Proteins In Plasma Samples Research Articles

Determination of febuxostat in human plasma by high performance liquid chromatography (HPLC) with fluorescence-detection

Febuxostat prevents gout attacks by lowering serum urate. Aspects of the pharmacokinetic-pharmacodynamic relationship of febuxostat concentrations to urate in gout patients need further elucidation. In order to undertake these studies, the assay methodology for febuxostat has been enhanced and validated to meet FDA standards. An HPLC method with fluorescence-detection has been modified to increase sensitivity, reduce complexity, shorten the sample preparation process and improve the inter-day coefficient of variation of the lowest quality control sample (0.03 μg/L). Protein in plasma samples (200 μL) is now precipitated with acetonitrile (400 μL) containing the internal standard (2-naphthoic acid). The supernatant is analysed at excitation and emission wavelengths of 320 and 380 nm, respectively as in the previous method. A Luna C18 column (Phenomenex, Australia) at 40 °C with mobile phase of glacial acetic acid (0.032%) in acetonitrile:water (60:40, v:v), an injection volume of 10 μL and a flow rate of 1.5 mL/min is employed. Analysis time is 8 min. Calibration curves in drug-free plasma range from 0.005 to 10.00 μg/mL. Data points are fitted using linear regression with a weighting factor of 1/concentration. The inter-day accuracy and imprecision of the quality control samples (0.0075, 0.015, 3.00 and 9.80 μg/mL) is 90–115% and ≤ 14.5%, respectively.

Determination of plasma protein binding rates of nine compounds of Inula cappa extraction based on method of equilibrium dialysis

To determine the plasma protein binding rate of the nine compounds in Inula cappa extraction by the method of equilibrium dialysis. The proteins in plasma samples were precipitated by methanol, and the ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS) was developed for determination of the concentrations of the nine active compounds, namely chlorogenic acid, scopolin, neochlorogenic acid, cryptochlorogenic acid, 1,3-O-dicaffeoylquinic acid, galuteolin, 3,4-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, 4,5-O-dicaffeoylquinic acid, with the internal standard of puerarin. We found that all components have a good linearity(r≥0.999), and accuracy, precision, extraction recovery and stability conformed to the requirements of determination, without endogenous compounds disturbing within the range of optimum concentration. This suggested that the method was stable and reliable, and could be used for the determination of the plasma protein binding rates of the nine active compounds in rat and human plasma of I. cappa. The plasma protein binding rates of the nine active compounds in rat and human plasma respectively were(41.07±0.046)%-(94.95±0.008)%, and(37.66±0.043)%-(97.46±0.013)%. According to the results, there were differences in the plasma protein binding rates of the nine compounds in I. cappa extraction between rat and human.

Protein signatures linking history of miscarriages and metabolic syndrome: a proteomic study among North Indian women.

BackgroundMetabolic syndrome (MeS), a constellation of metabolic adversities, and history of miscarriage make women at a higher risk for cardiovascular diseases (CVDs). However, molecular evidence indicating a link between the two phenotypes (history of miscarriage and MeS) among women would offer an opportunity to predict the risk factor for CVDs at an early stage. Thus, the present retrospective study attempts to identify the proteins signatures (if any) to understand the connection between the history of miscarriage and MeS.MethodsAge-matched 80 pre-menopausal women who were not on any medical intervention or drugs were recruited from a Mendelian population of the same gene pool. Recruited women were classified into four groups—(a) Group A—absolute cases with history of miscarriage and MeS, (b) Group B—absolute controls without any history of miscarriage and MeS, (c) Group C—cases with MeS but lack any history of miscarriage, (d) Group D—cases with history of miscarriage but lack MeS. Differentially expressed proteins in plasma samples of women from four groups were identified using 2-D gel electrophoresis and mass spectrometry.ResultsThree case groups (A, C, and D) showed 18 differentially expressed proteins. Nearly 60% of proteins (11/18) were commonly dysregulated in Group C (only with MeS) and Group D (only with miscarriage history). Nearly 40% of proteins (7/18) were commonly dysregulated in the three case groups (Groups A, C, and D), indicating a shared pathophysiology. Four proteins were exclusive but shared by case groups C and D indicating the independent routes for CVDs through MeS or miscarriages. In absolute cases, transthyretin (TTR) showed exclusive upregulation, which was further validated by Western blotting and ELISA. Networking analyses showed the strong association of TTR with haptoglobin, transferrin and ApoA1 hinting toward a cross-talk among these proteins which could be a cause or an effect of TTR upregulation.ConclusionThe study provides evidence for molecular link between the history of miscarriage and MeS through a putative role of TTR. However, longitudinal follow-up studies with larger sample size would further help to demonstrate the significance of TTR and other targeted proteins in risk stratification and the onset of CVDs.

Quantification of protein markers monitoring the pre-analytical effect of blood storage time before plasma isolation using 15 N metabolically labeled recombinant proteins.

In the hospital, blood samples are collected to monitor patients' health states, and thus various protein-based clinical methods have been developed. However, some proteins are found to change in abundances during the process of blood collection and storage. In order to account such pre-analytical effects, we performed liquid chromatography multiple reaction monitoring mass spectrometry (LC-MRM-MS) on 15 selected proteins in plasma samples prepared by varying storage time and temperature of whole blood prior to plasma isolation. Two cytosolic proteins, profilin-1 (PFN1) and thymosin beta-4 (TMSB4X), were absolutely quantified using 15 N-labeled recombinant proteins spiked externally. The other 13 proteins were quantified in a relative way compared with the two reference proteins. Triplicated LC-MRM-MS measurements showed that the median CV of MRM peak areas was 5.7%. The amounts of PFN1 and TMSB4X increased rapidly depending on the storage time between blood collection and plasma preparation. It indicates the leakage of cellular components into the plasma fraction. Relative quantification further revealed that five proteins including PFN1, S10A8, S10A9, S10A11, and TMSB4X showed significant difference (P<0.05). We further monitored PFN1 and TMSB4X on 40 samples collected for protein diagnostics under a typical clinical study condition. Compared with the plasma samples prepared within a day, the level of both PFN1 and TMSB4X increased in the plasma samples prepared from the blood collected the day before and kept overnight at 4°C (0.51 to 3.11μg/mL for PFN1 and 0.98 to 5.36μg/mL for TMSB4X in average). Our result suggests an effort of assuring plasma quality for accurate protein-based diagnosis or biomarker discovery and validation.

Proteomic Identification of Novel Plasma Biomarkers and Pathobiologic Pathways in Alcoholic Acute Pancreatitis.

Acute pancreatitis (AP) is a painful and potentially life-threatening disorder with the potential for therapeutic interventions. Biomarkers that characterize cases by severity and pathogenic mechanisms involved are not yet available but needed for the implementation of rational therapies. Here, we used shotgun proteomics to obtain information from plasma samples about local and systemic pathologies taking place during cases of alcoholic AP. Plasma was obtained at Kaunas University of Medicine Hospital (Lithuania) from 12 AP patients of alcohol related etiology (median age of 40) within 24 h of presentation, and 12 age-matched, healthy controls. Patients entered into the study had moderately severe AP with the following characteristics: mean blood lactate dehydrogenase level of 1127 mg/dl; median APACHEII score of 5.5 and mean IMRIE score of 3.5. For proteomic analysis, less-abundant proteins in plasma samples were enriched using Top 12 abundant protein depletion columns. Further processing was performed by a modified filter-assisted sample preparation combined with tandem mass tag labeling for quantitation. Samples were analyzed using an Orbitrap Elite mass spectrometer for high resolution liquid chromatography–tandem mass spectrometry (LC–MS/MS). Our analysis revealed 31 proteins that exhibited significant 1.5-fold or higher increases in the AP compared to control patients, and six that were significantly decreased. Gene ontology analysis indicated a strong correlation with exosomal origin in the elevated proteins, with 29/31 (93.5%) associated with this extracellularly-secreted compartment. Elevated proteins included established and proposed biomarkers of AP including C-reactive protein, LPS-binding protein, intercellular adhesion molecule-1, and von Willebrand factor, as well as several novel potential biomarkers. These results provide the methodology for proteomic analysis of plasma samples to discover novel biomarkers that characterize pancreatitis cases by pathogenic mechanism as well as disease activity at an early stage that is highly informative for routine clinical practice and clinical trials.

Differential protein profiling as a potential multi-marker approach for obese patients with heart failure: A retrospective study

Identification of novel circulating biomarkers predicting death and major cardio-metabolic events in obese patients with heart failure (HF) remains a research priority. In this study, we compared multi-marker profile of non-obese (NOB) and obese (OB) HF patients in relation to mortality outcome. The new multiplex proximity extension assay technology was used to analyze the levels of 92 proteins in plasma samples from HF patients according to body mass index (BMI) categories. At 2-year follow-up, all-cause mortality rates were significantly greater in NOB patients (BMI < 30 kg/m2) compared to the OB patients (BMI > 30 kg/m2) with HF (odds ratio 26; 95% CI: 1.14–624, p < 0,04). Quantitative proteomic analysis revealed thirteen distinct proteins expression profiles of OB and NOB HF patients. Among these proteins, RAGE, CXCL6, CXCL1, CD40, NEMO, VEGF-A, KLK6, PECAM1, PAR1, MMP1, BNP and NTproBNP were down-regulated, whereas leptin was up-regulated in OB HF patients. In addition, an inverse correlation between plasma BNP levels and leptin in OB HF patients was observed (r = −0.58 p = 0.02). This study identifies specific plasma protein signature in OB and NOB patients with HF in relation to mortality outcome.

LC–MS/MS bioassay of four proton pump inhibitors

A new validated bio-analytical LC–MS/MS method was developed for the simultaneous extraction and determination of four proton pump inhibitors: esomeprazole, lansoprazole, pantoprazole and rabeprazole in human plasma using escitalopram as an internal standard. The proteins in plasma samples were precipitated using acetonitrile for the extraction of analytes which is a simple economic method. The separation was accomplished using a mobile phase composed of 10 mM ammonium formate: acetonitrile: methanol (20:40:40% v/v) at a flow rate of 0.8 mL/min in isocratic mode on a reversed phase C18 INERTSIL ODS-3 (5 μm, 150 × 4.6 mm) and column temperature of 40 °C. Positive mode electrospray ionization source was used prior to multiple reaction monitoring (MRM) detection using parent and daughter ions: m/z 346.2 → 198.1 for esomeprazole, m/z 370.1 → 252 for lansoprazole, m/z 384.2 → 200.2 for pantoprazole, m/z 360.1 → 242.1 for rabeprazole and m/z 325.2 → 109 for escitalopram. The calibration curves were constructed, and the method was linear in the range of 20–5000 ng/mL applying weighted (1/X2) linear regression coefficient for all drugs. The method was fully validated following US-FDA and EMA guidelines.

Plasma vascular non-inflammatory molecule 3 is associated with gastrointestinal acute graft-versus-host disease in mice

BackgroundGastrointestinal acute graft-versus-host disease (GI aGVHD) is a lethal complication following allogeneic hematopoietic stem cell transplantation (HSCT). However, it is still very difficult to make a diagnosis of GI aGVHD in practice. To date, no consensus plasma biomarker of GI aGVHD can be used to help make a diagnosis. Here, we attempted to identify GI aGVHD associated plasma proteins in murine model, which can help make a diagnosis of GI aGVHD.MethodsWe used 8-plex isobaric tags for relative and absolute quantitation (8-plex iTRAQ) to screen out proteins in plasma samples taken from murine models before and after allogeneic HSCT. Next mRNA expressions were validated by quantitative real-time polymerase chain reaction in mouse intestinal epithelial samples.ResultsWe found that five proteins were increased at least 2-fold in the allogeneic group at day 7 compared with days 0, 3 and 15 (after Cyclosporin A treatment) and the syngeneic group at day 7. These 5 proteins were VNN3, ZNF746, C4BP, KNG1 and FETUB, and they were consistent with results from negative labeling with 8-plex iTRAQ. Furthermore, increase in mRNA level of VNN3 was confirmed in murine intestinal epithelial samples with aGVHD.ConclusionsOur results demonstrate that plasma VNN3 protein is associated with GI aGVHD in murine model.

Diagnostic and Prognostic Plasma Biomarkers for Idiopathic Pneumonia Syndrome after Hematopoietic Cell Transplantation

Idiopathic pneumonia syndrome (IPS) is a noninfectious pulmonary complication after hematopoietic cell transplantation (HCT) and is difficult to diagnose. In 41 patients with IPS, we evaluated 6 candidate proteins in plasma samples at day 7 post-HCT and at onset of IPS to identify potential diagnostic or prognostic biomarkers for IPS. Samples at similar times from 162 HCT recipients without documented infections and 37 HCT recipients with respiratory viral pneumonia served as controls. In multivariable models, a combination of Stimulation-2 (ST2; odds ratio [OR], 2.8; P < .001) and IL-6 (OR, 1.4; P = .025) was the best panel for distinguishing IPS at diagnosis from unaffected controls, whereas tumor necrosis factor receptor 1 (TNFR1; OR, 2.9; P = .002) was the best marker when comparing patients with IPS and viral pneumonia. The areas under the curve of the receiver operating characteristic (ROC) curves for discriminating between IPS and unaffected controls at day 7 post-HCT were .8 for ST2, .75 for IL-6, and .68 for TNFR1. Using estimated sensitivity and specificity values from cutoffs determined with the ROC analysis (cutoff level: ST2, 21 ng/mL; IL-6, 61 pg/mL; TNFR1, 3421 pg/mL), we calculated positive predictive values (PPV) for a range of estimated population prevalence values of IPS. Among the 3 markers, ST2 showed the highest PPV for IPS occurrence. Based on an assumed prevalence of 8%, a positive ST2 test increased likelihood of IPS to 50%. We conclude that a prospective validation study is warranted to determine whether a plasma biomarker panel can aid the noninvasive diagnosis and prognosis of IPS.

Sandwich NP-based biobarcode assay for quantification C-reactive protein in plasma samples

A NP-based biobarcode for C-reactive protein (CRP) quantification in plasma samples is reported for the first time. The assay uses capture antibody functionalized magnetic beads (pAbCRP2-MP), multifunctional oligonucleotide encoded probes modified with a detection antibody (pAbCRP1-ePSP), and a fluorescent DNA microarray. Thus, magnetic beads are added to the sample to form immunocomplexes that will be isolated, to then add the codified particles to form a sandwich complex with both particles and the target protein, subsequently the complexes are treated to release the oligonucleotide codes, which are finally hybridized in a fluorescent DNA microarray. The assay has been implemented to the analysis of plasma samples being able to quantify this biomarker within 900 ng mL−1 to 12500 ng mL−1 with an excellent accuracy (mean of recovery of 99.5 ± 4.2%, N = 3). The CRP biobarcode has been used on a small pilot clinical study in which plasma samples from patients suffering different pathologies, most of them related to cardiovascular diseases (CVDs). The samples have been analyzed and the results compared to a reference method demonstrating that the assay can be useful for monitoring this biomarker on patients being suspicious to be under risk of suffering CVDs or other diseases involving inflammatory processes.

COPD Exacerbation Biomarkers Validated Using Multiple Reaction Monitoring Mass Spectrometry.

BackgroundAcute exacerbations of chronic obstructive pulmonary disease (AECOPD) result in considerable morbidity and mortality. However, there are no objective biomarkers to diagnose AECOPD.MethodsWe used multiple reaction monitoring mass spectrometry to quantify 129 distinct proteins in plasma samples from patients with COPD. This analytical approach was first performed in a biomarker cohort of patients hospitalized with AECOPD (Cohort A, n = 72). Proteins differentially expressed between AECOPD and convalescent states were chosen using a false discovery rate <0.01 and fold change >1.2. Protein selection and classifier building were performed using an elastic net logistic regression model. The performance of the biomarker panel was then tested in two independent AECOPD cohorts (Cohort B, n = 37, and Cohort C, n = 109) using leave-pair-out cross-validation methods.ResultsFive proteins were identified distinguishing AECOPD and convalescent states in Cohort A. Biomarker scores derived from this model were significantly higher during AECOPD than in the convalescent state in the discovery cohort (p<0.001). The receiver operating characteristic cross-validation area under the curve (CV-AUC) statistic was 0.73 in Cohort A, while in the replication cohorts the CV-AUC was 0.77 for Cohort B and 0.79 for Cohort C.ConclusionsA panel of five biomarkers shows promise in distinguishing AECOPD from convalescence and may provide the basis for a clinical blood test to diagnose AECOPD. Further validation in larger cohorts is necessary for future clinical translation.

Fibronectin on circulating extracellular vesicles as a liquid biopsy to detect breast cancer

Extracellular vesicles (EVs) secreted from cancer cells have potential for generating cancer biomarker signatures. Fibronectin (FN) was selected as a biomarker candidate, due to the presence in surface on EVs secreted from human breast cancer cell lines. A subsequent study used two types of enzyme-linked immunosorbent assays (ELISA) to determine the presence of these proteins in plasma samples from disease-free individuals (n=70), patients with BC (n=240), BC patients after surgical resection (n=40), patients with benign breast tumor (n=55), and patients with non-cancerous diseases (thyroiditis, gastritis, hepatitis B, and rheumatoid arthritis; n=80). FN levels were significantly elevated (p<. 0001) at all stages of BC, and returned to normal after tumor removal. The diagnostic accuracy for FN detection in extracellular vesicles (ELISA method 1) (area under the curve, 0.81; 95% CI, 0.76 to 0.86; sensitivity of 65.1% and specificity of 83.2%) were also better than those for FN detection in the plasma (ELISA method 2) (area under the curve, 0.77; 95% CI, 0.72 to 0.83; sensitivity of 69.2% and specificity of 73.3%) in BC. The diagnostic accuracy of plasma FN was similar in both the early-stage BC and all BC patients, as well as in the two sets. This liquid biopsy to detect FN on circulating EVs could be a promising method to detect early breast cancer.

General intelligence is associated with subclinical inflammation in Nepalese children: A population-based plasma proteomics study

Improving child cognition in impoverished countries is a public health priority. Yet, biological pathways and associated biomarkers of impaired cognition remain poorly understood and largely unknown, respectively. This study aimed to explore and quantify associations between functional plasma protein biomarkers and childhood intellectual test performance. We applied proteomics to quantify proteins in plasma samples of 249 rural Nepalese children, 6–8years of age who, 1year later at 7–9years of age, were administered the Universal Nonverbal Intelligence Test (UNIT). Among 751 plasma proteins quantified, 22 were associated with UNIT scores, passing a false discovery rate threshold of 5.0% (q<0.05). UNIT scores were higher by 2.3–9.2 points for every 50% increase in relative abundance of two insulin-like growth factor binding proteins (IGFBPs), six subclasses of apolipoprotein (Apo) and transthyretin, and lower by 4.0–15.3 points for each 50% increase in relative abundance of 13 proteins predominantly involved in inflammation. Among them, IGFBP-acid labile subunit, orosomucoid 1 (ORM1), Apo C-I, and pyruvate kinase isoenzymes M1/M2 jointly explained 37% of the variance in UNIT scores. After additional adjustment for height-for-age Z-score and household socio-economic status as indicators of long-term nutritional and social stress, associations with 6 proteins involved in inflammation, including ORM1, α-1-antichymotrypsin, reticulocalbin 1, and 3 components of the complement cascade, remained significant (q<0.05). Using untargeted proteomics, stable, constitutive facets of subclinical inflammation were associated with lower developmental test performance in this rural South Asian child population. Plasma proteomics may offer opportunities to identify functional, antecedent biomarkers of child cognitive development.

Pretreatment Albumin/Globulin Ratio Predicts the Prognosis for Small-Cell Lung Cancer.

The pretreatment albumin/globulin ratio (AGR) has been used as a prognostic factor in various cancers. This study aimed to evaluate the predictive value of AGR in small-cell lung cancer (SCLC).We tested albumin and total proteins in plasma samples from 276 SCLC patients from our cancer center between January 2003 and December 2006. The AGR was defined by the formula: albumin/(total proteins–albumin). The correlation between AGR and overall survival (OS) was examined by Kaplan–Meier and Cox regression methods. For validation, AGR was used to evaluate the prognosis of SCLC in another independent group.Total 276 patients (testing) and 379 patients (validation) were finally enrolled. The median OS was 15.31 months for testing patients and 15.06 months for validation patients, respectively. We determined 1.29 as the cutoff value by using the biostatistical tool (Cutoff Finder), then the patients in the testing group were classified into 2 groups. Kaplan–Meier curves showed high AGR group had significantly longer OS than low AGR group (P = 0.026). According to multivariate analyses, AGR was an independent prognostic factor for OS of SCLC patients in the testing group (HR, 1.35, 95% CI: 1.01–1.81, P = 0.046). In the validation group, AGR was also verified as a predictive factor for OS (P < 0.001), and the risk of SCLC in the low AGR group was 1.43 times higher than that in the high AGR group (HR, 1.43, 95% CI: 1.05–1.94, P = 0.022).AGR is an independent prognostic marker in SCLC patients. Furthermore, it could be of great value in the management of SCLC patients.

Affinity proteomics discovers decreased levels of AMFR in plasma from Osteoporosis patients.

PurposeAffinity proteomic approaches by antibody bead arrays enable multiplexed analysis of proteins in body fluids. In the presented study, we investigated blood plasma within osteoporosis to discovery differential protein profiles and to propose novel biomarkers candidates for subsequent studies.Experimental designStarting with 4608 antibodies and plasma samples from 22 women for an untargeted screening, a set of 72 proteins were suggested for further analysis. Complementing these with targets from literature and other studies, a targeted bead array of 180 antibodies was built to profile for 92 proteins in plasma samples of 180 women from two independent population‐based studies.ResultsDifferential profiles between osteoporosis patients and matched controls were discovered for 12 proteins in at least one of the two study sets. Among these targets, the levels of autocrine motility factor receptor (AMFR) were concordantly lower in plasma of female osteoporosis patients. Subsequently, verification of anti‐AMFR antibody selectivity was conducted using high‐density peptide and protein arrays, and Western blotting.Conclusions and clinical relevanceFurther validation in additional study sets will be needed to determine the clinical value of the observed decrease in AMFR plasma levels in osteoporosis patients, but AMFR may aid our understanding of disease mechanisms and could support existing tools for diagnosis and monitoring of patient mobility within osteoporosis.

Carbonylated plasma proteins as potential biomarkers of obesity induced type 2 diabetes mellitus.

Protein carbonylation is a common nonenzymatic oxidative post-translational modification, which is often considered as biomarker of oxidative stress. Recent evidence links protein carbonylation also to obesity and type 2 diabetes mellitus (T2DM), though the protein targets of carbonylation in human plasma have not been identified. In this study, we profiled carbonylated proteins in plasma samples obtained from lean individuals and obese patients with or without T2DM. The plasma samples were digested with trypsin, carbonyl groups were derivatized with O-(biotinylcarbazoylmethyl)hydroxylamine, enriched by avidin affinity chromatography, and analyzed by RPC-MS/MS. Signals of potentially modified peptides were targeted in a second LC-MS/MS analysis to retrieve the peptide sequence and the modified residues. A total of 158 unique carbonylated proteins were identified, of which 52 were detected in plasma samples of all three groups. Interestingly, 36 carbonylated proteins were detected only in obese patients with T2DM, whereas 18 were detected in both nondiabetic groups. The carbonylated proteins originated mostly from liver, plasma, platelet, and endothelium. Functionally, they were mainly involved in cell adhesion, signaling, angiogenesis, and cytoskeletal remodeling. Among the identified carbonylated proteins were several candidates, such as VEGFR-2, MMP-1, argin, MKK4, and compliment C5, already connected before to diabetes, obesity and metabolic diseases.

Plasma-based proteomics reveals lipid metabolic and immunoregulatory dysregulation in post-stroke depression.

Post-stroke depression (PSD) is the most common psychiatric complication facing stroke survivors and has been associated with increased distress, physical disability, poor rehabilitation, and suicidal ideation. However, the pathophysiological mechanisms underlying PSD remain unknown, and no objective laboratory-based test is available to aid PSD diagnosis or monitor progression. Here, an isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomic approach was performed to identify differentially expressed proteins in plasma samples obtained from PSD, stroke, and healthy control subjects. The significantly differentiated proteins were primarily involved in lipid metabolism and immunoregulation. Six proteins associated with these processes--apolipoprotein A-IV (ApoA-IV), apolipoprotein C-II (ApoC-II), C-reactive protein (CRP), gelsolin, haptoglobin, and leucine-rich alpha-2-glycoprotein (LRG)--were selected for Western blotting validation. ApoA-IV expression was significantly upregulated in PSD as compared to stroke subjects. ApoC-II, LRG, and CRP expression were significantly downregulated in both PSD and HC subjects relative to stroke subjects. Gelsolin and haptoglobin expression were significantly dysregulated across all three groups with the following expression profiles: gelsolin, healthy control>PSD>stroke subjects; haptoglobin, stroke>PSD>healthy control. Early perturbation of lipid metabolism and immunoregulation may be involved in the pathophysiology of PSD. The combination of increased gelsolin levels accompanied by decreased haptoglobin levels shows promise as a plasma-based diagnostic biomarker panel for detecting increased PSD risk in post-stroke patients.

Discovery of novel biomarker candidates for liver fibrosis in hepatitis C patients using proteomics

Liver biopsy is the reference standard for assessing liver fibrosis and no reliable non-invasive diagnostic approach is available to discriminate between the intermediate stages of fibrosis. Therefore suitable serological biomarkers of liver fibrosis are urgently needed. We used proteomics to identify novel fibrosis biomarkers in hepatitis C patients with different degrees of liver fibrosis. Proteins in plasma samples from healthy control individuals and patients with hepatitis C virus (HCV) induced cirrhosis were analysed using a proteomics technique: two dimensional gel electrophoresis (2-DE). This technique separated the proteins in plasma samples of control and cirrhotic patients and by visualizing the separated proteins we were able to identify proteins which were increasing or decreasing in hepatic cirrhosis. Identified markers were validated across all Ishak fibrosis stages and compared to the markers used in FibroTest, Enhanced Liver Fibrosis (ELF) test, Hepascore and FIBROSpect by Western blotting. Forty four candidate biomarkers for hepatic fibrosis were identified of which 20 were novel biomarkers of liver fibrosis. Western blot validation of all candidate markers using plasma samples from patients across all Ishak fibrosis scores showed that the markers which changed with increasing fibrosis most consistently included lipid transfer inhibitor protein, complement C3d, corticosteroid-binding globulin, apolipoprotein J and apolipoprotein L1. These five novel fibrosis markers which are secreted in blood showed a promising consistent change with increasing fibrosis stage when compared to the markers used for the FibroTest, ELF test, Hepascore and FIBROSpect. These markers are currently being further validated using a large clinical cohort. This study identifies 20 novel fibrosis biomarker candidates. The proteins identified may help to assess hepatic fibrosis and eliminate the need for invasive liver biopsies.

Proteomic profiles among asymptomatic HTLV-1 carriers and HAM/TSP patients in Peru

High proviral loads have been described in HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients in comparison to asymptomatic HTLV-1 carriers (AC). However, biomarkers related to HAM/TSP progression have not been identified. We analyzed differential proteome changes by two-dimensional gel electrophoresis (2D-electrophoresis) to identify spots of proteins in plasma samples of three groups of patients: five AC, nine HAM/TSP patients: five patients with EDSS scores of 1.0-5.0 (=mild HAM/TSP) and 4 patients with EDSS scores of 5.5-9.0 (=severe HAM/TSP). Proteins were extracted from pooled plasma samples from each group of patients. Protein separations were performed by electrofocusing with 11 cm strips at 3-10 pH range, electrophoresis with 12% poliacrilamide gel electrophoresis and silver staining. Analyses were performed in Progenesis Same Spot software; ANOVA was used to compare the profiles of the groups. Six spots of proteins were differentially expressed among these groups: five proteins increased their expression according to HAM/TSP disease progression (highest fold change= 6.1, p=1.671 x10-4; lowest fold change= 1.5, p= 0.014) while one protein was highly expressed in AC and decreased according to HAM/TSP progression (fold change= 5.8, p= 0.002). Further studies to confirm the expression of these proteins in a larger set of samples are still in progress, the identification of these proteins will be performed by mass spectrometry. These results might be promissory to identify biomarkers for HAM/TSP progression.

Highly sensitive and quantitative human thrombospondin-1 detection by an M55 aptasensor and clinical validation in patients with atherosclerotic disease

Aptamer-based biosensors (aptasensor) are powerful tools for rapid and sensitive biomarker detection. In this study, we report a DNA aptamer probe evolved from cell-SELEX that can recognize thrombospondin-1 protein in human plasma samples. The KD value of the aptamer M55 binding to thrombospondin-1 was determined as 0.5±0.2μM with an R2 of 0.9144. A horseradish peroxidase-linked short oligo was complementarily bound onto the 3' end of the aptamer sequence to facilitate the ‘smart’ design of an M55-aptasensor for quantifying thrombospondin-1 protein in plasma samples. The limit of detection was 6.96fM. Thrombospondin-1 is a glycoprotein with multiple biological functions, including inflammation, platelet aggregation and endothelial cell apoptosis, and is involved in the pathology of atherosclerosis. In total, 118 plasma subjects were analyzed by using the aptasensor measurement with 1μL sample volume and 5min incubation time. The thrombospondin-1 concentrations in ST-Elevation Myocardial Infarction patients with severe atherosclerotic plaque burden were statistically significantly higher than in the healthy volunteers without atherosclerosis conditions, suggesting that thromboposnidn-1 is a potential plasma biomarker for atherosclerosis progression.