Analysis Of Serum Proteins Research Articles

Dual detection high-speed capillary electrophoresis for simultaneous serum protein analysis and immunoassays

Serum protein electrophoresis (SPE) separates serum proteins into bands whose shape and amplitude can alert clinicians to a range of disorders. This is followed by more specific immunoassays to quantify important antigens and confirm a diagnosis. Here we develop a high-speed capillary electrophoresis (HSCE) platform capable of simultaneous SPE and immunoassay measurements. A single laser excitation source is focused into the detection zone of the capillary to measure both refractive index (SPE) and fluorescence signals (immunoassays). The refractive index signal measures characteristic SPE profiles for human serum separated in 100 mM boric acid (pH 10), 100 mM arginine (pH 11), and 20 mM CHES (pH 10). For the immunoassay, the fluorescence electropherograms reveal that CHES provides the optimal buffer for measuring the immunocomplex and separating it from the free antigen. Immunoassays in CHES yield a LOD of 23 nM and a LOQ of 70 nM for the detection of fluorescein. The high pH reduces protein adsorption but reduces antibody affinity. Preliminary studies carried out in 50 mM barbital at pH 8 show improved stability of the immunocomplex and better separation for immunoassay quantification. Further optimization will open new capabilities for measuring orthogonal diagnostic signals in seconds with HSCE.

A comprehensive and comparative proteomic analysis of horse serum proteins in colitis

Equine colitis is a diarrhoeal disease caused by inflammation of the large bowel and can potentially be life-threatening due to its rapid progression. Pathogenesis is multifactorial and pathophysiology is highly complicated, therefore, reliable diagnostic biomarkers are needed in the veterinary field. Serum is one of the most commonly used diagnostic tools in equine clinical investigation. To discover diagnostic or prognostic protein markers for colitis in horse serum, comprehensive and comparative proteomic analysis was conducted using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Case-control study. Serum samples were collected from 36 healthy Thoroughbreds and 12 Thoroughbreds with colitis. Serum from each horse suffering from colitis was collected daily until death or recovery. Collected sera were digested with trypsin. Peptides obtained from serum proteins were measured by Q-Exactive HF Orbitrap mass spectrometer. The identification and quantification of peptides were performed using Proteome Discoverer version 2.2. On day 1 of treatment, eight proteins in the colitis group were upregulated (P<.05, more than a twofold change) compared with the healthy group. Among the eight proteins, biliverdin reductase B was significantly upregulated (P<.05) in the non-survivor group (n=5) compared with the survivor group (n=7). On the last day of the treatment, haemoglobin subunit alpha, clusterin, glyceraldehyde-3-phosphate dehydrogenase, lipopolysaccharide-binding protein, and biliverdin reductase B showed significant increases (P<.05) in the non-survivor group. The number of the identified proteins is limited due to the existence of abundant proteins. Measuring the changes of these proteins together may enable a potential prognosis or early diagnosis of horses suffering from colitis.

Comparative evaluation of darbepoetin therapy in non-regenerative anaemia associated with Babesia gibsoni infection in dogs

The present study describes a comparative evaluation of haemato-therapeutic response to darbepoetin therapy in non-regenerative anaemia associated with Babesia gibsoni infection in dogs. A total of 68 dogs, presented at Teaching Veterinary Clinical Complex, Mannuthy with signs of anaemia, weakness, anorexia and pallor of mucous membranes were screened for nonregenerative anaemia. Twenty dogs with a reticulocyte count of &lt; 60,000/μL, haematocrit of &lt; 30 per cent and positive for B. gibsoni were selected for detailed study. The twenty dogs were divided into two groups of ten animals and were subjected to estimation of complete blood count and analysis of serum protein and iron status. Ten dogs brought to the hospital for vaccination or health check-up served as healthy control. Haematological examination revealed significant anaemia and thrombocytopenia in both the groups. Serum total protein, albumin and total iron binding capacity revealed no difference between diseased groups with the healthy control. Serum iron and percentage transferrin saturation were similar in Group I and II, but significantly higher than healthy control. To compare darbepoetin efficacy, Group I dogs were administered with clindamycin and doxycycline for three weeks, whereas Group II was given with darbepoetin at the dose rate of 0.5 μg/kg body weight at weekly intervals for three weeks in addition to clindamycin-doxycycline as in Group I. Dogs of both groups showed complete remission of clinical signs within 10 days of therapy. Statistical analysis revealed no significant difference in haematological parameters between the treatment groups. It can be concluded from this study that there is no added advantage of darbepoetin therapy over conventional therapies against non-regenerative anaemia associated with B. gibsoni infection in dogs.

Proteomic analysis of serum proteins in children with brain death.

BackgroundBrain death (BD) is a catastrophic physiological outcome that can occur in individuals with terminal illness and can adversely affect the graft quality after donation of their organs. As BD has no specific symptoms, it can be difficult to diagnose in a timely manner. The present study was designed to investigate the serum protein expression profiles of children affected by BD in an effort to define diagnostic biomarkers for this condition.MethodsBlood samples were collected from 8 patients with BD and 8 healthy controls during the same time period. Tandem mass tags and mass spectrometry were used to conduct a proteomic analysis of serum extracted from the samples. The potential regulatory roles of the top 5 upregulated and downregulated proteins identified through the analysis were then explored using bioinformatics analyses and a review of the related literature.ResultsThe top 5 upregulated proteins in the serum samples from patients with BD were lipopolysaccharide-binding protein (LBP), α1-acid glycoprotein (α1-AGP), α1-antichymotrypsin (α1-ACT), leucine-rich α1-glycoprotein (LRG1), and lactate dehydrogenase B heavy chain (LDHB), and the 5 most downregulated proteins in these samples were actin-binding protein 2 (transgelin-2), platelet basic protein (PBP), tropomyosin α4 chain (TPM4), tropomyosin α3 chain (TPM3), and peptidase inhibitor 16 (PI16). Literature searches indicated that several of the identified proteins influence the pathogeneses of various diseases, with LBP, α1-AGP, α1-ACT, LRG1, transgelin-2, and PBP all being related to inflammatory activity.ConclusionsThrough a proteomics-based analysis, several differentially expressed proteins were identified in patients with BD relative to healthy controls. Most of these proteins are associated with inflammatory responses that have the potential to persist after the occurrence of BD. Further clinical work is needed to clarify the functional roles of the identified proteins.

Proteomic identification of serum proteins to induce osteoconductivity of hydroxyapatite.

We performed proteomic analysis of rat serum proteins adsorbed on hydroxyapatite (HAp) and α-alumina (α-Al2O3) in order to identify proteins that specifically adsorb onto HAp and control cellular responses. Proteins with either or both molecular weight of 22-32 kDa and computed isoelectric point of 5.0-5.5 were preferentially adsorbed on HAp. In total, 182 proteins were adsorbed on both HAp and α-Al2O3, of which 14 were highly enriched on HAp, whereas 68 were adsorbed only on HAp. Therefore, 82 (14+68) proteins were further evaluated by bioinformatics and literature-based analyses. We predicted that hepatocyte growth factor and angiopoietin-like protein 3 (ANGPTL3) are candidate proteins responsible for the osteoconductivity of HAp. Although ANGPTL3 promoted the attachment and spreading of MC3T3-E1 cells, it did not promote their proliferation and differentiation. Our results suggest that specific adsorption of ANGPTL3 on HAp induced osteoconductivity by enhancing the attachment and spreading of osteoblasts.

Proteomic analysis of serum protein during early pregnancy

Proteomic analysis of serum protein during early pregnancy

Proteomics Analysis of Serum Proteins in Gestational Diabetes.

Objective The purpose of this study was to screen serum proteins for biomarkers of gestational diabetes mellitus (GDM) and to investigate its pathogenesis by analyzing the differences in serum proteomics between pregnant women with GDM and healthy pregnant women. Methods Patients who were admitted to the First Affiliated Hospital of Fujian Medical University from June 2019 to January 2020 were included. According to the medical history and the results of the 75 g oral glucose tolerance test (OGTT), they were divided into the normal pregnant women group and GDM pregnant women group. The serum of two groups of patients was collected. High performance liquid chromatography-mass spectrometry was used to identify differentially expressed serum proteins between pregnant women with GDM and healthy pregnant women, and bioinformatics analysis was then performed on the identified proteins. Results A total of 1152 quantifiable proteins were detected; among them, 15 were upregulated in serum of GDM pregnant women, while 26 were downregulated. The subsequent parallel reaction monitoring (PRM) assay validated the expression levels of 12 out of 41 differentially expressed proteins. Moreover, bioinformatics analysis revealed that the differentially expressed proteins are involved in multiple biological processes and signaling pathways related to the lipid metabolism, glycan degradation, immune response, and platelet aggregation. Conclusions This study identified 41 serum proteins with differential expression between pregnant women with GDM and healthy pregnant women, providing new candidate molecules for elucidating GDM pathogenesis and screening therapeutic targets.

Comparative Analysis of Serum Proteins Between Hepatitis B Virus Genotypes B and C Infection by DIA-Based Quantitative Proteomics.

PurposeIn clinical practice, the clinicopathological profiles and outcomes of patients infected with hepatitis B virus (HBV) are different between genotypes B and C. However, little is known about the potential mechanism and differences in specific biological pathways associated with the different genotype. This study aimed to compare the serum protein profile between patients infected with HBV genotype B and those infected with HBV genotype C.Patients and MethodsA total of 54 serum samples from patients with chronic HBV genotype B infection and those with chronic HBV genotype C infection, and healthy controls were used for the proteomic analysis (n = 18 samples in per group). Serum proteomic profiles were analyzed using data-independent acquisition (DIA)-based liquid chromatography-mass spectrometry to identify differentially expressed proteins (up- or downregulation of at least 1.5-fold) between serum samples from HBV patients infected with HBV genotype B and those infected with genotype C.ResultsWe identified 1010 proteins, 53 of which were differentially expressed between the serum samples of the healthy controls and those of HBV genotype B infected patients, and 59 that were differentially expressed between the samples of the healthy controls and those of HBV genotype C infected patients. Furthermore, our results indicated that two proteins identified as being differentially expressed (VWF and C8B) have potential as biomarkers for distinguishing genotype B infected HBV patients from those infected with genotype C.ConclusionThe results of our DIA-based quantitative proteomic analysis revealed that HBV genotypes B and C are associated with different molecular profiles and may provide fundamental information for further detailed investigations of the molecular mechanism underlying these differences.

ASSESSING THE PREVALENCE OF DRUG-INDUCED NEPHRITIS IN HOSPITAL BASED SETTING OF CENTRAL INDIA: A CLINICAL CROSS-SECTIONAL RETROSPECTIVE STUDY

Background: One of the most common etiological factors leading to chronic kidney disease and acute renal failure in the present clinical scenario is drug-induced renal disease. By direct toxicity and immunologic mechanism virtue, certain stereotyped renal responses are initiated by various drugs.&#x0D; Objectives: The present study was conducted to retrospectively assess the prevalence and incidence of drug-induced nephrotoxicity at the Department of Pathology, Sri Shankaracharya Institute of Medical Sciences, Bhilai, and Chhattisgarh. The study was conducted for 6 months on 120 subjects having drug-induced nephritis. The study subjects were within the age range of 30-70 years and had 50% females.&#x0D; Methods: The study screened 500 subjects of a defined age group where anthropometric and demographic records were obtained followed by serum creatinine measurement and protein analysis using the dipstick method. Glomerular filtration rate was estimated (eGFR) using the 4-variable modification of diet in renal disease (MDRD) equation and Cockcroft-Gault equation corrected to the body surface area (CG-BSA).&#x0D; Results: In 2.8% of subjects proteinuria was seen with DIN in 6.3% (n=120) subjects using MDRD for GFR assessment. The DIN prevalence was found to be 24% using the CG-BSA method. DIN was found to be significantly associated with hypertension, diabetes, smoking, abdominal obesity, advanced age, and gender. The large difference in Din prevalence between CG-BSA equations and MDRD shows that there is a need for having better measures for assessing the kidney function in the population of central India. Also, CG-BSA equations suggest a similar need for having better measures for assessing the kidney function in the population of central India.&#x0D; Keywords: Body mass index (BMI), Cockcroft-Gault (CG), chronic kidney disease (CKD), drug-induced nephrotoxicity (DIN), Proteinuria, Glomerular filtration rate (GFR),

The Effect Of Koya Reboned Powder Consumption On Protein Levels Of Pregnant Women In Prevention Of Stunting In Babies

Linear growth retardation in childhood has decreased over the past few decades, but has not had a positive effect. In Indonesia, the change in the prevalence of stunting is around 37%. The nutritional status of pregnant women plays an important role in the incidence of stunting, which will be seen at the age of &lt;5 years in 1000 days of their life. Pregnant women with poor nutrition and significant infection can give birth to a baby with stunting. For this reason, nutritional intake is needed, one of the animal foods that are high in protein is rebon shrimp about 62.4 g / 100 grams of dried shrimp. Protein has an important component so it is necessary for the physical development of children. The purpose of this study was to analyze serum protein levels before and after consuming rebon shrimp based PMT powder in the intervention group and control group and to analyze differences in serum protein levels for pregnant women before and after consuming PMT Koya powder made from rebon shrimp between the control and intervention groups. . This type of research is a quantitative study with a quasi-experimental method using the pre post test control group design approach. The number of respondents was 30 people who were divided into 2 groups, namely the intervention group and the control group. The intervention group was given koya powder as much as 100 grams / day for 7 days, assuming the fulfillment of 70 KKal. Data analysis using Data analysis using the Mann-Whitney test, if the data is not normally distributed. If the data are not normally distributed using the free sample t2 test. The conclusion of the analysis results to answer the research hypothesis. The results of the study were significant values P 0.000 &lt;0.05. Conclusion There was a significant difference in serum protein levels of pregnant women before and after consuming PMT Koya powder made from boiled shrimp between the control and intervention groups.

Label-free diagnosis of breast cancer based on serum protein purification assisted surface-enhanced Raman spectroscopy

Serum protein is generally used to assess the severity of disease, as well as cancer progression and prognosis. Herein, a simple and rapid serum proteins analysis method combined with surface-enhanced Raman spectroscopy (SERS) technology was applied for breast cancer detection. The cellulose acetate membrane (CA) was employed to extract human serum proteins from 30 breast cancer patients and 45 healthy volunteers and then extracted proteins were mixed with silver nanoparticles for SERS measurement. Additionally, we also mainly assessed the use of different ratios of proteins-silver nanoparticles (Ag NPs) mixture to generate maximum SERS signal for clinical samples detection. Two multivariate statistical analyses, principal component analysis-linear discriminate analysis (PCA-LDA) and partial least square-support vector machines (PLS-SVM) were used to analyze the obtained serum protein SERS spectra and establish the diagnostic model. The results demonstrate that the PLS-SVM model provides superior performance in the classification of breast cancer diagnosis compared with PCA-LDA. This exploratory work demonstrates that the label-free SERS analysis technique combined with CA membrane purified serum proteins has great potential for breast cancer diagnosis.

Molecular basis for clinical diversity between autoantibody subsets in diffuse cutaneous systemic sclerosis

ObjectivesClinical heterogeneity is a cardinal feature of systemic sclerosis (SSc). Hallmark SSc autoantibodies are central to diagnosis and associate with distinct patterns of skin-based and organ-based complications. Understanding molecular differences...

Abstract 522: Immunogenic chemotherapy and immune checkpoint inhibition (ICI) in microsatellite-stable (MSS) metastatic colorectal cancer (mCRC): Biomarkers indicative of durable treatment response

Abstract Purpose: Cancer immune therapy has revolutionized patient outcome for the small mCRC subgroup with highly immunogenic disease. The majority of mCRC cases, however, are MSS with inherently low susceptibility to immune therapy. In our ongoing METIMMOX study (NCT03388190), MSS-mCRC patients are randomized to oxaliplatin-based immunogenic chemotherapy followed by ICI (experimental study arm) or the oxaliplatin-based standard-of-care (control arm). A subgroup of experimental arm patients has obtained durable major partial or complete responses. Such remarkable outcomes call for biomarkers that may help identifying MSS-mCRC patients who will benefit from such sequential therapy. Experimental procedures: Study enrollment began August 2018. Patients with unresectable, previously untreated MSS-mCRC have been randomized to the Nordic FLOX regimen (standard-of-care) or repeat sequential 2 FLOX cycles and 2 cycles of nivolumab (240 mg Q2W). At analysis October 2020, 48 patients were evaluable for progression-free survival (PFS). Serum collected at baseline and after the first 2 FLOX and 2 nivolumab cycles were analyzed for 42 immune factors with the Luminex multiplex immunoassay. The Significance Analysis for Microarrays method was applied to select the most significant proteins. Diffusion-weighted magnetic resonance imaging (DW-MRI) of the liver was performed at baseline and after the first 2 FLOX cycles. Using a b-value of 1000 s/mm2, the intensity ratio of a representative metastatic lesion relative to normal parenchymal tissue in the liver was calculated at each recording. Results: At this early time of analysis, with median follow-up of 8.6 (range, 1-21) months, median PFS for the entire groups of control and experimental arm patients was identical (6.4 and 6.6 months, respectively). We selected 13 experimental arm patients with long (median 13.6 months) or short (median 5.6 months) PFS for the serum protein analysis and 9 patients with liver DW-MRI. High serum CD40L levels (&amp;gt; median 47.6 ng/ml) at baseline or CD23 levels (&amp;gt; median 15.7 ng/ml) after the first 2 FLOX and 2 nivolumab cycles were both associated with improved PFS (p &amp;lt; 0.001 and p &amp;lt; 0.007; log-rank test). The baseline DW-MRI intensity of the peripheral hyperintense rim of the metastatic lesion (2.0- to 2.8-fold higher than the corresponding parenchymal value) diminished towards the parenchymal intensity (1.2- to 1.6-fold higher) after the first 2 FLOX cycles in patients with longer PFS than the median for all study patients. The rim intensity did not change in patients with short PFS. Conclusions: Three potential response markers in serum or at DW-MRI (one at baseline, one following the induction immunogenic chemotherapy, and one following the subsequent ICI) were identified for MSS-mCRC at this early time of the METIMMOX study conduct. Citation Format: Anne Hansen Ree, Sebastian Meltzer, Kine M. Bakke, Hanne M. Hamre, Christian Kersten, Eva Hofsli, Marianne Grønlie Guren, Halfdan Sorbye, Christin Johansen, Anne Negård, Kathrine Røe Redalen, Kjersti Flatmark. Immunogenic chemotherapy and immune checkpoint inhibition (ICI) in microsatellite-stable (MSS) metastatic colorectal cancer (mCRC): Biomarkers indicative of durable treatment response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 522.

POS1353 SERUM PROTEOMICS REVEALS INSULIN-LIKE GROWTH FACTOR BINDING PROTEINS-1 AS BIOMARKERS FOR IDIOPATHIC MULTICENTRIC CASTLEMAN’S DISEASE

Background:Castleman’s disease (CD) is a lymphoproliferative disorder1 and presents as two distinct clinical entities: the localized form, unicentric CD (UCD) and the multicentric form, multicentric CD (MCD)2. MCD without human herpesvirus-8 (HHV-8) infection is defined idiopathic MCD (iMCD)3 and most MCDs in Japan are iMCDs. TAFRO syndrome is a group of disease that present with T: thrombocytopenia, A: anasarca, F: fever, R: reticulin fibrosis/renal dysfunction, O: organomegaly4. iMCD-TAFRO is present in the TAFRO syndrome and it is thought that pathology of lymph node biopsy shows an MCD-like appearance, and some clinical features overlap with MCD5. That do not belong to any of the above categories is classified as iMCD-not otherwise specified (iMCD-NOS). No biomarkers stratifying iMCD-NOS and iMCD-TAFRO have been identified, and no biomarkers defining treatment response have been identified for iMCD.Objectives:We will identify biomarkers that discriminate iMCD-NOS and iMCD-TAFRO or predict the treatment responsiveness.Methods:We performed a comprehensive analysis of serum proteins using the L-Series Human Antibody Array L-507 on the 4 iMCD-NOS and 2 iMCD-TAFRO patients from which pre- and post-tocilizumab treatment samples were obtained. An analysis by L-507 identified insulin-like growth factor binding proteins-1 (IGFBP-1) as a protein with a high rate of reduction post treatment. Sera from 28 healthy controls, 8 patients with iMCD-NOS, and 6 patients with iMCD-TAFRO were used to validate IGFBP-1 by ELISA. The mean ages of healthy controls, iMCD-NOS, and iMCD-TAFRO used in the validation ELISA were 50, 56, and 47 years, respectively, with no significant differences among the groups. The ratio of male to female was almost 1:1.Results:The 4 patients who responded well to treatment with tocilizumab all had a high rate of IGFBP-1 reduction by L-507 serum protein arrays. In ELISA, serum IGFBP-1 was significantly higher (p=0.0016) before the introduction of treatment in iMCD patients than healthy controls. In addition, serum IGFBP-1 level of iMCD-TAFRO was significantly higher than iMCD-NOS (p=0.024). Furthermore, post-treatment serum IGFBP-1 was decreased in many cases.Conclusion:Serum IGFBP-1 may play a particularly important role in the pathogenesis of iMCD-TAFRO and may be useful in discriminating between iMCD-NOS and iMCD-TAFRO. In the future, we will accumulate more cases, compare it with other inflammatory diseases, and examine the difference in response to treatment.

Identification and Validation of Nutrient State-Dependent Serum Protein Mediators of Human CD4+ T Cell Responsiveness.

Intermittent fasting and fasting mimetic diets ameliorate inflammation. Similarly, serum extracted from fasted healthy and asthmatic subjects’ blunt inflammation in vitro, implicating serum components in this immunomodulation. To identify the proteins orchestrating these effects, SOMAScan technology was employed to evaluate serum protein levels in healthy subjects following an overnight, 24-h fast and 3 h after refeeding. Partial least square discriminant analysis identified several serum proteins as potential candidates to confer feeding status immunomodulation. The characterization of recombinant IGFBP1 (elevated following 24 h of fasting) and PYY (elevated following refeeding) in primary human CD4+ T cells found that they blunted and induced immune activation, respectively. Furthermore, integrated univariate serum protein analysis compared to RNA-seq analysis from peripheral blood mononuclear cells identified the induction of IL1RL1 and MFGE8 levels in refeeding compared to the 24-h fasting in the same study. Subsequent quantitation of these candidate proteins in lean versus obese individuals identified an inverse regulation of serum levels in the fasted subjects compared to the obese subjects. In parallel, IL1RL1 and MFGE8 supplementation promoted increased CD4+ T responsiveness to T cell receptor activation. Together, these data show that caloric load-linked conditions evoke serological protein changes, which in turn confer biological effects on circulating CD4+ T cell immune responsiveness.

Regulation of cell locomotion by nanosecond-laser-induced hydroxyapatite patterning

Hydroxyapatite, an essential mineral in human bones composed mainly of calcium and phosphorus, is widely used to coat bone graft and implant surfaces for enhanced biocompatibility and bone formation. For a strong implant–bone bond, the bone-forming cells must not only adhere to the implant surface but also move to the surface requiring bone formation. However, strong adhesion tends to inhibit cell migration on the surface of hydroxyapatite. Herein, a cell migration highway pattern that can promote cell migration was prepared using a nanosecond laser on hydroxyapatite coating. The developed surface promoted bone-forming cell movement compared with the unpatterned hydroxyapatite surface, and the cell adhesion and movement speed could be controlled by adjusting the pattern width. Live-cell microscopy, cell tracking, and serum protein analysis revealed the fundamental principle of this phenomenon. These findings are applicable to hydroxyapatite-coated biomaterials and can be implemented easily by laser patterning without complicated processes. The cell migration highway can promote and control cell movement while maintaining the existing advantages of hydroxyapatite coatings. Furthermore, it can be applied to the surface treatment of not only implant materials directly bonded to bone but also various implanted biomaterials implanted that require cell movement control.

Elevated expression of sepiapterin reductase, regulator of G protein signaling 1, hypothetical protein CXorf58 homolog, and zinc finger and BTB domain-containing protein 21 isoform X2 is associated with progression of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common cancers associated with high mortality rate. Understanding of events leading to HCC pathophysiology is essential for its better management. We earlier reported development of a novel rodent model by administrating chemical carcinogens, DEN, and 2-AAF for study of HCC at very early stage. 2D-Electrophoresis analysis of total serum proteins identified several differentially expressed proteins in animals undergoing tumorigenesis. MALDI-TOF-MS/MS analyses were performed to characterize the differentially expressed proteins. Further real-time PCR analyses were taken place to quantify the transcript expression for the identified proteins at HCC initiation and tumor stages. Considering protein-protein interactions among the experimentally identified proteins and their interacting neighbors, a protein network has been analyzed that provided further insight into molecular events taking place during HCC development. Histological changes confirmed HCC initiation and hepatotumorigenesis at 1 and 4 months post carcinogen treatment, respectively. Four differentially expressed proteins were identified which were further characterized as regulator of G protein signaling 1 (RGS1), sepiapterin reductase (SPR), similar to zinc finger and BTB domain-containing protein 21 isoform X2 (ZNF295), and a hypothetical protein CXorf58 homolog. Quantification of transcripts for these proteins revealed elevation in their expression both at initiation and tumorigenesis stages. The study deciphers the regulatory role of these proteins during HCC progression.

Exploring molecular pathology of chronic kidney disease in systemic sclerosis by analysis of urinary and serum proteins.

Objective.Renal involvement is common in systemic sclerosis (scleroderma; SSc) and includes chronic kidney disease (CKD). We have performed analysis of urinary proteins to gain insight into local molecular pathology of CKD in SSc and identify candidate markers for use in clinical trials.Methods.To evaluate urinary proteins that might specifically reflect SSc-related CKD, patients were recruited with confirmed SSc and stratified for the presence or absence of CKD. Controls included patients with CKD and no SSc, in addition to healthy volunteers. Candidate markers were measured in serum and urine by multiplex immunoassay testing for IL6, IL18, TNF-α, monocyte chemoattractant protein 1 (MCP1), monocyte chemoattractant protein 3 (MCP3), VEGF and the soluble adhesion molecules vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1). Results.One hundred and two subjects were examined, including patients with SSc with no evidence of CKD (n = 40), SSc with CKD (n = 39), non-SSc CKD (n = 11) and healthy volunteers (n = 12). Urinary levels of IL6, MCP1, TNF-α, MCP3, IL18 and ICAM-1 were elevated in SSc patients compared with healthy controls. The most significant differences were for MCP1 and ICAM-1 (both P < 0.0001), and these analytes also showed the most significant differences between groups overall (P = 0.003 for MCP1 and P < 0.0001 for ICAM-1). These markers showed a trend (MCP1, P = 0.0868) or a significant difference (ICAM-1, P = 0.0134) between SSc–CKD and SSc with normal renal function.Conclusion.Urinary levels of candidate molecular markers appear to reflect SSc–CKD more than serum markers. MCP1 and ICAM-1 are promising molecular markers for SSc–CKD and might be potential biomarkers of SSc renal involvement. This might be explored in future prospective analyses.

Rapid and Inexpensive Colorimetric Detection of Total Serum Protein Using Microzone Plates Wax-Printed on Polyester Films

This report describes a rapid and inexpensive colorimetric method based on biuret assay for total serum protein analysis using wax printed microzone plates and a desktop scanner. The colorimetric assay was performed in 96-microzone plates fabricated on polyester films through a wax printing protocol. The image scanning mode and the concentration of the biuret reagents were optimized to achieve the highest detectability levels. The best analytical response was obtained using the slide scanning mode as well as 4% CuSO4 and 150 mmol L-1 potassium sodium tartrate solutions. The proposed method offered linear relationship for the bovine serum albumin concentration range between 25 and 200 µmol L-1 with a limit of detection of 9.7 µmol L-1. The colorimetric approach revealed satisfactory precision (relative standard deviation values between 0.6 and 4.4%). The bioanalytical feasibility was demonstrated through the total protein analysis in serum samples collected from dogs previously diagnosed with kidney disease. The total serum protein concentrations varied from 960 ± 19 to 1661 ± 38 µmol L-1 and the achieved values revealed good accordance with those obtained through a reference method. Based on the reported results, wax printed microzone plates have demonstrated simplicity and potential to be adopted as alternative platforms for bioassays.

Rapid Sample Preparation Workflow for Serum Sample Analysis with Different Mass Spectrometry Acquisition Strategies.

Fast, robust, and high-throughput mass spectrometry-based serum proteomic pipelines have great potential to yield information for biomarker discovery and daily clinical practice. Here, we developed a simple and rapid sample preparation (RSP) workflow by reducing the classical pretreatment time from overnight to less than 1.5 h in an ordinary system. In HeLa cell lysates and serum samples, the number of proteins and tryptic peptides generated using the RSP was comparable to that generated using conventional methods. For fast scanning of the serum proteome, the RSP-supported pipeline could complete a test in less than 2 h with 30 min of LC-MS/MS analysis. Nearly 390 proteins spanning 8 magnitudes of abundance range were identified with high reproducibility, containing over 90 cancer-associated proteins and over 50 FDA-approved biomarkers. For fast assay development, eight candidate biomarker peptides for cardiovascular disease (CVD) were quantified by MRM with high accuracy (CV% <10). After a simple highly abundant protein removal, a deep serum proteome of over 1400 proteins was reached. By analyzing the depleted serum in DIA acquisition mode, over 700 proteins were quantified. The differentially expressed proteins could help us unambiguously distinguish the serum samples from healthy people and patients with pancreatic cancer (PC). Potential biomarkers for PC were also found. The new RSP method, which is rapid and simple, meets the demands of both deep mining and fast analysis of serum proteins. We believe that it will be widely used in serum protein studies and accelerate the transformation from biomarker discovery to clinical application.