Protein Markers Of Disease Research Articles

B-249 Assessing Targeted Mass Spectrometry Assay Performance for Putative Biomarker Proteins on a New Hybrid Nominal Mass Instrument

Abstract Background The development of targeted mass spectrometry-based proteomics assays that monitor biomedically relevant proteins is an important step in bridging discovery experiments to highly multi-analyte clinical studies. Targeted assays are currently unable to scale to thousands of peptide targets from hundreds of proteins, requiring extensive refinement down assays for the minimum useful analytes. Large parallel reaction monitoring (PRM) assays are now possible with a new hybrid nominal-mass instrument. The scale of assay is achievable with this instrument by using real-time alignment, while being sensitive and fast enough to handle many concurrent targets. To assess the performance of this new instrument we constructed a large scale PRM assay for proposed neurodegenerative disease marker proteins, and an assay for proteins associated with amyloid tissue typing. Methods To evaluate the performance of this instrument on proteins of interest in cerebrospinal fluid, we performed an analysis on a pool from individuals diagnosed as Alzheimer’s, Parkinson’s, or cognitively normal. A calibration curve of cerebrospinal fluid diluted into chicken serum at 14 dilutions and individual human cerebrospinal fluid were digested with a protein aggregation and capture based digestion protocol. CSF peptides were separated by 60 minute gradient on C18 IonOpticks column, and amyloid tissue peptides separated by 30 minute gradient on C18 PepMap column with a Vanquish Neo UHPLC and analyzed by data-independent acquisition (DIA) and PRM with a new hybrid nominal mass instrument designed for targeted tandem mass spectrometry (tMS2). Scheduled methods were generated using a Skyline external tool, transitions were refined to minimize interference and maximize the LLOQ. DIA data was searched, and data extracted and analyzed in Skyline. PRM data was extracted and quantified with Skyline; CSF calibration curve LLOQ is calculated by bilinear fit. Results For the large scale cerebrospinal fluid (CSF) assay the selection of protein targets was based on literature from neurodegenerative disease cohort studies. We target 1065 peptides from 104 proteins, including proteins currently measured by ELISA in clinical research, e.g.: APP, TAU, and APOE. Sensitivity and reproducibility of the assay is assessed using figures of merit from replicate matched-matrix calibration curves. Over 80% of proteins have at least one peptide with a LLOQ below 30% dilution in the matched matrix, and of all targeted peptides over 55% had a LLOQ below 30% dilution. To demonstrate the application of the real-time alignment on the new instrument we constructed an assay for samples where not every analyte is shared. An assay for amyloidosis associated proteins was constructed for a total of 180 peptides mapping to 52 proteins. This includes 11 proteins most commonly associated with amyloid deposition; APCS, APOA4, APOE, CLU, LECT2, IGKC, IGLC2/4/7, SAA1, TTR, and VTN. From the preliminary set of 12 previously typed samples we observe peptide abundances consistent with the assigned type. Conclusions This novel instrument provides a new approach for building large targeted mass spectrometry assays. We demonstrate the ability of the real-time alignment to adjust scheduled retention time windows, ensuring that chromatographic shifts do not lead to a loss of peptide detection and quantification.

Janus-Type Immunofluorescent Probes and a Quantitative Immunoassay System.

To realize highly sensitive immunoassays, high optical density probes conjugated with antibodies for target antigens have been demanded in order to increase the visibility of antigen-antibody complex formation. We herein demonstrate the development of an immunoassay system using magnetic and fluorescent Janus particles as probes in conjunction with an antibody-immobilized microfluidic device. The concentration of the detection limit at which there was a significant difference between SARS-CoV-2 and human coronavirus 229E antigens was 3.1 ng/mL, and the standard deviation of the signal was less than 5%. The immunofluorescent probe and immunoassay system developed in this study are expected to be applicable not only to SARS-CoV-2 but also to the quantitative measurement of various other disease marker proteins and biomolecules.

Aggregation-induced emission luminogen based ELISA for highly sensitive protein detection

Enzyme-linked immunosorbent assay (ELISA), as the most commonly used immunological detection technique, has been widely used in the clinical diagnosis of disease marker proteins. The key of the traditional ELISA is the probe, the horseradish peroxidase (HRP), which influences the sensitivity. However, the application of traditional ELISA in the biomedical field is limited due to low sensitivity. In this work, an aggregation-induced emission luminogen (AIEgen) was designed as a replacement of HRP in ELISA. It was synthesized by functionalizing a tetraphenylethylene (TPE) fluorogen with phosphorylated peptides (TPE-phenylalanine-phenylalanine-tyrosine(H2PO3)-OH, TPE-FFYp). The TPE-FFYp fluoresced weakly in an aqueous solution. Upon addition of the target protein, the phosphate group was hydrolyzed by alkaline phosphatase (ALP) on the immune complex while forming highly emissive AIE aggregates. Utilizing alpha-fetoprotein (AFP) as the model target, this proposed method has a detection limit as low as 0.78 ng/mL. Besides, the detection of AFP with TPE-FFYp was not only uninterrupted by other proteins, but also performed well in human serum. More importantly, it could be used to detect other targets by changing the corresponding antibodies, which further extended the application of AIEgen in bioassays.

Technetium Nitrido Complexes of Tetradentate Thiosemicarbazones: Kit-Based Radiolabeling, Characterization, and In Vivo Evaluation.

Bis(thiosemicarbazone) and pyridylhydrazone-thiosemicarbazone chelators have demonstrated utility in nuclear medicine. In particular, the 64Cu2+ complexes have been extensively developed for hypoxia imaging and molecular imaging of peptide and protein markers of disease. However, the chemistry and application of bis(thiosemicarbazone) and pyridylhydrazone-thiosemicarbazone chelators in combination with 99mTc, the most widely used radionuclide in nuclear medicine, is underexplored. Herein, a series of bis(thiosemicarbazone) and pyridylhydrazone-thiosemicarbazone chelators were radiolabeled with nitrido-technetium-99m in an optimized one-pot synthesis from [99mTc]TcO4-. Optimization of the radiochemical syntheses allowed for production of the complexes in >90% radiochemical conversion with apparent molar activities of 3.3-5 GBq/μmol. Competition experiments demonstrated the excellent stability of the complexes. The nitrido-technetium-99 complexes were synthesized, and the chemical identities were investigated using mass spectrometry, spectroscopy, and density functional theory calculations. Complexation of nitrido-rhenium(V) was achieved with the N4-dialkylated bis(thiosemicarbazones). Planar imaging and ex vivo biodistribution studies of the five 99mTc complexes were conducted on healthy BALB/c mice to determine in vivo behavior. The lipophilic nature of the complexes resulted in uptake of 1.6-5.7% ID g-1 in the brain at 2 min postinjection and retention of 0.4-1.7% ID g-1 at 15 min postinjection. The stability of the complexes and the biodistribution data demonstrate that these chelators are ideal platforms for future production of radiopharmaceutical candidates.

Comparative proteomic analysis of glomerular proteins in primary and bucillamine-induced membranous nephropathy

BackgroundAnti-phospholipase A2 receptor autoantibody (PLA2R Ab)-associated membranous nephropathy (MN) is the most common form of primary MN (pMN). On the other hand, bucillamine (BCL), an antirheumatic drug developed in Japan, was reported to cause a rare form of secondary MN (sMN). Between these MN forms, comparative proteomic analysis of glomerular proteins has not been performed.MethodsWe used renal biopsy specimens from 6 patients with PLA2R Ab (+) pMN, 6 patients with PLA2R Ab (‒) pMN, 6 patients with BCL-induced sMN, and 5 control cases (time 0 transplant biopsies). Proteins were extracted from laser-microdissected glomeruli and analyzed using mass spectrometry. The quantification values of protein abundance in each MN group were compared with those in the control group.ResultsMore than 800 proteins with high confidence were identified. Principal component analysis revealed a different distribution between the pMN and sMN groups. For further analysis, 441 proteins matched with ≥ 3 peptides were selected. Among the pMN and sMN groups, we compared the profiles of several protein groups based on the structural and functional characteristics, such as immunoglobulins, complements, complement-regulating proteins, podocyte-associated proteins, glomerular basement membrane proteins, and several proteins that are known to be associated with kidney diseases, including MN. In all MN groups, increased levels of immunoglobulins (IgG, IgA, and IgM), complements (C3, C4, and C9), complement factor H-related protein 5, type XVIII collagen, calmodulin, polyubiquitin, and ubiquitin ligase were observed. For some proteins, such as type VII collagen and nestin, the fold-change values were significantly different between the pMN and sMN groups.ConclusionsBetween the pMN and BCL-induced sMN groups, we observed common and different alterations in protein levels such as known disease-associated proteins and potential disease marker proteins.

Polyvalent Biotinylated Aptamer Scaffold for Rapid and Sensitive Detection of Tau Proteins.

Biomimetic construction of artificial scaffolds has attracted increasing attention. However, the construction methods usually require redundant materials and procedures, which is inconvenient for further application. Herein, inspired by the polyvalent multifunctional structure in nature, we have designed a polyvalent biotinylated aptamer scaffold (PBAS) which can conduct analytical performance with high sensitivity and simplified procedures. To construct a PBAS, the aptamers are designed to hybridize with prepared linker probes to form polyvalent biotinylated scaffolds, which contain both multiple aptamers and signal labels. Therefore, multifunctional scaffolds can be constructed with high recognition and capture efficiency as well as significant signal amplification. Furthermore, the scaffold can be used for the assay of some disease marker proteins. By taking tau proteins as an example, the proposed aptasensor can exhibit excellent performance with a low detection limit of 153 pg mL-1 and a short assay time of 50 min, which is much better than most of the previous methods. By assays of tau proteins in both serum and artificial cerebro spinal fluid, the PBAS-based aptasensor can work well. Therefore, the scaffold may be expected to be a powerful analytical tool which may have wide applications in the detection of a variety of analytes.

Circular RNA NF1-419 enhances autophagy to ameliorate senile dementia by binding Dynamin-1 and Adaptor protein 2 B1 in AD-like mice.

Recent studies have demonstrated circular RNAs (circRNAs) to be widely expressed and to have important physiological functions. However, the expression, regulation, and function of circRNAs in neuroglial cells are unknown. Herein, we characterized the expression, regulation, and function of circRNAs in astrocytes. Astrocyte circRNAs were identified by computational analysis of newborn SD rat primary astrocytes cultured with 20 g/L D-galactose. In this manner, 7376 circRNAs were identified, among which most circRNAs (5754) were derived from annot_exons, whereas 27 were antisense, 853 were exon/intron, 329 were intergenic, 41 were intronic, and 372 were one exon. Among these, circNF1-419 was demonstrated to regulate autophagy, in over-expressing circNF1-419 transfected astrocytes, through the PI3K-I/Akt-AMPK-mTOR and PI3K-I/Akt-mTOR signaling pathways. An adenovirus associated virus packaging system (virus titer 1 ×1012), over-expressing circNF1-419 and injected into mouse cerebral cortex, showed autophagy enhancing activity by binding the proteins Dynamin-1 and Adaptor protein 2 B1 (AP2B1). This binding regulated aging markers (p21, p35/25, and p16) and inflammatory factors (TNF-α and NF-κB), and reduced the expression of Alzheimer’s disease marker proteins (Tau, p-Tau, Aβ1-42, and APOE), which delayed senile dementia. Transcriptome analysis of the brain showed that circNF1-419 improved other signaling pathways, especially those related to the synapses of SAMP8 mice. These findings provide novel insights into circNF1-419 and its potential usefulness for the diagnosis and treatment of dementia by regulating Dynamin-1 and AP2B1 mediated autophagy.

Hypoxia-inducible factor 1 alpha and nuclear-related receptor 1 as targets for neuroprotection by albendazole in a rat rotenone model of Parkinson's disease.

Hypoxia-inducible factor-1 alpha (HIF-1α) and nuclear receptor related-1 (Nurr1) play pivotal roles in the development and survival of dopaminergic neurons, and deficiencies in these genes may be involved in Parkinson's disease (PD) pathogenesis. Recently, anthelminthic benzimidazoles were shown to promote HIF-1α transcription in vitro and were proposed to activate Nurr1 via their benzimidazole group. Therefore, the aim of this study was to explore the neuroprotective effects of albendazole (ABZ), an anthelminthic benzimidazole, in a rotenone model of Parkinson's disease (PD). Rotenone (1.5mg/kg) was subcutaneously injected into rats every other day for a period of 21days, resulting in the development of the essential features of PD. In addition to rotenone, ABZ (10mg/kg) was administered orally starting from the 11th day. Treatment of rats with ABZ markedly mitigated rotenone-induced histological alterations in substantia nigra (SN), restored striatal dopamine (DA) level and motor functions and decreased the expression of α-synuclein (a disease marker protein). ABZ also enhanced expression of Hypoxia-inducible factor-1 alpha (HIF-1α) in the SN along with its downstream target, vascular endothelial growth factor, promoting neuronal survival. Similarly, ABZ augmented nuclear receptor related-1 (Nurr1) expression in the SN and increased transcriptional activation of Nurr1-controlled genes, which are essential for regulation of DA synthesis; additionally, expression of neurotoxic proinflammatory cytokines that induce neuronal death was suppressed. In conclusion, the present study suggests that ABZ exerts a neuroprotective effect in a rotenone-induced PD model associated with HIF-1α and Nurr1 activation and thus may be a viable candidate for treating PD.

Thermally imprinted microcone structure-assisted lateral-flow immunoassay platforms for detecting disease marker proteins.

Although various types of on-site immunoassay platforms have been developed, facile and reliable sample-to-answer immunoassay systems are still under development. In this study, we proposed a lateral-flow immunoassay system utilizing a polymer sheet with microcone array structures fabricated by thermal imprinting. By adding a surfactant to the running/washing buffer, we were able to dispense with complicated chemical modification protocols, which are usually necessary to enhance antibody adsorption. We investigated three types of polymeric materials and confirmed that polycarbonate is most suitable as an imprinted polymer substrate. Experiments using microcone arrays with different distances revealed that the increased surface area with nanometer-scale surface roughness was key to achieving stable immobilization of antibodies. To assess the applicability of this assay to clinical diagnosis, C-reactive protein in a pure buffer and in a serum sample was analyzed as a model, with a ∼0.1 μg mL-1 lower limit of detection. The presented approach would open a new path to the development of immunoassay-based on-site point-of-care testing by virtue of its simplicity of operation, high sensitivity, and versatility.

Anti-Parkinson's disease effects of ketamine via inhibiting protein kinase B/mammalian target of rapamycin signal pathway and triggering autophagy in rat B103 neuroblastoma cells

Objective To investigate the effect and molecular mechanism of ketamine on anti-Parkinson's disease(PD) on rat B103 neuroblastoma cells. Methods B103 cells were used as the experimental models. After the treatment with ketamine at the concentration of 0, 100, 200, 400 μmol/L and 400 μmol/L ketamine+5 mmol/L 3-methyladenine (3-MA), cell proliferation effects of ketamine were detected using 3-(4,5-dimenthyl-2-thiazolyl) -2,5-diphenyl-2-H-tetrazolium bromide(MTT) assay. Autophagy was detected with acridine orange staining and Lyso-Tracker Red staining using fluorescence microscopy. Expression of the proteins associated with Beclin-1, microtubulesas sociated protein light(LC3), protein kinase B(Akt), p70S6K, mammalian target of rapamycin (mTOR), α-synuclein (α-syn) and β-synuclein (β-syn) was evaluated using Western blot. Results The proliferation of B103 cells had no significant influence after treatment with ketamine (100, 200, 400 μmol/L) vs 0 μmol/L for 7 d (P>0.05). Acridine orange staining and Lyso-Tracker Red staining showed that ketamine triggered B103 cell autophagy in a dose-dependent manner (P<0.05). Western blot results showed that the expression levels of LC3-Ⅰ, phospho(p)-Akt, p-p70S6K and p-mTOR were lower in 400 μmol/L than the levels in 0 μmol/L. The expression levels of LC3-Ⅱand Beclin-1 were higher in 400 μmol/L than expression levels in 0 μmol/L (P<0.05). Expression levels of Parkinson's disease marker protein α-syn decreased and β-syn were higher in 400 μmol/L than 0 μmol/L (P<0.05). After pretreatment with 3-MA(5 mmol/L), the anti-PD effect of ketamine on B103 cells was reversed (400 μmol/L+3-MA and 400 μmol/L, P<0.05). Conclusions Ketamine exerts anti-PD effect by inhibiting Akt/mTOR signaling pathway, inducing autophagy in B103 cells and degrading α-syn. Key words: Ketamine; Parkinson's disease; Autophagy; Protein kinase B/mammalian target of rapamycin signaling transduction pathway

Applications of a novel biodetection system to saliva using protein fingerprints with data processing

A fundamental method has been developed focusing on a facile and rapid examination of periodontal disease. Periodontal disease is an oral disease thought to affect 80% of adults, and early detection with treatment is desirable for the improvement of the quality of life. Unfortunately conventional methods are not consistent as the disease is caused by a number of undefined bacteria and detection relies on the skills of the dentist. Thus an objective detection system is required. We have performed an experiment on saliva using a novel biodetection system, designated PepTenChip®. A disease model for saliva was prepared using a specimen from a healthy subject and a mixture of hemoglobin (f-Hb) and lactate dehydrogenase (LDH), which is used as a periodontal disease marker protein with healthy saliva. PepTenChip® is a peptide microarray in which fluorescent labelled structured peptides are immobilized on a novel amorphous carbon substrate. Since the peptides used as capture molecules are fluorescently labelled, labeling of analytes is not necessary. The fluorescence intensity change before and after application of analytes are detected rather than the ON/OFF detection common to conventional microarrays using a set of antigen–antibody. The fluorescence intensity value changes according to the concentration of captured protein allowing the generation of protein fingerprint (PFP) and dendrograms. The present method does not rely on a “one to one” interaction, unlike conventional biodetection, and advantages can be envisaged in the case of an undefined or unknown cause of disease. The statistical analyses, such as multivariate analyses, allow classification of the type of proteins added in saliva as mimetics of disease. PepTenChip® system is useful and convenient for examination of periodontal disease in health care.

Characterization of Behavioral, Neuropathological, Brain Metabolic and Key Molecular Changes in zQ175 Knock-In Mouse Model of Huntington's Disease.

Huntington’s disease (HD) is caused by an expansion of the trinucleotide poly (CAG) tract located in exon 1 of the huntingtin (Htt) gene leading to progressive neurodegeneration in selected brain regions, and associated functional impairments in motor, cognitive, and psychiatric domains. Since the discovery of the gene mutation that causes the disease, mouse models have been developed by different strategies. Recently, a new model, the zQ175 knock-in (KI) line, was developed in an attempt to have the Htt gene in a context and causing a phenotype that more closely mimics HD in humans. The behavioral phenotype was characterized across the independent laboratories and important features reminiscent of human HD are observed in zQ175 mice. In the current study, we characterized the zQ175 model housed in an academic laboratory under reversed dark-light cycle, including motor function, in vivo longitudinal structural MRI imaging for brain volume, MRS for striatal metabolites, neuropathology, as well as a panel of key disease marker proteins in the striatum at different ages. Our results suggest that homozygous zQ175 mice exhibited significant brain atrophy before the motor deficits and brain metabolite changes. Altered striatal medium spiny neuronal marker, postsynaptic marker protein and complement component C1qC also characterized zQ175 mice. Our results confirmed that the zQ175 KI model is valuable in understanding of HD-like pathophysiology and evaluation of potential therapeutics. Our data also provide suggestions to select appropriate outcome measurements in preclinical studies using the zQ175 mice.

Immunoassay-based proteome profiling of 24 pancreatic cancer cell lines

Pancreatic ductal adenocarcinoma is one of the most deadly forms of cancers, with a mortality that is almost identical to incidence. The inability to predict, detect or diagnose the disease early and its resistance to all current treatment modalities but surgery are the prime challenges to changing the devastating prognosis. Also, relatively little is known about pancreatic carcinogenesis. In order to better understand relevant aspects of pathophysiology, differentiation, and transformation, we analysed the cellular proteomes of 24 pancreatic cancer cell lines and two controls using an antibody microarray that targets 741 cancer-related proteins. In this analysis, 72 distinct disease marker proteins were identified that had not been described before. Additionally, categorizing cancer cells in accordance to their original location (primary tumour, liver metastases, or ascites) was made possible. A comparison of the cells' degree of differentiation (well, moderately, or poorly differentiated) resulted in unique marker sets of high relevance. Last, 187 proteins were differentially expressed in primary versus metastatic cancer cells, of which the majority is functionally related to cellular movement.

A highly sensitive “switch-on” fluorescent probe for protein quantification and visualization based on aggregation-induced emission

A highly sensitive and water-soluble "switch-on" fluorescent probe with aggregation-induced emission characteristics was developed for protein quantification and visualization. It offers a rapid, economic and effective way for the assay of complete serum proteins and disease-marker proteins.

Differential protein expression profiling in BSE disease

Bovine spongiform encephalopathy (BSE) is a fatal neurodegenerative disease affecting cattle. Current tests for the detection of BSE are based solely on the only definitive marker of the disease, an abnormal conformer (PrPd), of the host encoded prion protein (PrPc). Recent evidence that other transmissible spongiform encephalopathy diseases can be present in the absence of PrPd, coupled with the need to establish pre-mortem diagnostic assays have led to a search for alternative diagnostic approaches. In this study we apply differential protein expression profiling for the prediction of BSE disease in post-mortem bovine brain tissue. The protein profiles of groups of 27 BSE diseased cattle were compared with 28 control animals. Analysis using statistical learning (and linear discriminant analysis) techniques established protein markers of disease with good predictive power (sensitivity 85% and specificity 71%). Further work will be required to test the predictive markers in a wider range of diseases, particularly other neurological conditions.

One-chip biosensor for simultaneous disease marker/calibration substance measurement in human urine by electrochemical surface plasmon resonance method

We have developed a miniaturized electrochemical surface plasmon resonance biosensor for measuring two biomolecules that have very different molecular sizes, one is transferrin (MW = 75 kDa) as a disease marker protein, the other is creatinine (MW = 113) as a calibration marker for the accurate measurement of human urinary samples. The sensor has a PDMS based microchannel that is 2 mm wide and 20 μm deep. Two gold films were integrated in the microchannel; one was modified with anti-transferrin antibody for immuno-reaction, and the other was modified with osmium-poly-vinylpyridine wired horseradish peroxidase (Os-gel-HRP). We further immobilized a tri-enzyme layer of creatininase, creatinase and sarcosine oxidase in order to measure creatinine by converting it to hydrogen peroxide in the upstream channel. We measured the transferrin concentration from the refractive index change involved in an immuno-complex formation, and we were simultaneously able to measure creatinine by employing the refractive index change in the Os-gel-HRP caused by oxidation with the hydrogen peroxide produced from creatinine by the tri-enzyme. The effects of ascorbic acid and uric acid in urine samples were sufficiently eliminated by adding ascorbate oxidase and uricase to the urine samples during sampling. We were able to measure two analyte concentrations within 15 min by one simple injection of 50 μL of diluted human urine into our sensor. The detectable transferrin and creatinine ranges were 20 ng/mL to 10 μg/mL, and 10 μM to 10 mM, respectively, which are sufficient levels for clinical tests. Finally, we compared the results obtained using our sensor with those obtained with a conventional immunoassay and the Jaffe method. We obtained a similar trend that can reduce the fluctuation in the urinary transferrin concentration from three different samples by calibrating the creatinine concentration.

In/Ex-situ Detection of HBV DNA Using Dynamic Microcantilever

The microcantilevers have emerged as a versatile biosensor, and showed excellent performance such as high sensitivity, high selectivity, and label-free detection. They have been successfully used for the detection of nucleic acids, disease marker proteins, cells, and pathogens including small molecules. So far, our group has successfully demonstrated the marker protein detection using the actuating layer (PZT)-embedded microcantilevers for the last decade. Here, we introduce in/ex-situ monitoring of the DNA binding events using performance improved actuating layer-embedded microcantilever sensors. To obtain the stable and reliable resonant frequency shifts, the microcantilevers were passivated with parylene-C film for in-situ detection and perfluorosilane (PF-Si) film for ex-situ detection. To achieve the recognition layer, the probe DNA (37-mer including T10 spacers) specific to HBV DNA was immobilized on the gold-coated microcantilever, and followed by backfilling of ethylene glycol spacer (HSC11-EG3-OH) to increase the DNA binding efficiency. After the surface treatment, the detection of HBV DNA (27-mer) was performed through two manners, in-situ and ex-situ. Target DNA in the range of 1 to 20 M and 10 nM to 5 M were applied for the in-situ and ex-situ detection respectively, and the resonant frequency shifts according to the concentration was examined quantitatively. From the results, we explained the relationship between the DNA hybridization and the nanomechanical response. In addition, we presented a hypothesis on the different tendency of in-situ and ex-situ results.

DSA affinity glycoproteome of human liver tissue

Due to the critical roles of glycoproteins in the activities of cells to tissues, mapping of liver glycoproteome may provide valuable basic information for finding disease marker proteins. In this study, Datura Stramonium Agglutinin (DSA) was chosen to enrich N-linked glycoproteins for its broader specificity with tri- or tetra-antennary complex type. DSA affinity glycoproteins’ profiles of human liver tissue were generated by two-dimensional electrophoresis (2-DE) followed by glycoprotein staining based on multiplexed proteomics (MP) technology. 64 ± 3 ( n = 3) protein spots were detected and 41 of glycoproteins were identified via peptide mass fingerprinting (PMF) using MALDI-TOF-MS/MS and annotated to IPI databases. Identified glycoproteins definitely take part in the regulation of cell cycle and metabolic processes. The detailed carbohydrate moiety of some glycoproteins might be concluded according to the literatures. The construction of DSA affinity glycoprotein database would contribute to the subsequent research.

Simultaneous On-chip Surface Plasmon Resonance Measurement of Disease Marker Protein and Small Metabolite

Disease marker protein and a correction marker were simultaneously measured in a poly-dimethylsiloxane based microchannel. Two gold films in the microchannel were modified with antibody and redox polymer containing horseradish peroxidase (HRP). Creatininase, creatinase, and sarcosine oxidase were immobilized in the upstream. At first, marker protein and creatinine were simultaneously measured by surface plasmon resonance (SPR) and electrochemical methods, respectively. Then, we measured two analytes only by SPR method. We could measure glycoprotein, prostate specific antigen (PSA) (MW=33 kDa) and creatinine (MW= 113) both by SPR angle shifts caused by immuno- and redox reactions, indicating that urinary PSA concentration could be normalized with creatinine concentration by single injection.

Simultaneous On-chip Surface Plasmon Resonance Measurement of Disease Marker Protein and Small Metabolite

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