Complement C3 Precursor Research Articles

Prototype Salivary Assay for Quantification of Two Biomarkers for In Vitro Diagnosis of Endometriosis.

Endometriosis, a very common disease in women, is characterized by endometrial structures outside the uterine cavity. The lack of a reliable noninvasive diagnostic test and the often nonspecific symptoms of this pathology are responsible for the delay in definitive diagnosis of this disease. Recently, through a proteomics approach, our research group has identified two potential diagnostic markers for endometriosis in serum (Zn-alpha2-glycoprotein and complement C3 precursor). In this article, we describe the experimental conditions of a simple ELISA for rapid quantification of these two biomarkers in the saliva of patients with endometriosis. Finally, preliminary experiments on a small cohort of patients and controls have confirmed the potential diagnostic value of this assay.

Identification of potential serum biomarkers in pigs at early stage after Lipopolysaccharide injection

The identification of useful biological indicators to monitor the body response before the presentation of clinical diseases has practical value in livestock production. To identify potential biomarkers in pigs at the early stage during inflammation, 12 pigs were intramuscularly injected with 2mL of Lipopolysaccharide (LPS, 15μg/kg BW) or saline. Serum protein expression profiles were detected with two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) techniques. Serum biochemical indicators and acute phase protein (APP) concentrations were analyzed with an automatic biochemical analyzer and ELISA, respectively. Of the serum biochemical indicators, creatinine concentration significantly increased 6h post infection, whereas albumin showed a decreased tendency. The 2-DE and MALDI-TOF mass spectrometry technique detected 17 protein spots representing 10 proteins: α-1-antichymotrypsin, albumin, bovine lactoferrin, serotransferrin, serpin A3-6, immunoglobulin light chain (κ chain, mu chain), complement C3 precursor, zinc-α-2-glycoprotein (ZAG), and ceruloplasmin. Two proteins were selected to confirm the mass spectrometry results, and resulting differences accorded with the proteomics results. Of the four typical acute phase protein (APPs) measured, the C-reactive protein (CRP) and haptoglobin (HP) concentrations increased significantly, while no differences were observed in the pig-major acute phase protein (Pig-MAP) and transthyretin (TTR) contents. The results showed that serum creatinine, CRP, HP, and ten other proteins content changed significantly after LPS injection. Of these proteins, ZAG was first reported in pigs during inflammation. These proteins show great promise as biomarkers to monitor the health status and welfare of pigs during the early stage of LPS-induced inflammation.

Prototype of Multiplex Bead Assay for Quantification of Three Serum Biomarkers for In Vitro Diagnosis of Endometriosis.

Endometriosis is a very common disease, affecting 10% of women in the reproductive age. To date, a significant delay between onset of the symptoms and definitive diagnosis is caused by the lack of a reliable non-invasive diagnostic test. Recently, the potential value as diagnostic markers for endometriosis of three proteins (Zn-alpha2-glycoprotein, serum albumin, and complement C3 precursor), has been showed. In this article, we have defined the experimental conditions for the development of a multiplex bead array assay for rapid and simultaneous quantification of these three biomarkers in the serum of patients with endometriosis. Finally, pivotal experiments on a small cohort of patients have confirmed the diagnostic value of this assay. J. Cell. Physiol. 231: 2622-2627, 2016. © 2016 Wiley Periodicals, Inc.

Supporting evidences for potential biomarkers of endometriosis detected in peripheral blood.

Incidence of endometriosis is very high in women in the reproductive age (around 10%). To date, a reliable non-invasive diagnostic test for early diagnosis of endometriosis is not available. In this article we describe the potential value as diagnostic markers for endometriosis of two proteins (serum albumin and complement C3 precursor), previously identified as differentially expressed in women with endometriosis respect to healthy control by 2D gel analysis. A detailed description of the results obtained with this proteomic approach can be found in Signorile and Baldi [1]. ELISAs were performed on a large cohort of endometriosis (n=100) and healthy patients (n=10) to establish the differential expression of the identified proteins. ROC analyses confirmed the statistical significance of the differential expression of these proteins: serum albumin (p=0.028) ad complement C3 precursor (p=0.082). Evaluation of these two proteins, together with the already described Zn-alpha2-glycoprotein [1], could help in the early identification of endometriosis patients

Remote ischemic preconditioning (RIPC) modifies the plasma proteome in children undergoing repair of tetralogy of fallot: a randomized controlled trial.

BackgroundRemote ischemic preconditioning (RIPC) has been applied in paediatric cardiac surgery. We have demonstrated that RIPC induces a proteomic response in plasma of healthy volunteers. We tested the hypothesis that RIPC modifies the proteomic response in children undergoing Tetralogy of Fallot (TOF) repair.Methods and ResultsChildren (n=40) were randomized to RIPC and control groups. Blood was sampled at baseline, after cardiopulmonary bypass (CPB) and 6, 12 and 24h post-CPB. Plasma was analysed by liquid chromatography mass spectrometry (LC-MS) in an untargeted approach. Peptides demonstrating differential expression (p<0.01) were subjected to tandem LC-MS/MS and protein identification. Corresponding proteins were identified using the NCBI protein database. There was no difference in age (7.3±3.5vs6.8±3.6 months)(p=0.89), weight (7.7±1.8vs7.5±1.9 kg)(p=0.71), CPB time (104±7vs94±7 min)(p=0.98) or aortic cross-clamp time (83±22vs75±20 min)(p=0.36). No peptides were differentially expressed at baseline or immediately after CPB. There were 48 peptides with higher expression in the RIPC group 6h post-CPB. This was no longer evident at 12 or 24h, with one peptide down-regulated in the RIPC group. The proteins identified were: inter-alpha globulin inhibitor (42.0±11.8 vs 820.8±181.1, p=0.006), fibrinogen preproprotein (59.3±11.2 vs 1192.6±278.3, p=0.007), complement-C3 precursor (391.2±160.9 vs 5385.1±689.4, p=0.0005), complement C4B (151.5±17.8 vs 4587.8±799.2, p=0.003), apolipoprotein B100 (53.4±8.3 vs 1364.5±278.2, p=0.005) and urinary proteinase inhibitor (358.6±74.9 vs 5758.1±1343.1, p=0.009). These proteins are involved in metabolism, haemostasis, immunity and inflammation.ConclusionsWe provided the first comprehensive analysis of RIPC-induced proteomic changes in children undergoing surgery. The proteomic changes peak 6h post-CPB and return to baseline within 24h of surgery.Trial RegistrationACTR.org.au ACTRN12610000496011

Seminal plasma proteins of adult boars and correlations with sperm parameters

The present study was conducted to identify the major seminal plasma protein profile of boars and its associations with semen criteria. Semen samples were collected from 12 adult boars and subjected to evaluation of sperm parameters (motility, morphology, vitality, and percent of cells with intact acrosome). Seminal plasma was obtained by centrifugation, analyzed by two-dimensional SDS-PAGE, and proteins identified by mass spectrometry (electrospray ionization quadrupole time-of-flight). We tested regression models using spot intensities related to the same proteins as independent variables and semen parameters as dependent variables (P ≤ 0.05). One hundred twelve spots were identified in the boar seminal plasma gels, equivalent to 39 different proteins. Spermadhesin porcine seminal protein (PSP)-I and PSP-II, as well as spermadhesins AQN-1, AQN-3 and AWN-1 represented 45.2 ± 8% of the total intensity of all spots. Other proteins expressed in the boar seminal plasma included albumin, complement proteins (complement factor H precursor, complement C3 precursor and adipsin/complement factor D), immunoglobulins (IgG heavy chain precursor, IgG delta heavy chain membrane bound form, IgG gamma-chain, Ig lambda chain V-C region PLC3, and CH4 and secreted domains of swine IgM), IgG-binding proteins, epididymal-specific lipocalin 5, epididymal secretory protein E1 precursor, epididymal secretory glutathione peroxidase precursor, transferrin, lactotransferrin and fibronectin type 1 (FN1). On the basis of the regression analysis, the percentage of sperm with midpiece defects was related to the amount of CH4 and secreted domains of swine IgM and FN1 (r² = 0.58, P = 0.006), IgG-binding protein (r² = 0.41, P = 0.024), complement factor H precursor (r² = 0.61, P = 0.014) and lactadherin (r² = 0.45, P = 0.033). The percentage of sperm with tail defects was also related to CH4 and secreted domains of swine IgM and FN1 (r² = 0.40, P = 0.034), IgG-binding protein (r² = 0.35, P = 0.043) and lactadherin (r² = 0.74, P = 0.001). Sperm motility, in turn, had association with the intensities of spots identified as lactadherin (r² = 0.48, P = 0.027). In conclusion, we presently describe the major proteome of boar seminal plasma and significant associations between specific seminal plasma proteins and semen parameters. Such relationships will serve as the basis for determination of molecular markers of sperm function in the swine species.

PROTEOMIC ANALYSIS OF POST-HEMORRHAGIC SHOCK MESENTERIC LYMPH

Recent studies have documented the association of mesenteric lymphatic route with adult respiratory distress syndrome and multiple organ failure after hemorrhagic shock. However, the mediators and mechanisms of the toxic effects of mesenteric lymph remain unclear. This study aimed to identify mediators or biomarkers in the mesenteric lymph through comparative proteomic analysis. Fourteen mature male Sprague-Dawley rats were randomly divided and subjected to trauma (laparotomy) plus hemorrhagic shock or trauma plus sham shock. Mesenteric lymph samples were collected before shock and at 3 h after resuscitation from hemorrhagic shock (or sham shock). To investigate changes in proteome profiles between preshock and 3-h postshock (or 3-h post-sham shock) mesenteric lymph samples, two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry were performed. We found a more than 2-fold change in abundance of 31 protein spots in the lymph samples. Mass spectrometry analyses identified 12 distinct proteins. Four proteins were consistently upregulated in the 3-h postshock lymph samples, including serum albumin precursor, two isoforms of cytoplasmic actin, complement C3 precursor, and major urinary protein precursor. Two proteins, including haptoglobin and one unidentified protein, were consistently downregulated. The deregulation of these proteins was confirmed by Western blots. Most of these altered proteins are functionally implicated in tissue inflammation. The findings of this study provide a starting point for investigating the functions of these proteins in hemorrhagic shock-induced lung injury and hold great promise for the development of potential therapeutic interventions.

Impact of the storage temperature on human plasma proteomic analysis: Implications for the use of human plasma collections in research

To analyze the impact of storage temperature on the 2D-DIGE profile of human plasma. 2D-DIGE and MS were used to identify the differences in the proteomic profiles of aliquots of eight human plasma samples stored at either - 30 or - 80°C for 18 months. After the depletion of albumin and IgG, 2D-DIGE identified four spots significantly and reproducibly increased, and five spots that were decreased, in samples stored at - 30°C compared with those stored at - 80°C. These nine spots were manually excised, digested ingel and analyzed by MALDI-TOF/TOF. MASCOT database search using the PMF spectra allowed the identification of the proteins present in eight of the nine spots. All the spots corresponded to the complement C3 precursor protein. Our present results indicate that plasma collections stored at -30°C, and not only those stored at - 80°C, may be used for 2D-DIGE analyses of albumin-and IgG-depleted human plasma without loosing the essential information about highly abundant proteins. This finding expands the applicability of 2D-DIGE to the study of human disease by permitting the analysis of human plasma samples stored at temperatures between - 30 and - 80°C.

Characteristics and Molecular Mechanism of Adhesion Proteins on Reused Hemodialysis Membranes

In order to study the mechanism of protein adhesion on the Fresenius F6 polysulfone membrane dialyzer, two-dimensional gel electrophoresis, LC-ESI-MS/MS and bioinformatics methods were used to analyze the protein which adhered to the dialyzer membrane. Six of the adhered proteins account for more than 50% of the total 179 proteins, i.e. ficolin precursor, complement C3 precursor, 3 variants of MASP1 and albumin. The results also showed that easily adhered proteins have a greater percentage of acidic amino acids (p < 0.01). The isoelectric point of the 20 proteins with the most deposits is 6.2 ± 1.08, which is obviously lower than of those with the least deposits (7.56 ± 1.36, p < 0.01). The dipole moment of a polysulfone membrane molecule has a tendency to absorb molecules with a negative charge. These results are of significance in understanding and improving membrane protein interactions.

Proteomic Analysis of Differentially Expressed Proteins in Bovine Milk During Experimentally Induced Escherichia coli Mastitis

The objectives of the current study were to profile changes in protein composition using 2-dimensional gel electrophoresis on whey samples from a group of 8 cows before and 18h after infection with Escherichia coli and to identify differentially expressed milk proteins by peptide sequencing using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry post source decay. Only proteins present in whey fractions of all 8 cows were sequenced to avoid reporting a protein response unique to only a subset of infected cows. Despite the overwhelming presence of casein and β-lactoglobulin, the low abundance proteins transthyretin, lactadherin, β-2-microglobulin precursor, α-1-acid glycoprotein, and complement C3 precursor could be identified in whey samples from healthy cows. Whey samples at 18h postinfection were characterized by an abundance of serum albumin, in spots of varying mass and isoelectric point, as well as increased transthyretin and complement C3 precursor levels. Also detected at 18h postinoculation were the antimicrobial peptides cathelicidin, indolicidin, and bactenecin 5 and 7, and the proteins β-fibrinogen, α-2-HS-glycoprotein, S100-A12, and α-1-antiproteinase. Most notable was the detection of the acute phase protein α-1-acid glycoprotein in mastitic whey samples, a result not previously reported. In contrast to methods used in previous proteomic analyses of bovine milk, the methods used in the current study enabled the rapid identification of milk proteins with minimal sample preparation. Use of a larger sample size than previous analyses also allowed for more robust protein identification. Results indicate that examination of the protein profile of whey samples from cows after inoculation with E. coli could provide a rapid survey of milk protein modulation during coliform mastitis and aid in the identification of biomarkers of this disease.

Identification of putative serum glycoprotein biomarkers for human lung adenocarcinoma by multilectin affinity chromatography and LC‐MS/MS

Glycoproteins in human serum play fundamental roles in many biological processes, and also have clinical value as biomarkers for disease progression and treatment. In this study, we isolated glycoproteins from the sera of three healthy individuals and three lung adenocarcinoma patients using multilectin affinity chromatography. The recovered glycoproteins were subjected to treatment with peptide-N-glycosidase F (PNGase F) and in-gel digestion by trypsin. Tryptic peptides were analyzed by nano-LC coupled to ESI-MS/MS and the MS/MS spectra were processed by Bioworks 3.2 and an in-house bioinformatics tool, ProtAn. Approximately 90% of the proteins identified contained more than one potential glycosylation site. Comparison of the serum glycoproteome of healthy and adenocarcinoma individuals revealed 38 cancer-selective proteins. Among them, 60% have previously been reported as low abundance proteins in human sera. We identified several cancer-selective proteins that have been previously characterized as potential indicators of lung cancer in serum or plasma, including haptoglobin (HP), inter-alpha-trypsin inhibitor heavy chain 4 (ITI-H4), complement C3 precursor, and leucine-rich alpha-2-glycoprotein. In addition, plasma kallikrein (KLKB1) and inter-alpha-trypsin inhibitor heavy chain 3 (ITI-H3) were identified as being potentially elevated in the lung cancer group, and were validated by Western blot analysis. Furthermore, approximately 18 kDa plasma kallirein protein fragment was detected at high levels in 25 out of 28 adenocarcinoma patients, while one of the eight normal individuals showed moderate positive. The results suggest that KLKB1 represents a potential candidate serum biomarker of lung cancer.

Direct chemiluminescent imaging detection of human serum proteins in two‐dimensional polyacrylamide gel electrophoresis

A novel chemiluminescence (CL)-based imaging method capable of directly detecting proteins in polyacrylamide gels after electrophoresis is proposed. Human serum proteins are presently detected by a direct CL imaging method after native 2-D PAGE. As a consequence, some proteins, including haptoglobin (Hp), Hp precursor, hemopexin (Hpx) precursor, Ig alpha-1 chain C region, and Complement C3 precursor can be detected and identified by MS and MS/MS techniques. These proteins are all acute phase proteins, which have been defined as biomarkers for certain diseases. Moreover, serum proteins from healthy people and cirrhotic patients were analyzed. A decrease in Hp spots for cirrhotic patients could be confirmed. The CL imaging conditions were optimized, including the concentrations of H(2)O(2) and luminol. The process of CL detection of proteins is simple, and there is no need for specialized equipment. In comparison with the traditional CBB-R250 staining method, the detection sensitivity was improved and the detection period decreased about 70 times. Hence, this technique possesses potentials as a rapid, convenient, and inexpensive analytical technique for protein detection and for the diagnosis of diseases.

A Pilot Application of SELDI Mass Spectrometry To Study Proteomic Patterns in Plasma from Patients with T-Cell Large Granular Lymphocyte Leukemia.

T-cell large granular lymphocyte (T-LGL) leukemia is a rare chronic lymphoproliferative disorder of cytotoxic T cells of unknown etiology. Chronic antigenic stimulation of T-lymphocytes in autoimmune diseases or viral infection has been postulated to play a role in the pathogenesis of this condition. Surface-enhanced laser desorption/ionization (SELDI) mass spectrometry of plasma and serum specimens was applied as a useful screening method to determine pathophysiologic protein/peptide markers, which may be useful in the diagnosis of occult malignancies. In contrast to traditional diagnostic principles, the SELDI method enables screening of protein patterns without knowing the identity of targets. Patterns of up- and down-modulation of peptides and proteins (resulting from the presence of abnormal products of diseased cells, pathogens or immunogenetic factors) may signal various clinical features and diagnostic clues. We used SELDI mass spectrometry to study proteomic patterns in the plasma of patients with T-LGL leukemia. After initial screening and optimization we selected a hydrophobic H4 ProteinChip surface for further systematic screening. Deviation of mass values and peak intensities were <0.5% and <20%, respectively.Reproducibility was monitored by random duplication of samples with an average of 70 peaks per spectrum (mass/charge ratio from 1500–50,000 Da). Our study included plasma samples from 28 LGL patients, 9 patients with idiopathic neutropenia (without the presence of LGL clone) and 36 healthy blood donors. The diagnosis of T-LGL leukemia included the presence of monoclonal TCR rearrangement and flow cytometric detection of abnormal CD8+CD3+CD57+ cell population. Spectra were analyzed using Biomarker Patterns Software, which allows for internal cross validation for the prediction of the accuracy in a blinded test set. To separate LGL patients from patients with chronic neutropenia, intensity cut-off for 2050 Da peak was found in 22/28 LGL patients, resulting in 100% specificity and 67% sensitivity. Based on the intensities of 3 mass peaks (2274, 5870, 2660 Da), the sensitivity and specificity of LGL distinction from healthy controls was 96% and 61%, respectively. By direct digestion with carboxypeptidase Y on the ProteinChip surface, the C-terminal sequence of the 2050 Da mass peak has been identified. From the sequencing analysis and the search in the ExPASy protein database, we concluded that the 2050 Da mass peak corresponded to the fragment of complement C3 precursor. Additional 2 kDa mass peaks may represent various other C3 fragments. Interestingly, the intensity of the mass peak corresponding to the C3 fragments decreased with the application of immunosuppressive therapy when plasma samples from 3 LGL patients were serially analyzed. The pathophysiologic significance of the identified peak remains unclear, but increased expression of the C3 receptor on LGL cells has been reported. Our data in LGL leukemia indicate that SELDI mass spectrometry can be used to identify immune biomarkers for malignant lymphoproliferations.

Peripheral detection of S100β during cardiothoracic surgery: what are we really measuring?

BackgroundS100β has been used in cardiac surgery to identify patients with postoperative neurologic complications. However, extracranial proteins may falsely elevate measurements of serum S100β;. Objectives of this study were (1) to quantify S100β levels in serum and pericardial cavity during coronary artery bypass grafting (CABG), and (2) to identify proteins recognized by standard immunodetection as S100β. MethodsSystemic and pericardial cavity blood from 5 patients undergoing CABG were sampled before, during, and after cardiopulmonary bypass (CPB). A commercially available enzyme-linked immunosorbent assay (ELISA) kit was used to quantify S100β. Two-dimensional gel electrophoresis, Western blot, and mass spectroscopy were also performed to identify S100â and other proteins. ResultsMean S100β levels measured by ELISA, systemic and pericardial cavity blood were (in ng · mL−1) 1.0 ± 0.46 and 111 ± 71 before CPB, 0.6 ± 0.11 and 113 ± 54 during CPB, and 1.7 ± 0.64 and 101 ± 42 after CPB, respectively. However, gel electrophoresis and Western blot analysis revealed proteins other than S100β to be present in the pericardial cavity giving a falsely elevated serum S100â levels measured by immunoassay. Mass spectroscopy of identified potential candidates revealed contaminants including haptoglobin I precursor, apolipoprotein A-1 precursor, complement factor B precursor, and complement C3 precursor. ConclusionsS100β immunoassays are not specific for S100â and give a falsely elevated reading due to contaminants from the surgical field that cross react with the assay's antibody. This does not appear to be an issue in nonsurgical patients. Caution must be exerted when evaluating immunodetection results for low-abundance proteins under conditions where contamination of the sample is likely.