Ig Kappa Chain Research Articles

Proteome Analysis of Bevacizumab Intervention in Experimental Central Retinal Vein Occlusion.

Bevacizumab is a frequently used inhibitor of vascular endothelial growth factor (VEGF) in the management of macular edema in central retinal vein occlusion (CRVO). Studying retinal protein changes in bevacizumab intervention may provide insights into mechanisms of action. In nine Danish Landrace pigs, experimental CRVO was induced in both eyes with argon laser. The right eyes received an intravitreal injection of 0.05 mL bevacizumab (n = 9), while the left control eyes received 0.05 mL saline water (NaCl). Retinal samples were collected 15 days after induced CRVO. Label-free quantification nano-liquid chromatography-tandem mass spectrometry identified 59 proteins that were regulated following bevacizumab treatment. Following bevacizumab intervention, altered levels of bevacizumab components, including the Ig gamma-1 chain C region and the Ig kappa chain C region, were observed. Changes in other significantly regulated proteins ranged between 0.58-1.73, including for the NADH-ubiquinone oxidoreductase chain (fold change = 1.73), protein-transport protein Sec24B (fold change = 1.71), glycerol kinase (fold change = 1.61), guanine-nucleotide-binding protein G(T) subunit-gamma-T1 (fold change = 0.67), and prefoldin subunit 6 (fold change = 0.58). A high retinal concentration of bevacizumab was achieved within 15 days. Changes in the additional proteins were limited, suggesting a narrow mechanism of action.

Abstract P2-19-01: Characterization of fully human internalizing anti-HER2 monoclonal antibodies that compete with Trastuzumab for binding to HER2

Abstract The human epidermal growth factor receptor (HER) family of receptors plays a significant role in the pathogenesis of several human cancers. Among them, HER2 is overexpressed in several cancers. In particular, overexpression with or without gene amplification occurs in 15-30% of breast cancers and this has both prognostic and predictive implications. HER2 overexpression and amplification is associated with shorter disease-free and overall survivals and with resistance to certain hormonal agents as well as increased risk of metastasis to the brain. Two types of treatment targeting HER2 biological activity have been developed: anti-Her2 monoclonal antibodies and small molecule tyrosine kinase inhibitors such as lapatinib and neratinib targeting HER2 and EGFR and Afatinib targeting all HER family members. Trastuzumab is a humanized anti-HER2 monoclonal antibody that binds to domain IV of HER2 extracellular segment and blocks its signaling. It is the first therapeutic antibody targeting Her2 overexpression approved by the FDA. It is administered in combination with standard of care chemotherapy. More recently, since it was shown that Trastuzumab was an internalizing antibody, two antibody drug conjugates using trastuzumab have been approved and used in the standard of care: Ado-Trastuzumab-Emtansine (Kadcyla) consisting of trastuzumab conjugated to the drug mertansine DM1 and fam-trastuzumab-deruxtecan-nhki (Enhertu). Enhertu is another antibody drug conjugate using Trastuzumab to deliver a topoisomerase inhibitor deruxtecan. Our laboratory has been focused in developing fully human internalizing anti-Her2 antibodies that compete or not with trastuzumab for binding to Her2. These antibodies have been developed by immunizing fully human mice with recombinant Her2 protein. Human Ab producing Tc mice (TC-mAb mice) stably maintain a mouse-derived engineered chromosome containing the entire human Ig heavy and kappa chain loci in a mouse Ig knockout background. After production of hybridoma secreting fully human immunoglobulins, the screening process included inhibition of binding of trastuzumab to Her2 by enzyme linked immunoassay and by Octet epitope binning as well as internalization assay. Several internalizing antibodies with Kd ranging from 10-9 M to 10-12 M were selected. They were stratified by their ability to compete or not with trastuzumab. They were further characterized for their ability to deliver a cytotoxic payload in Her2 overexpressing cells as well as for their efficacy to inhibit proliferation, signaling and migration of Her2 overexpressing breast cancer cells. Data related to these antibodies will be presented here. In conclusion, the use of fully human TC mice in our laboratory represents an attractive approach to develop fully human monoclonal antibodies to high value cancer targets that can by-pass the need for humanization and affinity maturation of antibodies. Citation Format: Ginette Serrero, Jianping Dong, Binbin Yue, Udaya Yerramalla, Jun Hayashi. Characterization of fully human internalizing anti-HER2 monoclonal antibodies that compete with Trastuzumab for binding to HER2 [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-19-01.

Efficient human-like antibody repertoire and hybridoma production in trans-chromosomic mice carrying megabase-sized human immunoglobulin loci

Trans-chromosomic (Tc) mice carrying mini-chromosomes with megabase-sized human immunoglobulin (Ig) loci have contributed to the development of fully human therapeutic monoclonal antibodies, but mitotic instability of human mini-chromosomes in mice may limit the efficiency of hybridoma production. Here, we establish human antibody-producing Tc mice (TC-mAb mice) that stably maintain a mouse-derived, engineered chromosome containing the entire human Ig heavy and kappa chain loci in a mouse Ig-knockout background. Comprehensive, high-throughput DNA sequencing shows that the human Ig repertoire, including variable gene usage, is well recapitulated in TC-mAb mice. Despite slightly altered B cell development and a delayed immune response, TC-mAb mice have more subsets of antigen-specific plasmablast and plasma cells than wild-type mice, leading to efficient hybridoma production. Our results thus suggest that TC-mAb mice offer a valuable platform for obtaining fully human therapeutic antibodies, and a useful model for elucidating the regulation of human Ig repertoire formation.

Cord Blood Proteomic Biomarkers for Predicting Adverse Neurodevelopmental Outcomes in Monoamniotic Twins.

Monoamniotic twins have a high risk of mortality and perinatal morbidity due to cord entanglement and vascular anastomosis. Despite efforts to reduce the mortality rate through intensive fetal surveillance and timed delivery, poor long-term neurodevelopmental outcomes remain an unsolved problem. This study aimed to identify novel biomarkers predicting abnormal neurodevelopmental outcomes in monoamniotic twins with cord blood samples taken at the time of delivery. Abnormal neurodevelopmental outcomes were defined as (1) a severe brain lesion on neonatal brain ultrasound, (2) developmental delay, (3) cerebral palsy, and/or (4) blindness or deafness. Cord blood was analyzed with mass spectrometry-based proteomics according to the neurodevelopmental outcomes. Statistical analysis was performed to determine the differentially expressed proteins between neonates with normal and abnormal neurodevelopmental outcomes. Several candidate proteins were further validated with enzyme-linked immunosorbent assays. A total of 20 neonates (10 pairs) of monoamniotic twins were included in the proteomic analysis, of which 25% had abnormal neurodevelopmental outcomes. Eighteen proteins were differentially expressed in neonates with abnormal neurodevelopmental outcomes. The upregulated proteins in the neonates with adverse neurodevelopmental outcome were immunoglobulin (Ig)-gamma-4 chain C region, apolipoprotein E, and alpha-fetoprotein. In contrast, Ig-lambda chain V region 4A, Ig-heavy variable 3, Ig-kappa chain C region, Ig-mu chain C region, C1q, ceruloplasmin, and Ig-lambda chain V-I region were decreased. In the validation experiment, the cord blood concentration of ceruloplasmin was significantly lower in neonates with adverse neurodevelopmental outcomes than in those without. Therefore, ceruloplasmin could be a useful predictive biomarker of adverse neurodevelopmental outcomes in monoamniotic twins.

Predictive protein markers for depression severity in mood disorders: A preliminary trans-diagnostic approach study

Depression is a common symptom of many mental disorders, especially major depressive disorder (MDD) and bipolar disorder (BD). Previous studies have reported that these diseases share common pathophysiological pathways; therefore, this study elucidated whether the plasma levels of protein markers related to common depressive symptoms differed between patients with BD and those with MDD. Plasma samples of 71 patients with mood disorders and clinical manifestations were analyzed in this study. After depleting the abundant proteins, liquid chromatography–tandem mass spectrometry and label-free quantification were performed. Five proteins, viz., cholesteryl ester transfer protein (CETP), apolipoprotein D (APOD), mannan-binding lectin serine protease 2 (MASP2), Ig lambda chain V-II region BO (IGLV2-8) and Ig kappa chain V-III region NG9 (IGKV3-20) were negatively associated with the total scores of the Hamilton depression rating scale (HAM-D), after adjusting for the covariates. CETP and APOD also showed significant negative correlations with the anhedonia/retardation and guilt/agitation scores of the HAM-D. Four proteins, namely, Ig kappa chain V-II region TEW (IGKC; IGKV2D-28), Ig lambda variable 5–45 (IGLV5-45), complement factor H (CFH) and attractin (ATRN), showed significant associations with anhedonia/retardation after adjusting for covariates. Proteins that significantly correlated with the symptoms could predict the remission state of depression (area under the curve [AUC], 0.83) and anhedonia/retardation (AUC, 0.80). Bioinformatics analysis revealed that complement activation, immune response, and lipid metabolism were significantly enriched pathways. Although our study design was cross-sectional and no controls were included, protein markers identified in this preliminary study will be further investigated in our subsequent longitudinal study.

Isotypes of autoantibodies against novel differential 4-hydroxy-2-nonenal-modified peptide adducts in serum is associated with rheumatoid arthritis in Taiwanese women

BackgroundRheumatoid arthritis (RA) is an autoimmune disorder with systemic inflammation and may be induced by oxidative stress that affects an inflamed joint. Our objectives were to examine isotypes of autoantibodies against 4-hydroxy-2-nonenal (HNE) modifications in RA and associate them with increased levels of autoantibodies in RA patients.MethodsSerum samples from 155 female patients [60 with RA, 35 with osteoarthritis (OA), and 60 healthy controls (HCs)] were obtained. Four novel differential HNE-modified peptide adducts, complement factor H (CFAH)1211–1230, haptoglobin (HPT)78–108, immunoglobulin (Ig) kappa chain C region (IGKC)2–19, and prothrombin (THRB)328–345, were re-analyzed using tandem mass spectrometric (MS/MS) spectra (ProteomeXchange: PXD004546) from RA patients vs. HCs. Further, we determined serum protein levels of CFAH, HPT, IGKC and THRB, HNE-protein adducts, and autoantibodies against unmodified and HNE-modified peptides. Significant correlations and odds ratios (ORs) were calculated.ResultsLevels of HPT in RA patients were greatly higher than the levels in HCs. Levels of HNE-protein adducts and autoantibodies in RA patients were significantly greater than those of HCs. IgM anti-HPT78−108 HNE, IgM anti-IGKC2−19, and IgM anti-IGKC2−19 HNE may be considered as diagnostic biomarkers for RA. Importantly, elevated levels of IgM anti-HPT78−108 HNE, IgM anti-IGKC2−19, and IgG anti-THRB328−345 were positively correlated with the disease activity score in 28 joints for C-reactive protein (DAS28-CRP). Further, the ORs of RA development through IgM anti-HPT78−108 HNE (OR 5.235, p < 0.001), IgM anti-IGKC2−19 (OR 12.655, p < 0.001), and IgG anti-THRB328−345 (OR 5.761, p < 0.001) showed an increased risk. Lastly, we incorporated three machine learning models to differentiate RA from HC and OA, and performed feature selection to determine discriminative features. Experimental results showed that our proposed method achieved an area under the receiver operating characteristic curve of 0.92, which demonstrated that our selected autoantibodies combined with machine learning can efficiently detect RA.ConclusionsThis study discovered that some IgG- and IgM-NAAs and anti-HNE M-NAAs may be correlated with inflammation and disease activity in RA. Moreover, our findings suggested that IgM anti-HPT78−108 HNE, IgM anti-IGKC2−19, and IgG anti-THRB328−345 may play heavy roles in RA development.

Proteomic serum profile in menstrual-related and post menopause migraine

The aim of this pilot study was to analyze the serum proteomic profile of women suffering from menstrual-related migraine (MM group, n = 15) and migraine in post-menopause (PM group, n = 15) in comparison with non-headache control females (C group, n = 15). Serum samples were subjected to two-dimensional gel electrophoresis (2-DE) followed by mass spectrometry (MS) analysis for protein identification. Based on 2D-gel maps and PDQuest 2-D software, 13 differentially expressed spots, corresponding to 12 unique proteins identified by Liquid Chromatography-Electrospray Ionization-Quadrupole-Time of Flight/tandem mass spectrometry (LC-ESI-QToF-MS/MS), were detected in the MM and PM groups vs C group. Five inflammatory and regulatory of vascular integrity proteins (prothrombin, serum amyloid P-component, Ig kappa chain C region, apolipoprotein A-I, serum amyloid A-4 protein) were found deregulated in both MM and PM groups compared to C group; MM group showed the upregulation of other inflammatory protein fragments (inter-alpha-trypsin inhibitor heavy chain H4 and complement C4-A) compared to C group; PM group, in comparison with C group, displayed a noteworthy upregulation of transthyretin and other deregulated proteins (tetranectin, alpha-1-antitrypsin, haptoglobin, apolipoprotein A-IV) playing a role in anti-inflammatory and reparative processes. In conclusion, proteomic analysis was able to reveal differences in protein expression between migraine sufferers and non-headache women; as in other neurological diseases characterized by neuroinflammation, the serum proteome of migraine women presents an abundance of proteins indicative of cellular damage, oxidative stress and inflammation. This relevant inflammatory status, if confirmed in larger series, could represent a target for menstrual-related migraine treatment.

Identification of Differentially Expressed Proteins from Smokeless Tobacco Addicted Patients Suffering from Oral Squamous Cell Carcinoma.

Oral squamous cell carcinoma (OSCC) is the eight most common malignancy worldwide with an incidence rate of 40% in south-east Asia. Lack of effective diagnostic tools at early stage and disease recurrence despite extensive treatments are main reasons for high mortality and low survival rates. The aim of current study was to identify differentially expressed proteins to explore potential candidate biomarkers having diagnostic significance. We performed comparative proteomic analysis of paired protein samples (cancerous buccal mucosa and adjacent normal tissue) from OSCC patients using a combination of two dimensional gel electrophoresis and Mass spectrometric analysis. On the basis of spot intensity, seventeen proteins were found to be consistently differentially expressed among most of the samples which were identified through mass spectrometry. For validation of identified proteins, expression level of stratifin was determined using immuno-histochemistry and Western blot analysis. All identified proteins were analyzed by STRING to explore their interaction. Among uniquely identified proteins in this study, at least two candidate markers (Ig Kappa chain C region and Isoform 2 of fructose bisphosphate aldolase A) were found to be novel with respect to OSCC which can be explored further. Results presented in current study are likely to contribute in understanding the involvement of these molecules in carcinogenesis apart from their plausible role as diagnostic/prognostic markers.

Effect of iFISH defined 1q21 amplification/gain in multiple myeloma patients treated on total therapy protocols.

8023 Background: The amplification of the proximal 1q21 region has been reported by interphase FISH (iFISH) in about 40% of newly diagnosed patients, and in 70% of MM patients at relapse. We hereby report the prognostic value of 1q21 amplification/gain by iFISH at enrollment in subjects treated on total therapy (TT) 4, 5 and 6 protocols. Methods: TT4 protocol enrolled newly diagnosed patients with LR disease as defined GEP 70 model ≤ 0.66. TT5 was designed for newly diagnosed patients with HR MM.TT6 enrolled previously treated patients with both HR and LR disease. iFISH was performed on bone marrow samples obtained at the time of study enrollment. FISH probes were generated from specific BAC DNA clone for CKS1B gene locus (1q21) gene locus. MM cells were identified post hybridization using isotype specific antibody conjugated with 7-amino-4-methylcoumarin-3-acetic acid (AMCA) to stain Ig-Kappa or Ig-Lambda light chain in cytoplasm (cIg) of MM cells. The iFISH signals in 100 of MM cells were recorded. A 20% cutoff point was used for detection of significant abnormalities i.e. gain of 1q21 (≥ 3 copies). Results: 607 patients were included in this analysis. With a median age of 61 years, 39% had high risk and 88% low risk disease. 1q21 abnormalities were present in 55% of patients at enrollment. 591 patients (97%) received at least one HDT ASCT. The analysis included 382 TT4 patients with a median PFS of 7.2 years and OS not yet reached. 75 patients enrolled on TT5 protocol experienced median PFS of 2.2 years and median OS of 5.6 years.150 TT6 enrolled patients experienced median PFS of 4.1 years and median OS of 7.5 years. Statistically significant difference in PFS (p &lt; 0.0001) and OS (p &lt; 0.0001) was observed between 1q21 defined groups (figure 1). No differences in survival were observed based on the 1q21 copy number &gt; 3. Conclusions: This retrospective analysis clearly showed that 1q21 amplification/gain detected by interphase iFISH in different stages of disease can identify patients with significant shorter progressive free and overall survival even if exposed to total therapy regimens. Clinical trial information: NCT00734877, NCT00869232.

OR-007 Identification of Early Predictive Biomarkers for Exercise-induced Immunodepression by Urinary iTRAQ-proteomic Analysis

Objective Exercise-induced immunodepression is a common medical problem in competitive sports, leading to upper respiratory tract infection, affecting sports training and sports performance, and increasing athletes' sports disease and injury risk. Finding non-invasive early predictive biomarkers of exercise-induced immunodepression and giving corresponding preventive measures is therefore an important issue in sports training. iTRAQ is an important method that currently looking for and discovering disease-specific protein biomarkers of disease prevention, diagnosis, prognosis, and efficacy monitoring. In this study, early predictive biomarkers of exercise-induced immunodepression will be identified through the iTRAQ proteomics technique, which helps to prevent the occurrence of exercise-induced immunodepression.&#x0D; Methods Fifteen healthy males were recruited from the student cohort of Guangzhou Sport University. Subjects performed four-week incremental load running exercise. The weekly running load intensity was 60% VO2max, 70% VO2max, 80% VO2max, 90% VO2max respectively, 5d/w, and 1h/d. The fasting venous blood and urine of the subjects was collected in the morning before the start of the training intervention and at the end of each training week. The white blood cells of the whole blood and the levels of the lymphocyte subtypes CD4+ and CD8+ were tested to monitor the immune function status of the subjects. iTRAQ proteomics technology was used to test and identify differential proteins and their characteristics in urine.&#x0D; Results During the four weeks of increasing running load, the subject’s immune function was progressively reduced. Whole blood white blood cells, and CD4+ and CD8+ lymphocyte fell by more than 10% at the end of the fourth week, showing exercise-induced Immunodepression. Using iTRAQ to test urine proteomes, there were as many as 1854 proteins in the urine during the incremental loading process. The relative molecular weights of most of the proteins were between 10-80 kDa, and the isoelectric point was between 4.5 and 7. During the four weeks of incremental loading running, there were 89, 52, 77, and 148 differential proteins up-regulated, and 66, 27, 68, and 114 differential proteins downregulated respectively in the urine of each week. The differential proteins were mostly found in extracellular and plasma membranes. It is mainly involved in the in vivo biological process, the immune system process, the material transport, and is related to the positive regulatory pathways and immune regulatory pathways for stress response. The up-regulation multiples of four up-regulated proteins such as Semenogelin-1, Prolactin-inducible protein, Platelet-derived growth factor receptor-like protein, and Nucleoside diphosphate kinase increased with increasing exercise intensity. The up-regulated multiples of Glycerol-3-phosphate phosphatase, Secretogranin-1, Prosaposin, and Nephronectin (Fragment) increased with increasing exercise intensity from the second week of exercise. The down-regulation multiples of Ig kappa chain C region, Immunoglobulin lambda variable 3-21 of CUB and EGF-like domain-containing protein 2 and Uromodulin decreased further with the increase of exercise intensity from the second week of exercise, which was consistent with the change of immune function.&#x0D; Conclusions Urine iTRAQ proteomics technique is an important method to identify early predictive biomarkers of exercise-induced immunodepression, which helps to prevent the occurrence of exercise-induced immunodepression. In this study, the differential proteins in urine, such as Semenogelin-1, Prolactin-inducible protein, Platelet-derived growth factor receptor-like protein, and Nucleoside diphosphate kinase can be considered as early predictive biomarkers of exercise-induced immunodepression.

Decreased Neuro-Axonal Proteins in CSF at First Attack of Suspected Multiple Sclerosis.

The pathology of multiple sclerosis is located in the central nervous system, therefore cerebrospinal fluid (CSF) is an attractive biofluid for biomarker research for proteins related to the early stages of this disease. In this study, the CSF proteome of patients with a clinically isolated syndrome of demyelination (CIS, a first attack of multiple sclerosis) is compared to the CSF proteome of control patients to identify differentially abundant proteins. CSF samples of 47 CIS patients and 45 control subjects are enzymatically digested and subsequently measured by LC-MS/MS (LTQ-Orbitrap). Following mass spectrometry differential abundances of the identified proteins between groups are investigated. A total of 3159 peptides are identified, relating to 485 proteins. One protein is significantly more abundant in CSF of CIS patients than in controls: Ig kappa chain C region. In contrast, 35 proteins are significantly lower in CIS patients than controls, most of them with functions in nervous system development and function, such as amyloid-like protein 1 (validated by ELISA in an independent sample set (p<0.01)), contactin 1, contactin 2 and neuronal cell adhesion molecule. A remarkably lower abundance of neuro-axonal proteins is observed in patients with a first demyelinating event compared to controls.

Isotypes of autoantibodies against differentially expressed novel malondialdehyde-modified peptide adducts in serum of Taiwanese women with rheumatoid arthritis

This study identified and validated four differentially expressed novel malondialdehyde (MDA)-modified peptide adducts and evaluated autoantibodies against native and MDA-modified peptides among Taiwanese women patients with rheumatoid arthritis (RA), osteoarthritis (OA) and healthy controls (HCs). Ig kappa chain C region76–99, alpha-1-antitrypsin284–298, alpha-2-macroglobulin824–841, and apolipoprotein B-1004022–4040 exhibiting 2-fold differences in relative modification ratios were identified by concanavalin A (Con A) affinity chromatography, 1D SDS-PAGE, in-gel digestion, nano-LC/MS/MS and nano-LC/MS using pooled serum-derived Con A-captured proteins from 9 RA and 9 age-matched HCs. Furthermore, the levels of proteins, serum MDA, and MDA-modified protein adducts were further validated against individual serum from 20 RA and 20 HCs, and autoantibodies against native and their MDA-modified peptides used 45 RA, 30 OA and 45 HCs. Levels of serum MDA and MDA-modified protein adducts were significantly higher in RA than HCs but protein levels were not significantly different. Serum Igs G and M against MDA-modified peptides showed better diagnostic performance in differentiating among patients with RA, OA and HCs, with an area under the receiver operating characteristic curve of 0.96–0.98, sensitivity of 88.9%–97.8%, and specificity of 88.9%–100%. Autoantibodies against MDA-modified epitopes become useful clinical biomarkers for RA. Biological significanceBy using a label-free relative quantitative proteomic analysis of concanavalin A (Con A)-bound serum samples, the current study discovered and validated malondialdehyde (MDA)-modified peptide adducts as novel biomarkers for differentiating between rheumatoid arthritis (RA) patients and healthy controls (HCs). In addition, the serum levels of MDA, proteins, and MDA-modified protein adducts as well as the MDA modification of proteins were determined. Isotypes of autoantibodies against MDA-modified peptide adducts can be used as serological biomarkers for further discriminating among RA patients, osteoarthritis patients and HCs. This strategy can become the basis for identifying potential diagnostic and pathological biomarkers for RA.

Analysis of Urine Composition in Type II Diabetic Mice after Intervention Therapy Using Holothurian Polypeptides.

Hydrolysates and peptide fractions (PF) obtained from sea cucumber with commercial enzyme were studied on the hyperglycemic and renal protective effects on db/db rats using urine metabolomics. Compared with the control group the polypeptides from the two species could significantly reduce the urine glucose and urea. We also tried to address the compositions of highly expressed urinary proteins using a proteomics approach. They were serum albumins, AMBP proteins, negative trypsin, elastase, and urinary protein, GAPDH, a receptor of urokinase-type plasminogen activator (uPAR), and Ig kappa chain C region. We used the electronic nose to quickly detect changes in the volatile substances in mice urine after holothurian polypeptides (HPP) fed, and the results show it can identify the difference between treatment groups with the control group without overlapping. The protein express mechanism of HPP treating diabetes was discussed, and we suggested these two peptides with the hypoglycemic and renal protective activity might be utilized as nutraceuticals.

Fluoxetine Regulates Ig Kappa Chain C Region Expression Levels in the Serum of Obsessive-Compulsive Disorder Patients: A proteomic Approach.

Obsessive-Compulsive Disorder (OCD) is one of the most common mental conditions. Proteome profiling may help identifying important proteins and finally shed lights to complexity of OCD underlying mechanisms. Here, by the application gel-based proteomic approach the proteome profile of patients with washing subtype of OCD before and after treatment with Fluoxetine (positive responders) are compared to healthy matched controls. However, only one of the differentially expressed proteins is examined and introduced in this paper. Proteomic analysis was done by the application of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), combined with (MALDI-TOF-TOF MS)-based. Furthermore, network analysis and biological annotation were handled by Cytoscape Plug-in and CluePedia. The proteome comparison between groups identified protein with the significant expression changes (p<0.05 and fold change ≥ 1.5). While the expression level of Ig Kappa Chain C Region is significantly decreased in OCD patients before any treatments, the trend is almost normalized after treatment with Fluoxetine in positive responders. In addition, interaction profile of IGKC shows that the interacting proteins may be affected as the expression pattern of IGKC changes in OCD patients. In conclusion, IGKC may be introduced as potential biomarker in our study; yet, investigation in bigger sample size and application of validation methods is a requirement.

Comparative Proteomic Analysis of Breast Cancer Tissue and the Adjacent Normal Tissue in Iranian Patients with HER2 Negative Ductal Carcinoma of Breast

Background: There is very limited evidence about the differences between proteome of breast cancer tissue and the adjacent normal tissue. Given the importance of identifying factors involved in the tumorogenesis of breast cancer; herein we aimed to compare the proteome of HER2 negative invasive ductal carcinoma and their adjacent normal tissues. Methods: During the removal surgery, 50 mg of cancer tissue as well as 50 mg of adjacent normal tissue were obtained from 5 female patients with ductal carcinoma in stage 3. The total protein content of the tissues was extracted and subjected to two-dimensional gel electrophoresis and MALDI-TOF MS/MS mass spectrometry. Results: A total of 1300 spots were found in the cancer tissues and 751 spots in the adjacent normal tissues, 46 of which were matched between the groups. Cytoskeletal keratin types I and II showed a 4-fold increase (P < 0.001); C region of Ig kappa chain, 2 folds increase (P < 0.001); serum albumin and Collagen VI, 16 fold decrease (P = 0.007); and tropomyosin alpha-4 and alpha-3 chains, 41 fold decrease (P = 0.04) in the cancer tissues compared to the normal tissues. In addition, calreticulin, galectin-1, and constant region of Ig gamma-1 chain were only identified in the former, and alpha-1-antitrypsin and hemoglobin subunit beta only in the latter. Conclusions: It seems that overexpression of galectin-1, the types I and II keratin, as well as down-regulation of collagen VI and alpha 1-antitrypsin in the cancer nest might be implicated in the progression of advanced breast carcinoma, and therefore, be used as independent biomarkers for the disease. However, much more studies are suggested to confirm our results.

Proteomics analysis of pleomorphic adenoma of the human parotid gland.

1b.

Abstract B11: Proteomic analysis of serum-derived exosomes: Identification of novel protein signature associated with African-American prostate cancer

Abstract African-American men face a stark prostate cancer (PCA)-related health disparity in the United States, with the highest incidence and mortality rate compared to all other races. African-American men are more frequently diagnosed with high Gleason grade PCA at a younger age and with a poorer prognosis. In the past three decades, there has been little improvement in reducing this health disparity, demanding additional and innovative measures. Here, we focused on the identification of novel serum exosome-based ‘protein signature' for potential use in the early detection and better prognosis of PCA in African-American men. Analysis of clinical serum samples showed that compared to healthy individuals, exosome concentration was increased by ~3.2 fold (p=0.047) in the sera of African-American men with PCA (Gleason score 7). Mass spectrometry based proteomic analysis of serum exosomal proteins showed 55 common proteins in African-American men with PCA (PAA) and normal African-American men without PCA (NAA). Interestingly, 7 unique proteins [Isoform 2 of Coiled-coil and C2 domain-containing protein 1A, Keratin type I cytoskeletal 10, UPF0728 protein C10orf53, DnaJ homolog subfamily C member 13, Prothrombin, Apolipoprotein(a) and Coiled-coil domain-containing protein 172] were present only in PAA, and not in NAA. The top upregulated proteins in PAA, as compared to NAA, included Keratin type II cytoskeletal 2 epidermal (64 fold), Serum amyloid P-component (39 fold), Keratin type II cytoskeletal 1 (13 fold), Keratin type I cytoskeletal 9 (12 fold), Isoform 2 of Filamin-A (2.6 fold), Isoform 3 of Vitamin D-binding protein (2.4 fold), Ig kappa chain V-II region Cum (2.4 fold) and Isoform 2 of Eukaryotic translation initiation factor 2-alpha kinase 4 (1.8 fold). The top downregulated proteins in PAA included Apolipoprotein A-I (0.04 fold), Complement component C8 alpha chain (0.07 fold), Plasminogen (0.08 fold), Complement component C7 (0.1 fold), and Isoform 2 of Inter-alpha-trypsin inhibitor heavy chain H4 (0.2 fold). Importantly, Ingenuity pathway analysis (IPA) showed that the proteins detected by proteomic analysis in PAA exosomes belonged to the acute-phase response signaling pathway proteins, which have been reported for their potential use as diagnostic biomarkers for PCA. Next, we compared proteins loaded in PAA serum exosomes with Gleason score-matched Caucasian PCA (PCC) serum exosomes. PAA and PCC exosomes shared 57 common proteins, while PAA serum exosomes showed six unique proteins (Isoform 2 of Coiled-coil and C2 domain-containing protein 1A, UPF0728 protein C10orf53, Ig kappa chain V-I region EU, Ig kappa chain V-II region Cum, DnaJ homolog subfamily C member 13, Coiled-coil domain-containing protein 172). Furthermore, top upregulated proteins in PAA, as compared to PCC, included Isoform 2 of Eukaryotic translation initiation factor 2-alpha kinase 4 (34 fold), Keratin type I cytoskeletal 10 (10 fold), SCO-spondin (5.3 fold), Serum amyloid P-component (4 folds) and Apolipoprotein(a) (3.9 fold). The top down regulated proteins in PAA, as compared to PCC, included Ig lambda chain V-I region WAH (0.02 fold), alpha-1B-glycoprotein (0.03 fold), Apolipoprotein A-I (0.03 fold), Complement component C8 alpha chain (0.06 fold) and Isoform 2 of Haptoglobin (0.09 fold). Overall, we have identified several unique and differentially expressed proteins in the serum exosomes of African-American men with PCA. These results also support further development and application of serum exosome-based proteomic signature for early detection and better prognosis of PCA in African- American men. Citation Format: Gagan Deep, Gatikrushna Panigrahi, Rakesh Singh, Kathleen Torkko, Adrie Bokhoven. Proteomic analysis of serum-derived exosomes: Identification of novel protein signature associated with African-American prostate cancer. [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr B11.

A highly efficient modified human serum albumin signal peptide to secrete proteins in cells derived from different mammalian species

Signal peptides (SPs) are key elements in the production of recombinant proteins; however, little information is available concerning different SP in mammalian cells other than CHO. In order to study the efficiency of different SPs to direct the traffic along the secretory pathway of the green fluorescence protein (GFP) and a scFv-Fc fusion protein; CHO-K1, HEK293 and NS0 cell lines were transfected in a transient and stable way. SP of human azurocidin (AZ), modified human albumin (mSA), modified Cricetulus griseus Ig kappa chain V III region MOPC 63 like (mIgκ C) and modified human Ig kappa chain V III region VG (mIgκ H) were evaluated. The efficiency of SPs to translocate a propeptide across the ER membrane was evaluated by fluorescence microscopy and flow cytometry for the GFP inside the secretory pathway, and by antigen-specific indirect ELISA for the scFv-Fc outside the cell. The mSA SP was successful in directing the secretion of the active proteins in these different types of mammalian cells, regardless of the transgene copy number. The goal of this work was to demonstrate that a modified version of SA SP might be used in different mammalian cells employing the same expression vector.

Redox proteomic identification of carbonylated proteins in autism plasma: insight into oxidative stress and its related biomarkers in autism

BackgroundAutism is a severe childhood neurological disorder with poorly understood etiology and pathology. Currently, there is no authentic laboratory test to confirm the diagnosis of autism. Oxidative damage may play a central role in the pathogenesis of autism. Present study is an effort to search for possible biomarkers of autism and further clarify the molecular changes associated with oxidative stress that occurs in the plasma of autistic children.MethodsWe performed redox proteomics analysis to compare carbonylated proteins in the plasma of autistic subjects and healthy controls. Immunoprecipitation and Western blot analysis were used to validate carbonylated proteins identified by the redox proteomics.ResultsProtein carbonylation levels in two proteins, complement component C8 alpha chain and Ig kappa chain C were found to be significantly increased in autistic patients compared with controls. These two proteins were successfully validated via immunoprecipitation and Western blot analysis.ConclusionsThe results further highlight the role of oxidative stress in the pathogenesis of autism and provide some information for the diagnosis and/or monitoring of autism.

Proteomics Analysis to Identify and Characterize the Biomarkers and Physical Activities of Non-Frail and Frail Older Adults.

Globally, the proportion of older adults is increasing. Older people face chronic conditions such as sarcopenia and functional decline, which are often associated with disability and frailty. Proteomics assay of potential serum biomarkers of frailty in older adults. Older adults were divided into non-frail and frail groups (n = 6 each; 3 males in each group) in accordance with the Chinese-Canadian Study of Health and Aging Clinical Frailty Scale. Adults were measured for grip power and the 6-min walk test for physical activity, and venous blood was sampled after adults fasted for 8 h. Ultra-high-performance liquid chromatography-tandem mass spectrometry was used for proteomics assay. The groups were compared for levels of biomarkers by t test and Pearson correlation analysis. Non-frail and frail subjects had mean age 77.5±0.4 and 77.7±1.6 years, mean height 160.5±1.3 and 156.6±2.9 cm and mean weight 62.5±1.2 and 62.8±2.9 kg, respectively. Physical activity level was lower for frail than non-frail subjects (grip power: 13.8±0.4 vs 26.1±1.2 kg; 6-min walk test: 215.2±17.2 vs 438.3±17.2 m). Among 226 proteins detected, for 31, serum levels were significantly higher for frail than non-frail subjects; serum levels of Ig kappa chain V-III region WOL, COX7A2, and albumin were lower. The serum levels of ANGT, KG and AT were 2.05-, 1.76- and 2.22-fold lower (all p < 0.05; Figure 1A, 2A and 3A) for non-frail than frail subjects and were highly correlated with grip power (Figure 1B, 2B and 3B). Our study found that ANGT, KG and AT levels are known to increase with aging, so degenerated vascular function might be associated with frailty. In total, 226 proteins were revealed proteomics assay; levels of angiotensinogen (ANGT), kininogen-1 (KG) and antithrombin III (AT) were higher in frail than non-frail subjects (11.26±2.21 vs 5.09±0.74; 18.42±1.36 vs 11.64±1.36; 22.23±1.64 vs 9.52±0.95, respectively, p < 0.05). These 3 factors were highly correlated with grip power (p < 0.05), with higher correlations between grip power and serum levels of ANGT (r = -0.89), KG (r = -0.90), and AT (r = -0.84). In conclusion, this is the first study to demonstrate a serum proteomic profile characteristic of frailty in older adults. Serum ANGT, KG and AT levels could be potential biomarkers for monitoring the development and progression of frailty in older adults.