment strategies (i.e., hydrophilic and hydrophobic) and a liquid handling platform allowing for a high throughput and rapid enrichment of IGP in a 96-well plate format. The C18/MAX-Tip workflow reduced sample processing time and facilitated the selective enrichment of IGPs from complex samples. Furthermore, our approach enabled the analysis of deglycosylated peptides and glycans from enriched IGPs following PNGase F digest. Following development and optimization of the C18/MAX-Tip methodology using the standard glycoprotein, fetuin, we investigated normal urine samples to obtain N-linked glycoprotein information. Together, our method enables a high-throughput enrichment of glycan, glycosites, and IGPs from biological samples.

Recently, the rapid development and application of mass spectrometry (MS)-based technologies have markedly improved the comprehensive proteomic characterization of global proteome and protein post-translational modifications (PTMs). However, the current conventional approach for global proteomic analysis is often carried out separately from PTM analysis. In our study, we developed an integrated workflow for multiplex analysis of global, glyco-, and phosphor-proteomics using breast cancer patient-derived xenograft (PDX) tumor samples. Our approach included the following steps: trypsin-digested tumor samples were enriched for phosphopeptides through immobilized metal ion affinity chromatography (IMAC), followed by enrichment of glycopeptides through mixed anion exchange (MAX) method, and then the flow-through peptides were analyzed for global proteomics. Our workflow demonstrated an increased identification of peptides and associated proteins in global proteome, as compared to those using the peptides without PTM depletion. In addition to global proteome, the workflow identified phosphopeptides and glycopeptides from the PTM enrichment. We also found a subset of glycans with unique distribution profiles in the IMAC flow-through, as compared to those enriched directly using the MAX method. Our integrated workflow provided an effective platform for simultaneous global proteomic and PTM analysis of biospecimens.

In this study, we present a simple but effective fluorescent system for highly sensitive and versatile sensing of 2,4,6-trinitrotoluene (TNT) using few layered planar MoS2 quantum dots (QDs) as reporters. Excitation-independent emitting MoS2 QDs were first fabricated by using the proposed ultrasonic–hydrothermal-based top-down method assisted by carbon-free hydroxylamine hydrochloride. The obtained pristine MoS2 QDs were then modified with cysteine for introducing amino groups as TNT binding sites. The as-prepared MoS2 QDs possess a planar structure, which can more adequately interact with flat aromatic TNT molecules due to π–π attraction and decreased steric effects, compared with traditional spherical/quasi-spherical QDs. As a result, they exhibit extremely high sensitivity for TNT sensing (1 nM and 2 ng for solution and substrate assay, respectively). The common ions containing in environmental water samples do not interfere with the sensing. Furthermore, the QDs-decorated test paper shows an instantan...

Fucosylation (Fuc) of glycoproteins plays an important role in regulating protein function and has been associated with the development of several cancer types including prostate cancer (Pca). Therefore, the research of Fuc glycoproteins has attracted increasing attention recently in the analytical field. Herein, a strategy based on lectin affinity enrichments of intact glycopeptides followed by mass spectrometry has been established to evaluate the specificities of various Fuc-binding lectins for glycosite-specific Fuc analysis of nonaggressive (NAG) and aggressive (AG) Pca cell lines. The enrichment specificities of Fuc glycopeptides using lectins (LCA, PSA, AAL, LTL, UEA I, and AOL) and MAX extraction cartridges alone, or in tandem, were evaluated. Our results showed that the use of lectin enrichment significantly increased the ratio of fucosylated glycopeptides to total glycopeptides compared to MAX enrichment. Furthermore, tandem use of lectin followed by MAX increased the number of identifications of Fuc glycopeptides compared to using lectin enrichment alone. LCA, PSA, and AOL showed stronger binding capacity than AAL, LTL, and UEA I. Also, LCA and PSA bound specifically to core Fuc, whereas AOL, AAL, and UEA I showed binding to both core Fuc and branch Fuc. The optimized enrichment method with tandem enrichment of LCA followed by MAX (LCA-MAX) was then applied to examine the Fuc glycoproteomes in two NAG and two AG Pca cell lines. In total, 973 intact Fuc glycopeptides were identified and quantified from 252 Fuc proteins by using the tandem-mass-tags (TMT) labeling and nanoliquid chromatography-mass spectrometry (nanoLC-MS/MS) analysis. Further data analysis revealed that 51 Fuc glycopeptides were overexpressed more than 2-fold in AG cell lines compared to NAG cells. The analysis of protein core fucosylation has great potential for aiding our understanding of invasive activity of AG Pca and may lead to the development of diagnostic approaches for AG Pca.

Sialic acids play many important roles in several physiological and pathological processes, including cancers, infection and blood diseases. Sialic acids are fragile and prone to fragmentation under electrospray ionization and matrix-assisted laser desorption/ionization. It is crucial to modify sialic acids for qualitative and quantitative identification of their change in abundance in complex biological samples. Permethylation is a method of choice for sialic acid stabilization, but the harsh conditions during permethylation may lead to the decomposition of O-acetyl groups. Esterification or amidation in solution effectively protects sialic acids, yet, it is not trivial to purify glycans from their reagents. Quantitative analysis of glycans can be achieved by labeling their reducing end using fluorescent tags. Loss of sialic acids during labeling is a major concern. In this study, we demonstrated the utility of sialic acids modification for the analysis sialyl oligosaccharides and glycopeptides. Without modification, sialic acids are partially or completely lost during sample preparation, leading to the presence of false glycans or glycopeptides in the sample. The stabilized sialic acids not only result in accurate identification of sialylated glycans, but they also improve the characterization of intact glycopeptides. The modification of sialic acids on the solid support facilitates analysis of glycans and their intact glycoproteins.

Isobaric labeling quantification by mass spectrometry (MS) has emerged as a powerful technology for multiplexed large-scale protein profiling, but measurement accuracy in complex mixtures is confounded by the interference from coisolated ions, resulting in ratio compression. Here we report that the ratio compression can be essentially resolved by the combination of pre-MS peptide fractionation, MS2-based interference detection, and post-MS computational interference correction. To recapitulate the complexity of biological samples, we pooled tandem mass tag (TMT)-labeled Escherichia coli peptides at 1:3:10 ratios and added in ∼20-fold more rat peptides as background, followed by the analysis of two-dimensional liquid chromatography (LC)–MS/MS. Systematic investigation shows that quantitative interference was impacted by LC fractionation depth, MS isolation window, and peptide loading amount. Exhaustive fractionation (320 × 4 h) can nearly eliminate the interference and achieve results comparable to the MS3...

Modified cytosines are important epigenetic marks that exert critical influences in a variety of cellular processes in living organisms. However, biological functions of rare modified cytosines, especially certain functions of 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), are still unclear due to the extremely low abundance in biological samples. In this work, a series of novel hydrazine-based reagents, which held a hydrazine group as the reaction group, a hydrophobic triazine group, and two easily charged tertiary amine groups with different alkyl chains for adjusting the hydrophobicity of the labeling reagents, were first explored to label rare modified cytosines such as 5fC and 5caC. The derivatization reaction between 5fC and the labeling reagents was extremely fast, and more than 99% derivatization efficiency could be achieved only by vortexing without additional reaction time. The detection sensitivity of 5fC increased with the increase of the hydrophobicity of the labeling reagents, the best of which was dramatically enhanced by 125-fold. The limit of detection was as low as 10 amol, realizing the most sensitive genome-wide overall quantification for 5fC. Moreover, the labeling reagents were also successfully applied for the detection of 5caC with 100-fold improvement of sensitivity. With this method, we achieved the simultaneous detection of 5fC and 5caC in different mammalian tissues using only about 600 ng of genomic DNA, which was less than one-tenth of the sample consumption for other reported methods, providing an opportunity to monitor 5fC and 5caC in precious samples and biology processes that could not be investigated before.

Here, we fabricated a novel photoelectrochemical (PEC) aptasensor based on Br,N-codoped TiO

Biothiols are essential reactive sulfur species (RSS), which play crucial roles in various critical physiological activities. To clarify their complex correlations in signal transduction and metabolism pathways, methods to distinguish H

Proteomics at single-cell resolution can help to identify the heterogeneity among cell populations, shows more and more significance in current chemistry and biology. In this work, we demonstrated a new single cell chemical proteomic (SCCP) strategy with a membrane-permeable activity-based probe (ABP) to characterize the functional proteins in lysosome located in the cytosol. The ABP targeted to the cysteine cathepsin family protein, CpFABP-G, was designed for cysteine cathepsins labeling. The labeled HeLa cell of a cancer cell line was injected into a capillary and was lysed by SDS solution with heating. The lysate was then online readout by capillary electrophoresis-laser-induced fluorescence method. Due to the employment of highly specified ABP kicking out the uncorrelated proteins, the expression of cysteine cathepsins in individual HeLa cells was easily detected, and heterogeneity among those HeLa cells was readily discriminated. Further work was concentrated on SCCP analysis of the mouse leukemia cell of monocyte macrophage (RAW264.7). It was for the first time identifying two expression modes of cysteine cathepsins in RAW264.7, which could be undermined by the analysis of cell populations. We believed that SCCP would be one of the powerful alternatives for proteomics at single-cell resolution.

Protein glycosylation plays fundamental roles in many cellular processes, and previous reports have shown dysregulation to be associated with several human diseases, including diabetes, cancer, and neurodegenerative disorders. Despite the vital role of glycosylation for proper protein function, the analysis of glycoproteins has been lagged behind to other protein modifications. In this study, we describe the re-analysis of global proteomic data from breast cancer xenograft tissues using recently developed software package GPQuest 2.0, revealing a large number of previously unidentifiedN-linked glycopeptides. More importantly, we found that using immobilized metal affinity chromatography (IMAC) technology for the enrichment of phosphopeptides had co-enriched a substantial number of sialoglycopeptides, allowing for a large-scale analysis of sialoglycopeptides in conjunction with the analysis of phosphopeptides. Collectively, combined MS/MS analyses of global proteomic and phosphoproteomic datasets resulted in the identification of 6,724 N-linked glycopeptides from 617 glycoproteins derived from two breast cancer xenograft tissues. Next, we utilized GPQuest for the re-analysis of global and phosphoproteomic data generated from 108 human breast cancer tissues that were previously analyzed by Clinical Proteomic Analysis Consortium (CPTAC). Reanalysis of the CPTAC dataset resulted in the identification of 2,683 glycopeptides from the global proteomic data set and 4,554 glycopeptides from phosphoproteomic data set, respectively. Together, 11,292 N-linked glycopeptides corresponding to 1,731 N-linked glycosites from 883 human glycoproteins were identified from the two data sets. This analysis revealed an extensive number of glycopeptides hidden in the global and enriched in IMAC-based phosphopeptide-enriched proteomic data, information which would have remained unknown from the original study otherwise. The reanalysis described herein can be readily applied to identify glycopeptides from already existing data sets, providing insight into many important facets of protein glycosylation in different biological, physiological, and pathological processes.

Most serum proteins are N-linked glycosylated, and therefore the glycoproteomic profiling of serum is essential for characterization of serum proteins. In this study, we profiled serum N-glycoproteome by our recently developed N-glycoproteomic method using solid-phase extraction of N-linked glycans and glycosite-containing peptides (NGAG) coupled with LC-MS/MS and site-specific glycosylation analysis using GPQuest software. Our data indicated that half of identified N-glycosites were modified by at least two glycans, with a majority of them being sialylated. Specifically, 3/4 of glycosites were modified by biantennary N-glycans and 1/3 of glycosites were modified by triantennary sialylated N-glycans. In addition, two novel atypical glycosites (with N–X–V motif) were identified and validated from albumin and α-1B-glycoprotein. The widespread presence of these two glycosites among individuals was further confirmed by individual serum analyses.

Sensitive and specific detection of tumor exosomes is of great significance for early cancer diagnosis. In this paper, we report an aptamer strategy for exosome detection based on aptamer recognition-induced multi-DNA release and cyclic enzymatic amplification. First, we use aptamer–magnetic bead bioconjugates to capture tumor exosomes derived from LNCaP cells, leading to the release of three kinds of messenger DNAs (mDNAs). After magnetic separation, the released mDNAs hybridized with the probe DNAs immobilized on a gold electrode. Electroactive Ru(NH3)63+ was used as the signal reporter because of its electrostatic attraction to DNA. Subsequent Exo III cyclic digestion caused the electrochemical signal to “turn off”. Because the electrochemical signal reflects the concentration of Ru(NH3)63+ and the concentration of Ru(NH3)63+ is correlated with the mDNA concentration, which is correlated with the exosome concentration, the tumor exosomes can be detected by examining the decrease in the peak current of ...

In this study, we present a simple but effective fluorescent system for highly sensitive and versatile sensing of 2,4,6-trinitrotoluene (TNT) using few layered planar MoS

Triethylamine (TEA), a common coreactant for electrochemiluminescence (ECL), is first utilized as a coreactant for chemiluminescence (CL). The CL intensity of carbon dots/K2S2O8 could be increased by ∼20 times in the presence of TEA. On the basis of this fascinating phenomenon, a room temperature operated senor is constructed for the fast, selective, and sensitive determination of TEA. A wide linear relationship between CL intensity and TEA concentration from 1 μM to 1000 μM (R(2) = 0.9995) was found with the detection limit down to 1 μM. The enhancement mechanism of TEA to this CL system is carefully investigated. Experimental results reveal that the forming of TEA free radical is what indeed induced the enhancement of the CL efficiency of CDs.

Protein glycosylation is one of the most common protein modifications, and the quantitative analysis of glycoproteins has the potential to reveal biological functions and their association with disease. However, the high throughput accurate quantification of glycoproteins is technically challenging due to the scarcity of robust assays to detect and quantify glycoproteins. Here we describe the development of multiplexed targeted MS assays to quantify N-linked glycosite-containing peptides in serum using parallel reaction monitoring (PRM). Each assay was characterized by its performance metrics and criteria established by the National Cancer Institute's Clinical Proteomic Tumor Analysis Consortium (NCI CPTAC) to facilitate the widespread adoption of the assays in studies designed to confidently detect changes in the relative abundance of these analytes. An in-house developed software program, MRMPlus, was used to compute assay performance parameters including specificity, precision, and repeatability. We show that 43 selected N-linked glycosite-containing peptides identified in prostate cancer tissue studies carried out in our group were detected in the sera of prostate cancer patients within the quantitative range of the developed PRM assays. A total of 41 of these formerly N-linked glycosite-containing peptides (corresponding to 37 proteins) were reproducibly quantified based on their relative peak area ratios in human serum during PRM assay development, with 4 proteins showing differential significance in serum from nonaggressive (NAG) vs aggressive (AG) prostate cancer patient serum (n = 50, NAG vs AG). The data demonstrate that the assays can be used for the high throughput and reproducible quantification of a panel of formerly N-linked glycosite-containing peptides. The developed assays can also be used for the quantification of formerly N-linked glycosite-containing peptides in human serum irrespective of disease state.

Post-translational modifications of proteins can have a major role in disease initiation and progression. Incredible efforts have recently been made to study the regulation of glycoproteins for disease prognosis and diagnosis. It is essential to elucidate glycans and intact glycoproteins to understand the role of glycosylation in diseases. Sialylated N-glycans play crucial roles in physiological and pathological processes; however, it is laborious to study sialylated glycoproteins due to the labile nature of sialic acid residues. In this study, an integrated platform is developed for the analysis of intact glycoproteins and glycans using a chemoenzymatic approach for immobilization and derivatization of sialic acids. N-Glycans, deglycosylated proteins, and intact glycoproteins from heart tissues of wild type (WT) and transverse aortic constriction (TAC) mouse models were analyzed. We identified 291 unique glycopeptides from 195 glycoproteins; the comparative studies between WT and TAC mice indicate the overexpression of extracellular proteins for heart matrix remodeling and the down-regulation of proteins associated with energy metabolism in cardiac hypertrophy. The integrated platform is a powerful tool for the analysis of glycans and glycoproteins in the discovery of potential cardiac hypertrophy biomarkers.

Herein, we proposed a portable, easy-to-operate, and antifouling microcapsule array chip for target detection. This prepackaged chip was fabricated by innovative and cost-effective 3D ice printing integrating with photopolymerization sealing which could eliminate complicated preparation of wet chemistry and effectively resist outside contaminants. Only a small volume of sample (2 μL for each microcapsule) was consumed to fulfill the assay. All the reagents required for the analysis were stored in ice form within the microcapsule before use, which guaranteed the long-term stability of microcapsule array chips. Nitrite and glucose were chosen as models for proof of concept to achieve an instant quantitative detection by naked eyes without the need of external sophisticated instruments. The simplicity, low cost, and small sample consumption endowed ice-printing microcapsule array chips with potential commercial value in the fields of on-site environmental monitoring, medical diagnostics, and rapid high-throughput point-of-care quantitative assay.

Isobaric labeling quantification by mass spectrometry (MS) has emerged as a powerful technology for multiplexed large-scale protein profiling, but measurement accuracy in complex mixtures is confounded by the interference from co-isolated ions, resulting in ratio compression. Here we report that the ratio compression can be essentially resolved by the combination of pre-MS peptide fractionation, MS2-based interference detection and post-MS computational interference correction. To recapitulate the complexity of biological samples, we pooled tandem mass tag (TMT) labeled E. coli peptides at 1 : 3 : 10 ratios, and added in ∼20-fold more rat peptides as background, followed by the analysis of two dimensional liquid chromatography (LC)-MS/MS. Systematic investigation show that quantitative interference was impacted by LC fractionation depth, MS isolation window and peptide loading amount. Exhaustive fractionation (320 × 4 h) can nearly eliminate the interference and achieve results comparable to the MS3-based method. Importantly, the interference in MS2 scans can be estimated by the intensity of contaminated y1 product ions, and we thus developed an algorithm to correct reporter ion ratios of tryptic peptides. Our data indicate that intermediate fractionation (40 × 2 h) and y1 ion-based correction allow accurate and deep TMT profiling of more than 10,000 proteins, which represents a straightforward and affordable strategy in isobaric labeling proteomics.

Envelope protein gp120 of human immunodeficiency virus (HIV) is armored with a dense glycan shield, which plays critical roles in envelope folding, immune-evasion, infectivity, and immunogenicity. Site-specific glycosylation profiling of recombinant gp120 is very challenging. Therefore, glycoproteomic analysis of native viral gp120 is still formidable to date. This challenge promoted us to employ a Q-Exactive mass spectrometer to identify low abundant glycopeptides from virion-associated gp120. To search the HCD-MS data for glycopeptides, a novel spectral-aligning strategy was developed. This strategy depends on the observation that glycopeptides and the corresponding deglycosylated peptides share very similar MS/MS pattern in terms of b- and y-ions that do not contain the site of glycosylation. Moreover, glycopeptides with an identical peptide backbone show nearly resembling spectra regardless of the attached glycan structures. For the recombinant gp120, this "copy-paste" spectral pattern of glycopeptides facilitated identification of 2224 spectra using only 18 spectral templates, and after precursor mass correction, 1268 (57%) spectra were assigned to 460 unique glycopeptides accommodating 19 N-linked and one O-linked glycosylation sites (glycosites). Strikingly, we were able to observe five N- and one O-linked glycosites in native gp120. We further revealed that except for Asn276 in the C2 region, glycans were processed to contain both high mannose and hybrid/complex glycans; an additional four N-linked glycosites were decorated with high mannose type. Core 1 O-linked glycan Gal1GalNAc1 was seen for the O-linked glycosite at Thr499. This direct observation of site-specific glycosylation of virion-derived gp120 has implications in HIV glycobiology and vaccine design.

Glycosylation is one of the most common protein modifications and profoundly regulates many biological processes. Aberrant glycosylation is reported to associate with diseases such as cancers, human immunodeficiency virus, and immune disorders. It is considerably important to study protein glycosylation and the associated glycans for diagnostics and disease prognostics. Unlike other protein modifications, glycans attached to proteins are enormously complex. Therefore, the comprehensive analysis of glycans from biological or clinical samples is an unmet technical challenge. Development of the high-throughput method will facilitate the glycomics analysis. In this study, we developed a novel method for the high-throughput analysis of N-glycans from glycoproteins using glycoprotein immobilization for glycan extraction (GIG) coupled with liquid chromatography (LC) in an integrated microfluidic platform (chipLC). The separated glycans were then analyzed by mass spectrometry. Briefly, proteins were first immobilized on a solid support. Glycans on immobilized glycoproteins were modified on solid phase to increase the detection and structure analysis. N-Glycans were then enzymatically released and subsequentially separated by porous graphitized carbon particles packed in the same device. By applying the GIG-chipLC for glycomic analysis of human sera, we identified N-glycans with 148 distinct N-glycan masses. The platform was used to analyze N-glycans from mouse heart tissue and serum. The extracted N-glycans from tissues indicated that unique unsialylated N-glycans were detected in tissues that were missing from the proximal or distal serum, whereas common N-glycans from tissues and serum have mature and sialylated structures. The GIG-chipLC provides a simple and robust platform for glycomic analysis of complex biological and clinical samples.

Glycosylation is one of the most common protein modifications and is involved in many functions of glycoproteins. Investigating aberrant protein glycosylation associated with diseases is useful in improving disease diagnostics. Due to the non-template nature of glycan biosynthesis, the glycans attached to glycoproteins are enormously complex; thus, a method for comprehensive analysis of glycans from biological or clinical samples is needed. Here, we describe a novel method for glycomic analysis using glycoprotein Immobilization for glycan extraction (GIG). Proteins or peptides from complex samples were first immobilized on solid support, and other non-conjugated molecules were removed. Glycans were enzymatically or chemically modified on solid-phase before releasing from glycoproteins/glycopeptides for mass spectrometry analysis. The method was applied to the glycomic analysis of both N- and O-glycans.

Effective detection of DNA methyltransferase (DNMT) activity is significant for cancer research. Herein, we developed a sensitive electroanalytical method to detect human DNA (cytosine-5)–methyltransferase 1 (DNMT1) from crude lysates of cancer cells. In this assay, capture DNA having a preferred DNMT1 methylation site was immobilized on a gold electrode and then hybridized with gold nanoparticle (Au NP)–DNA complexes. The modified electrodes were equilibrated with the lysate and then incubated with methylation-sensitive restriction enzyme. If the lysate was negative for DNMT1 activity, the Au NP–DNA complexes would be cut by the restriction enzyme and released from the electrode. Conversely, restriction enzyme cleavage would be blocked by the fully methylated duplexes, and the Au NP–DNA complexes would remain on the electrode. Electroactive Ru(NH3)63+ was used as the signal reporter, because of its electrostatic attraction to DNA, resulting in an electrochemical signal. Since the electrochemical signal r...

Here, we report a redox-mediated indirect fluorescence immunoassay (RMFIA) for the detection of the disease biomarker α-fetoprotein (AFP) using dopamine (DA)-functionalized CdSe/ZnS quantum dots (QDs). In this immunoassay, tyrosinase was conjugated with the detection antibody and acted as a bridge connecting the fluorescence signals of the QDs with the concentration of the disease biomarkers. The tyrosinase label used for RMFIA catalyzed the enzymatic oxidation of DAs on the surface of functionalized QDs and caused fluorescence quenching in the presence of the analyte. Using this technique, we obtained a limit of detection as low as 10 pM for AFP. This assay's potential for clinical analysis was demonstrated by detecting the real sera of patients with hepatocellular carcinoma (HCC). This study makes the first use of RMFIA for the rapid detection of AFP, opening up a new pathway for the detection of disease biomarkers.

The aptamer (S2.2)-guided Ag-Au nanostructures (aptamer-Ag-Au) have been synthesized by photoreduction and validated by ultraviolet-visible light (UV-vis) spectra and transmission electron microscopy (TEM) images. Differential interference contrast (DIC), fluorescence, and TEM images, and surface-enhanced Raman scattering (SERS) spectra indicated that the aptamer-Ag-Au nanostructures can target the surface of human breast cancer cells (MCF-7) with high affinity and specificity. This targeting is completed via the specific interaction between S2.2 aptamer (a 25-base oligonucleotide) and MUC1 mucin (a large transmembrane glycoprotein, whose expression increased at least 10-fold at MCF-7 cells in primary and metastatic breast cancers). However, the nanostructures cannot target HepG2 (human liver cancer cells) or MCF-10A cells (human normal breast epithelial cells), because these cells are MUC1-negative expressed. Moreover, the synthesized nanostructures exhibited a high SERS activity. Based on these results, a new assay for specifically detecting MCF-7 cells has been proposed. This assay can also discriminate MCF-7 cells from MCF-10A cells and different cancer cell lines, such as HepG2 cells. In addition, the aptamer-Ag-Au nanostructures have a high capability of adsorpting near-infrared (NIR) irradiation and are able to perform photothermal therapy of MCF-7 cells at a very low irradiation power density (0.25 W/cm(2)) without destroying the healthy cells and the surrounding normal tissue. Therefore, the proposed assay is significant for the diagnosis of tumors in their nascent stage. The synthesized nanostructures could offer a protocol to specifically recognize and sensitively detect the cancer cells, and would have great potential for application in the photothermal therapy of the cancers.

Investigation into glycoproteins and their associated glycans is the key to understanding the function of glycoproteins in biological pathways and disease development. Current methods for glycan analysis are generally based on multiple preparation processes to separate glycans from proteins and other molecules prior to analysis. During the multistep purification processes, glycans are continuously lost and the procedure increases the difficulty for accurate quantitative analysis of glycans. Here we describe the development of a novel technique, which uses hydrazide beads to capture glycans. It is based on the conjugation of glycans to hydrazide beads through the formation of reversible hydrazone, washing out unbound nonglycans, then releasing captured glycans by acids. The results showed that the glycans were able to be isolated from concatenate peptides by using hydrazide beads. This technique was also applied to the analysis of glycans from sera sample. The integrated capture-release on the solid-phase simplifies the procedure for glycan preparation from a complex mixture and can be a powerful tool for glycan analysis.

Prostate cancer is the most common malignancy in men in the United States, and one in seven men with prostate cancer dies of the disease. A major issue of prostate diagnosis is that there is no good method to reliably distinguish aggressive prostate cancer from nonaggressive prostate cancer. This leads to significant unnecessary suffering among prostate cancer patients and massive unnecessary health care expenditures. In this study, we aim to identify glycoproteins associated with aggressive prostate cancer using optimal cutting temperature (OCT)-embedded frozen tissues obtained from patients with known clinical outcome. To eliminate the interference of mass spectrometric analysis by the compounds in OCT and identify extracellular proteins that are likely to serve as biomarkers in body fluids, we employed glycoproteomic analysis using solid-phase extraction of glycopeptides, which allowed the immobilization of glycopeptides to solid support and removal of OCT from sample proteins before releasing the glycopeptides from the solid support for mass spectrometry analysis. Tumor tissues were cryostat microdissected from four cases of aggressive and four cases of nonaggressive prostate tumors, and glycopeptides were isolated and labeled with iTRAQ reagents before the samples were analyzed with LTQ Orbitrap Velos. From the aggressive prostate cancer tissues, we identified the overexpression of three glycoproteins involved in an extracellular matrix remodeling and further examined two glycoproteins, cathepsin L and periostin, using Western blot and immunohistochemistry analyses. This is the first proteomic study to identify proteins potentially associated with aggressive prostate cancer using OCT-embedded frozen tissues. Further study of these proteins will be needed to understand the roles of extracellular matrix proteins in cancer progression and their potential clinical utility in improving diagnosis of aggressive prostate cancer.

Zebrafish (Danio rerio) is a model organism that is used to study the mechanisms and pathways of human disorders. Many dysfunctions in neurological, development, and neuromuscular systems are due to glycosylation deficiencies, but the glycoproteins involved in zebrafish embryonic development have not been established. In this study, a mass spectrometry-based glycoproteomic characterization of zebrafish embryos was performed to identify the N-linked glycoproteins and N-linked glycosylation sites. To increase the number of glycopeptides, proteins from zebrafish were digested with two different proteases--chymotrypsin and trypsin--into peptides of different length. The N-glycosylated peptides of zebrafish were then captured by the solid-phase extraction of N-linked glycopeptides (SPEG) method and the peptides were identified with an LTQ OrbiTrap Velos mass spectrometer. From 265 unique glycopeptides, including 269 consensus NXT/S glycosites, we identified 169 different N-glycosylated proteins. The identified glycoproteins were highly abundant in proteins belonging to the transporter, cell adhesion, and ion channel/ion binding categories, which are important to embryonic, organ, and central nervous system development. This proteomics data will expand our knowledge about glycoproteins in zebrafish and may be used to elucidate the role that glycosylation plays in cellular processes and disease. The glycoprotein data are available through the GlycoFish database (http://betenbaugh.jhu.edu/GlycoFish) introduced in this paper.

This work proposes a new strategy for the electrochemical detection of hepatitis C virus (HCV) RNA level and identification of HCV-1b genotype based on the site-specific cleavage of BamHI endonuclease combined with gold nanoparticles (AuNPs) signal amplification. The assay procedures include the reverse transcription, polymerase chain reaction (PCR) amplification, and electrochemical detection. The samples of 244 mer sequence of HCV RNA from the highly conserved region of HCV-1a, HCV-1b, HCV-1, and HCV-6a, respectively, were first reverse transcribed into complementary cDNA and amplified by PCR. The PCR-amplified samples were then analyzed using a synthetic 21 mer DNA probe, which has been assembled on the electrode surface via a bifunctional molecule of p-aminobenzoic acid (ABA). The results demonstrated that the developed approach can be used for specifically identification of the HCV-1b genotype and selective and sensitive detection of HCV-1b cDNA (244 mer) with a detection limit as low as (3.1 ± 0.8) × 10(-22) M (less than 200 molecules; the concentration refers to the one before PCR amplification). Moreover, the developed method has an ability to discriminate the HCV-1b cDNA sequence from even single-base mismatched DNA sequence, to assay the HCV-1b cDNA level precisely from the mixture of HCV-1, HCV-1b, HCV-1a, and HCV-6a, and to detect HCV in real clinical samples. The protocol has high potential application in molecular diagnostics of HCV in clinical environments.

Aberrant protein glycosylation has been shown to be associated with disease progression and can be potentially useful as a biomarker if disease-specific glycosylation can be identified. However, high-throughput quantitative analysis of protein glycosylation derived from clinical specimens presents technical challenges due to the typically high complexity of biological samples. In this study, a mass spectrometry-based analytical method was developed to measure different glycosylated forms of glycoproteins from complex biological samples by coupling glycopeptide extraction strategy for specific glycosylation with selected reaction monitoring (SRM). Using this method, we monitored glycosylated and sialylated prostate-specific antigen (PSA) in prostate cancer and noncancer tissues. Results of this study demonstrated that the relative abundance of glycosylated PSA isoforms were not correlated with total PSA protein levels measured in the same prostate cancer tissue samples by clinical immunoassay. Furthermore,...