Comprehensive N- and O-Glycoproteomic Analysis of Multiple Chinese Hamster Ovary Host Cell Lines.

Glycoproteomic analysis of three Chinese hamster ovary (CHO) suspension host cell lines (CHO-K1, CHO-S, and CHO-Pro5) commonly utilized in biopharmaceutical settings for recombinant protein production is reported. Intracellular and secreted glycoproteins were examined. We utilized an immobilization and chemoenzymatic strategy in our analysis. Glycoproteins or glycopeptides were first immobilized through reductive amination, and the sialyl moieties were amidated for protection. The desired N- or O-glycans and glycopeptides were released from the immobilization resin by enzymatic or chemical digestion. Glycopeptides were studied by Orbitrap Liquid chromatography-mass spectrometry (LC/MS), and the released glycans were analyzed by Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). Differences were detected in the relative abundances of N- and O-glycopeptide types, their resident and released glycans, and their glycoprotein complexity. Ontogeny analysis revealed key differences in features, such as general metabolic and biosynthetic pathways, including glycosylation systems, as well as distributions in cellular compartments. Host cell lines and subfraction differences were observed in both N- and O-glycan and glycoprotein pools. Differences were observed in sialyl and fucosyl glycan distributions. Key differences were also observed among glycoproteins that are problematic contaminants in recombinant antibody production. The differences revealed in this study should inform the choice of cell lines best suited for a particular bioproduction application.

Glycans, Glycosite, and Intact Glycopeptide Analysis of N-Linked Glycoproteins Using Liquid Handling Systems.

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.

An Integrated Workflow for Global, Glyco-, and Phospho-proteomic Analysis of Tumor Tissues.

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 phospho-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.

Sialidase NEU1 suppresses progression of human bladder cancer cells by inhibiting fibronectin-integrin α5ß1 interaction and Akt signaling pathway.

BACKGROUND: Sialic acids are widely distributed in animal tissues, and aberrantly expressed in a variety of cancer types. High expression of sialic acid contributes to tumor aggressiveness by promoting cell proliferation, migration, angiogenesis, and metastasis. Sialidases are responsible for removal of sialic acids from glycoproteins and glycolipids. METHODS: N-glycomics of bladder cancer cells were detected by MALDI-TOF mass spectrometry. Sialic acid modification in bladder cancer tissue was determined by lectin blot. The down-regulation of NEU1 in bladder cancer cells was determined by high resolution liquid chromatography mass spectrometry (HR LC-MS). The effects of sialidase NEU1 expression on proliferation and apoptosis of human bladder cancer cells were examined by western blot, RT-PCR, confocal imaging and flow cytometry. Moreover, the function of sialic acids on fibronectin-integrin α5ß1 interaction were assayed by immunoprecipitation and ELISA. The importance of NEU1 in tumor formation in vivo was performed using BALB/c-nu mice. Expression of NEU1 in primary human bladder cancer tissue samples was estimated using bladder cancer tissue microarray. RESULTS: (1) Downregulation of NEU1 was primarily responsible for aberrant expression of sialic acids in bladder cancer cells. (2) Decreased NEU1 expression was correlated with bladder cancer progression. (3) NEU1 overexpression enhanced apoptosis and reduced proliferation of bladder cancer cells. (4) NEU1 disrupted FN-integrin α5ß1 interaction and deactivated the Akt signaling pathway. (5) NEU1 significantly suppressed in vivo tumor formation in BALB/c-nu mice. CONCLUSIONS: Our data showed that NEU1 inhibited cancer cell proliferation, induced apoptosis, and suppressed tumor formation both in vitro and in vivo, by disrupting interaction of FN and integrin ß1 and inhibiting the Akt signaling pathway. Our observations indicate that NEU1 is an important modulator of the malignant properties of bladder cancer cells, and is a potential therapeutic target for prognosis and treatment of bladder cancer. Video Abstract.

Characterization of intact glycopeptides reveals the impact of culture media on site-specific glycosylation of EPO-Fc fusion protein generated by CHO-GS cells.

With the increasing demand to provide more detailed quality attributes, more sophisticated glycan analysis tools are highly desirable for biopharmaceutical manufacturing. Here, we performed an intact glycopeptide analysis method to simultaneously analyze the site-specific N- and O-glycan profiles of the recombinant erythropoietin Fc (EPO-Fc) protein secreted from a Chinese hamster ovary glutamine synthetase stable cell line and compared the effects of two commercial culture media, EX-CELL (EX) and immediate advantage (IA) media, on the glycosylation profile of the target protein. EPO-Fc, containing the Fc region of immunoglobulin G1 (IgG1) fused to EPO, was harvested at Day 5 and 8 of a batch cell culture process followed by purification and N- and O-glycopeptide profiling. A mixed anion exchange chromatographic column was implemented to capture and enrich N-linked glycopeptides. Using intact glycopeptide characterization, the EPO-Fc was observed to maintain their individual EPO and Fc N-glycan characteristics in which the EPO region presented bi-, tri-, and tetra-branched N-glycan structures, while the Fc N-glycan displayed mostly biantennary glycans. EPO-Fc protein generated in EX medium produced more complex tetra-antennary N-glycans at each of the three EPO N-sites while IA medium resulted in a greater fraction of bi- and tri-antennary N-glycans at these same sites. Interestingly, the sialylation content decreased from sites 1-4 in both media while the fucosylation progressively increased with a maximum at the final IgG Fc site. Moreover, we observed that low amounts of Neu5Gc were detected and the content increased at the later sampling time in both EX and IA media. For O-glycopeptides, both media produced predominantly three structures, N1F1F0SOG0, N1H1F0S1G0, and N1H1F0S2G0, with lesser amounts of other structures. This intact glycopeptide method can decipher site-specific glycosylation profile and provide a more detailed characterization of N- and O-glycans present for enhanced understanding of the key product quality attributes such as media on recombinant proteins of biotechnology interest.

Comprehensive Glycoproteomic Analysis of Chinese Hamster Ovary Cells.

The Chinese hamster ovary (CHO) cell line is a major expression system for the production of therapeutic proteins, the majority of which are glycoproteins, such as antibodies and erythropoietin (EPO). The characterization glycosylation profile of therapeutic proteins produced from engineered CHO cells and therapeutic functions, as well as side effects, are critical to understand the important roles of glycosylation. In this study, a large scale glycoproteomic workflow was established and applied to CHO-K1 cells expressing EPO. The workflow includes enrichment of intact glycopeptides from CHO-K1 cell lysate and medium using hydrophilic enrichment, fractionation of the obtained intact glycopeptides (IGPs) by basic reversed phase liquid chromatography (bRPLC), analyzing the glycopeptides using LC-MS/MS, and annotating the results by GPQuest 2.0. A total of 10 338 N-linked glycosite-containing IGPs were identified, representing 1162 unique glycosites in 530 glycoproteins, including 71 unique atypical N-linked IGPs on 18 atypical N-glycosylation sequons with an overrepresentation of the N-X-C motifs. Moreover, we compared the glycoproteins from CHO cell lysate with those from medium using the in-depth N-linked glycoproteome data. The obtained large scale glycoproteomic data from intact N-linked glycopeptides in this study is complementary to the genomic, proteomic, and N-linked glycomic data previously reported for CHO cells. Our method has the potential to monitor the production of recombinant therapeutic glycoproteins.

A pilot study of salivary N-glycome in HBV-induced chronic hepatitis, cirrhosis, and hepatocellular carcinoma.

Hepatitis B is a potentially life-threatening liver infection caused by the hepatitis B virus (HBV), which can lead to chronic liver disease and put people at high risk of death from cirrhosis of the liver and liver cancer. However, little is known about the correlation of salivary N-linked glycans related to HBV-infected liver diseases. Here we investigated N-linked glycome in saliva from 200 subjects (50 healthy volunteers (HV), 40 HBV-infected patients (HB), 50 cirrhosis patients (HC), and 60 hepatocellular carcinoma patients (HCC) using MALDI-TOF/TOF-MS. Representative MS spectra of N-glycans with signal-to-noise ratios >6 were annotated using the GlycoWorkbench program. A total of 40, 47, 29, and 33 N-glycan peaks were identified and annotated from HV, HB, HC, and HCC groups, respectively. There were 15 N-glycan peaks (e.g., m/z 1647.587, 1688.613 and 2101.755) were present in all groups. Three N-glycan peaks (m/z 2596.925, 2756.962, and 2921.031) were unique in HV group, 2 N-glycan peaks (m/z 1898.676 and 1971.692) were unique in HB group, 5 N-glycan peaks (m/z 1954.677, 2507.914, 2580.930, 2637.952, and 3092.120) were unique in HC group, and 3 N-glycan peaks (m/z 2240.830, 2507.914, and 3931.338) were unique in HCC group. The proportion of fucosylated N-glycans was apparently increased in the HCC group (84.8%) than in any other group (73.1% ± 0.01), however, the proportion of sialylated N-glycans was decreased in HCC group (12.1%) than in any other group (17.23% ± 0.003). Our data provide pivotal information to distinguish between HBV-associated hepatitis, cirrhosis and HCC, and facilitate the discovery of biomarkers for HCC during its early stages based on precise alterations of N-linked glycans in saliva.

Alteration of N-glycan expression profile and glycan pattern of glycoproteins in human hepatoma cells after HCV infection.

BACKGROUND: Hepatitis C virus (HCV) infection causes chronic liver diseases, liver fibrosis and even hepatocellular carcinoma (HCC). However little is known about any information of N-glycan pattern in human liver cell after HCV infection. METHODS: The altered profiles of N-glycans in HCV-infected Huh7.5.1 cell were analyzed by using mass spectrometry. Then, lectin microarray, lectin pull-down assay, reverse transcription-quantitative real time PCR (RT-qPCR) and western-blotting were used to identify the altered N-glycosylated proteins and glycosyltransferases. RESULTS: Compared to uninfected cells, significantly elevated levels of fucosylated, sialylated and complex N-glycans were found in HCV infected cells. Furthermore, Lens culinaris agglutinin (LCA)-binding glycoconjugates were increased most. Then, the LCA-agarose was used to precipitate the specific glycosylated proteins and identify that fucosylated modified annexin A2 (ANXA2) and heat shock protein 90 beta family member 1 (HSP90B1) was greatly increased in HCV-infected cells. However, the total ANXA2 and HSP90B1 protein levels remained unchanged. Additionally, we screened the mRNA expressions of 47 types of different glycosyltransferases and found that α1,6-fucosyltransferase 8 (FUT8) was the most up-regulated and contributed to strengthen the LCA binding capability to fucosylated modified ANXA2 and HSP90B1 after HCV infection. CONCLUSIONS: HCV infection caused the altered N-glycans profiles, increased expressions of FUT8, fucosylated ANXA2 and HSP90B1 as well as enhanced LCA binding to Huh7.5.1. GENERAL SIGNIFICANCE: Our results may lay the foundation for clarifying the role of N-glycans and facilitate the development of novel diagnostic biomarkers and therapeutic targets based on the increased FUT8, fucosylated ANXA2 and HSP90B1 after HCV infection.

Quantitative Analysis of Differential Proteome Expression in Epithelial-to-Mesenchymal Transition of Bladder Epithelial Cells Using SILAC Method.

Epithelial-to-mesenchymal transition (EMT) is an essential biological process involved in embryonic development, cancer progression, and metastatic diseases. EMT has often been used as a model for elucidating the mechanisms that underlie bladder cancer progression. However, no study to date has addressed the quantitative global variation of proteins in EMT using normal and non-malignant bladder cells. We treated normal bladder epithelial HCV29 cells and low grade nonmuscle invasive bladder cancer KK47 cells with transforming growth factor-beta (TGF-ß) to establish an EMT model, and studied non-treated and treated HCV29 and KK47 cells by the stable isotope labeling amino acids in cell culture (SILAC) method. Labeled proteins were analyzed by 2D ultrahigh-resolution liquid chromatography/LTQ Orbitrap mass spectrometry. Among a total of 2994 unique identified and annotated proteins in HCV29 and KK47 cells undergoing EMT, 48 and 56 proteins, respectively, were significantly upregulated, and 106 and 24 proteins were significantly downregulated. Gene ontology (GO) term analysis and pathways analysis indicated that the differentially regulated proteins were involved mainly in enhancement of DNA maintenance and inhibition of cell-cell adhesion. Proteomes were compared for bladder cell EMT vs. bladder cancer cells, revealing 16 proteins that displayed similar changes in the two situations. Studies are in progress to further characterize these 16 proteins and their biological functions in EMT.

Quantitative glycome analysis of N-glycan patterns in bladder cancer vs normal bladder cells using an integrated strategy.

Diagnosis of bladder cancer, one of the most common types of human cancer, at an early (nonmuscle-invasive) stage is the best way to reduce the mortality rate. Tumor malignancy in general is closely associated with alterations of glycan expression. Glycosylation status, particularly global glycomes, in bladder cancer has not been well studied. We integrated lectin microarray and mass spectrometry (MS) methods to quantitatively analyze and compare glycan expression in four bladder cancer cell lines (KK47, YTS1, J82, T24) and one normal bladder mucosa cell line (HCV29). Glycopattern alterations were analyzed using lectin microarray analysis and confirmed by lectin staining and lectin blotting. Associations of glycopatterns with diverging stages were evaluated by lectin histochemistry on tissue microarrays. N-Glycans were derivatized by amidation of sialylated glycans with acetohydrazide and reductive amination with the stable isotope tags [(12)C6]- and [(13)C6]-aniline, and were quantitatively analyzed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). N-Glycan biosynthesis-associated proteins were quantitatively analyzed by a stable isotope labeling by amino acids in cell culture (SILAC) proteomics method, which revealed significant differences in expression of 13 glycosyltransferases and 4 glycosidases. Our findings indicate that sialyl Lewis X (sLe(x)), terminal GalNAc and Gal, and high mannose-type N-glycans were more highly expressed in bladder cancer cells and tissues than in normal cells. Bladder cancer cells showed high expression of core-fucosylated N-glycans but low expression of terminally fucosylated N-glycans. Each of these glycome changes may be directly related to bladder cancer progression.

Altered N-Glycan expression profile in epithelial-to-mesenchymal transition of NMuMG cells revealed by an integrated strategy using mass spectrometry and glycogene and lectin microarray analysis.

Epithelial-to-mesenchymal transition (EMT) is an essential biological process that occurs in embryonic development, metastatic diseases, and cancer progression. Altered expression of glycans is known to be associated with cancer progression. No studies to date have presented global analysis of the precise variation of N-glycans in EMT. We describe here the profile of N-glycans and glycogene expression in the EMT process induced by transforming growth factor-ß1 (TGFß1) in a normal mouse mammary gland epithelial (NMuMG) cell model. An integrated strategy with a combination of mass spectrometry, glycogene microarray analysis, and lectin microarray analysis was applied, and results were confirmed by lectin histochemistry and quantitative real-time PCR. In TGFß-induced EMT, levels of high-mannose-type N-glycans were enhanced, antennary N-glycans, and fucosylation were suppressed, and bisecting GlcNAc N-glycans were greatly suppressed. The expression of seven N-glycan-related genes was significantly changed. The products of glycogenes ALG9, MGAT3, and MGAT4B appeared to contribute to the observed alteration of N-glycans. The findings indicate that dysregulation of N-glycan synthesis plays a role in the EMT process. Systematic glycomic analysis based on the combination of techniques described here is expected to facilitate the discovery of the aberrant N-glycosylation in tumor progression and provide essential information in systems glycobiology.

Alteration of N-glycans and expression of their related glycogenes in the epithelial-mesenchymal transition of HCV29 bladder epithelial cells.

The epithelial-mesenchymal transition (EMT) is an essential step in the proliferation and metastasis of solid tumor cells, and glycosylation plays a crucial role in the EMT process. Certain aberrant glycans have been reported as biomarkers during bladder cancer progression, but global variation of N-glycans in this type of cancer has not been previously studied. We examined the profiles of N-glycan and glycogene expression in transforming growth factor-beta (TGFß)-induced EMT using non-malignant bladder transitional epithelium HCV29 cells. These expression profiles were analyzed by mass spectrometry, lectin microarray analysis, and GlycoV4 oligonucleotide microarray analysis, and confirmed by lectin histochemistry and real-time RT-PCR. The expression of 5 N-glycan-related genes were notably altered in TGFß-induced EMT. In particular, reduced expression of glycogene man2a1, which encodes α-mannosidase 2, contributed to the decreased proportions of bi-, tri- and tetra-antennary complex N-glycans, and increased expression of hybrid-type N-glycans. Decreased expression of fuca1 gene, which encodes Type 1 α-L-fucosidase, contributed to increased expression of fucosylated N-glycans in TGFß-induced EMT. Taken together, these findings clearly demonstrate the involvement of aberrant N-glycan synthesis in EMT in these cells. Integrated glycomic techniques as described here will facilitate discovery of glycan markers and development of novel diagnostic and therapeutic approaches to bladder cancer.

Profiling of concanavalin A-binding glycoproteins in human hepatic stellate cells activated with transforming growth factor-ß1.

Glycoproteins play important roles in maintaining normal cell functions depending on their glycosylations. Our previous study indicated that the abundance of glycoproteins recognized by concanavalin A (ConA) was increased in human hepatic stellate cells (HSCs) following activation by transforming growth factor-ß1 (TGF-ß1); however, little is known about the ConA-binding glycoproteins (CBGs) of HSCs. In this study, we employed a targeted glycoproteomics approach using lectin-magnetic particle conjugate-based liquid chromatography-tandem mass spectrometry to compare CBG profiles between LX-2 HSCs with and without activation by TGF-ß1, with the aim of discovering novel CBGs and determining their possible roles in activated HSCs. A total of 54 and 77 proteins were identified in the quiescent and activated LX-2 cells, respectively. Of the proteins identified, 14.3% were glycoproteins and 73.3% were novel potential glycoproteins. Molecules involved in protein processing in the endoplasmic reticulum (e.g., calreticulin) and calcium signaling (e.g., 1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase ß-2 [PLCB2]) were specifically identified in activated LX-2 cells. Additionally, PLCB2 expression was upregulated in the cytoplasm of the activated LX-2 cells, as well as in the hepatocytes and sinusoidal cells of liver cirrhosis tissues. In conclusion, the results of this study may aid future investigations to find new molecular mechanisms involved in HSC activation and antifibrotic therapeutic targets.

Production of CA125 with Tn antigens using a glycosylphosphatidylinositol anchoring system.

Cancer antigen 125 (CA125) is a serum marker associated with ovarian cancer. Despite its widespread use, CA125 levels can also be elevated in benign conditions. Recent reports suggest that detecting serum CA125 that carries the Tn antigen, a truncated O-glycan containing only N-acetylgalactosamine on serine or threonine residues, can improve the specificity of ovarian cancer diagnosis. In this study, we engineered cells to express CA125 with a Tn antigen. To achieve this, we knocked out C1GALT1 and SLC35A1, genes encoding Core1 synthase and a transporter for cytidine-5'-monophospho-sialic acid respectively, in human embryonic kidney 293 (HEK293) cells. In ClGALT1-SLC35A1-knockout (KO) cells, the expression of the Tn antigen showed a significant increase, whereas the expression of the T antigen (galactose-ß1,3-N-acetylgalactosamine on serine or threonine residues) was decreased. Due to the inefficient secretion of soluble CA125, we employed a glycosylphosphatidylinositol (GPI) anchoring system. This allowed for the expression of GPI-anchored CA125 on the cell surface of ClGALT1-SLC35A1-KO cells. Cells expressing high levels of GPI-anchored CA125 were then enriched through cell sorting. By knocking out the PGAP2 gene, the GPI-anchored form of CA125 was converted to a secretory form. Through the engineering of O-glycans and the use of a GPI-anchoring system, we successfully produced CA125 with Tn antigen modification.

Protein Signatures for Distinguishing Colorectal Cancer Liver Metastases from Primary Liver Cancer Using Tissue Slide Proteomics.

BACKGROUND: Colorectal cancer liver metastasis (CRLM) and hepatocellular carcinoma (HCC) are both high incidence tumors in China. In certain poorly differentiated cases they can exhibit comparable imaging and pathological characteristics, which impedes accurate clinical diagnosis. The use of protein-based techniques with tissue slides offers a more precise means to assess pathological changes and has the potential to assist with tumor diagnosis. METHODS: A simple in situ protein digestion protocol was established for protein fingerprint analysis of paraffin-embedded tissue slide samples. Additionally, machine learning techniques were employed to construct predictive models for CRLM and HCC. The accuracy of these models was validated using tissue slides and a clinical database. RESULTS: Analysis of differential protein expression between CRLM and HCC groups reliably identified 977 proteins. Among these, 53 were highly abundant in CRLM samples and 57 were highly abundant in HCC samples. A prediction model based on the expression of six proteins (CD9, GSTA1, KRT20, COL1A2, AKR1C3, and HIST2H2BD) had an area under curve (AUC) of 0.9667. This was further refined to three proteins (CD9, ALDH1A1, and GSTA1) with an AUC of 0.9333. CONCLUSIONS: Tissue slide proteomics can facilitate accurate differentiation between CRLM and HCC. This methodology holds great promise for improving clinical tumor diagnosis and for identifying novel markers for challenging pathological specimens.

Highly Sensitive and Linear Resonator-Based Biosensor for White Blood Cell Counting: Feasible Measurement Method and Intrinsic Mechanism Exploration.

Since different quantities of white blood cells (WBCs) in solution possess an adaptive osmotic pressure of cells, the WBCs themselves and in solution have similar concentrations, resulting in them having similar dielectric properties. Therefore, a microwave sensor could have difficulty in sensing the quantity variation when WBCs are in solution. This paper presents a highly sensitive, linear permittivity-inspired microwave biosensor for WBCs, counting through the evaporation method. Such a measurement method is proposed to record measurements after the cell solution is dripped onto the chip and is completely evaporated naturally. The proposed biosensor consists of an air-bridged asymmetric differential inductor and a centrally located circular fork-finger capacitor fabricated on a GaAs substrate using integrated passive fabrication technology. It is optimized to feature a larger sensitive area and improved Q-factor, which increases the effective area of interaction between cells and the electromagnetic field and facilitates the detection of their changes in number. The sensing relies on the dielectric properties of the cells and the change in the dielectric constant for different concentrations, and the change in resonance properties, which mainly represents the frequency shift, corresponds to the macroscopic change in the concentration of the cells. The microwave biosensors are used to measure biological samples with concentrations ranging from 0.25 × 106 to 8 × 106 cells per mL in a temperature (26.00 ± 0.40 °C) and humidity (54.40 ± 3.90 RH%) environment. The measurement results show a high sensitivity of 25.06 Hz/cells·mL-1 with a highly linear response of r2 = 0.99748. In addition, a mathematical modeling of individual cells in suspension is performed to estimate the dielectric constant of individual cells and further explain the working mechanism of the proposed microwave biosensor.

Microwave biosensor for the detection of growth inhibition of human liver cancer cells at different concentrations of chemotherapeutic drug.

Cytotoxicity assays are crucial for assessing the efficacy of drugs in killing cancer cells and determining their potential therapeutic value. Measurement of the effect of drug concentration, which is an influence factor on cytotoxicity, is of great importance. This paper proposes a cytotoxicity assay using microwave sensors in an end-point approach based on the detection of the number of live cells for the first time. In contrast to optical methods like fluorescent labeling, this research uses a resonator-type microwave biosensor to evaluate the effects of drug concentrations on cytotoxicity by monitoring electrical parameter changes due to varying cell densities. Initially, the feasibility of treating cells with ultrapure water for cell counting by a microwave biosensor is confirmed. Subsequently, inhibition curves generated by both the CCK-8 method and the new microwave biosensor for various drug concentrations were compared and found to be congruent. This agreement supports the potential of microwave-based methods to quantify cell growth inhibition by drug concentrations.

Real-Time Detection of Yeast Growth on Solid Medium through Passive Microresonator Biosensor.

This study presents a biosensor fabricated based on integrated passive device (IPD) technology to measure microbial growth on solid media in real-time. Yeast (Pichia pastoris, strain GS115) is used as a model organism to demonstrate biosensor performance. The biosensor comprises an interdigital capacitor in the center with a helical inductive structure surrounding it. Additionally, 12 air bridges are added to the capacitor to increase the strength of the electric field radiated by the biosensor at the same height. Feasibility is verified by using a capacitive biosensor, and the change in capacitance values during the capacitance detection process with the growth of yeast indicates that the growth of yeast can induce changes in electrical parameters. The proposed IPD-based biosensor is used to measure yeast drop-added on a 3 mm medium for 100 h at an operating frequency of 1.84 GHz. The resonant amplitude of the biosensor varies continuously from 24 to 72 h due to the change in colony height during vertical growth of the yeast, with a maximum change of 0.21 dB. The overall measurement results also fit well with the Gompertz curve. The change in resonant amplitude between 24 and 72 h is then analyzed and reveals a linear relationship with time with a coefficient of determination of 0.9844, indicating that the biosensor is suitable for monitoring yeast growth. Thus, the proposed biosensor is proved to have potential in the field of microbial proliferation detection.

Selective isolation and analysis of glycoprotein fractions and their glycomes from hepatocellular carcinoma sera.

As one of the most important post-translational modifications, the discovery, isolation, and identification of glycoproteins are becoming increasingly important. In this study, a Con A-magnetic particle conjugate-based method was utilized to selectively isolate the glycoproteins and their glycomes from the healthy donor and hepatocellular carcinoma (HCC) case sera. The isolated glycoproteins and their N-linked glycans were identified by LC-ESI-MS/MS and MALDI-TOF/TOF-MS, respectively. A total of 93 glycoproteins from the healthy donors and 85 glycoproteins from the HCC cases were identified. There were 34 different glycoproteins shown between the healthy donors (21/34) and the HCC cases (13/34). Twenty-eight glycans from the healthy donors and 30 glycans from the HCC cases were detected and there were 22 different glycans shown between the healthy donors (10/22) and HCC cases (12/22). Among these glycoproteins, 50 were known to be N-linked glycoproteins and three novel glycopeptides from two predicted potential glycoproteins were discovered. Moreover, lectin blotting, Western blotting and lectin/glyco-antibody microarrays were applied to definitely elucidate the change of selective protein expressions and their glycosylation levels, the results indicated that the differences of the identified glycoproteins between the healthy donors and HCC cases were caused by the change of both protein expression and their glycosylation levels.

Isolation and identification of mannose-binding proteins and estimation of their abundance in sera from hepatocellular carcinoma patients.

The interaction of glycan-binding proteins (GBPs) and glycans plays a significant biological role that ranges from cell-cell recognition to cell trafficking, and glycoprotein targeting. The anomalies of GBPs related to the types and/or quantities were not clearly known in cancer incidence. It is imperative to identify and annotate the GBPs related with the canceration. Here the mannose-binding proteins (MBPs) from the clinical sera were isolated and identified by the mannose-magnetic particle conjugates and the high-accuracy MS analysis. Seventy-five MBPs from normal donors' sera and 79 MBPs from hepatocellular carcinoma patients' sera were identified and annotated. By using the stringent criteria of exponentially modified protein abundance index (emPAI) quantification, 12 MBPs were estimated to be significantly upregulated (emPAI ratio > 4) and nine MBPs were estimated to be significantly downregulated (emPAI ratio < 0.25) in the hepatocellular carcinoma sera. Real-time quantitative PCR, Western blotting, and protein microarrays were also used to confirm the altered MBPs expression level and the specific binding between the isolated MBPs and mannose. The sequence recognition motifs and structure preference of the isolated MBPs were characterized. The functional enrichment analysis revealed that over 57% of the isolated MBPs were binding protein and the upregulated MBPs were involved in cell death, tumor progression, and macromolecular complex remodeling.