scholarly journals Dramatic Improvement of Proteomic Analysis of Zebrafish Liver Tumor by Effective Protein Extraction with Sodium Deoxycholate and Heat Denaturation

2015 ◽  
Vol 2015 ◽  
pp. 1-11 ◽  
Author(s):  
Jigang Wang ◽  
Yew Mun Lee ◽  
Caixia Li ◽  
Ping Li ◽  
Zhen Li ◽  
...  
Keyword(s):  
Liver Tumor ◽  
Proteomic Analysis ◽  
Protein Extraction ◽  
Sodium Deoxycholate ◽  
Heat Denaturation ◽  
Dramatic Improvement ◽  
Tissue Samples ◽  
Highly Efficient ◽  
Extraction Buffer ◽  
Zebrafish Liver

Majority of the proteomic studies on tissue samples involve the use of gel-based approach for profiling and digestion. The laborious gel-based approach is slowly being replaced by the advancing in-solution digestion approach. However, there are still several difficulties such as difficult-to-solubilize proteins, poor proteomic analysis in complex tissue samples, and the presence of sample impurities. Henceforth, there is a great demand to formulate a highly efficient protein extraction buffer with high protein extraction efficiency from tissue samples, high compatibility with in-solution digestion, reduced number of sample handling steps to reduce sample loss, low time consumption, low cost, and ease of usage. Here, we evaluated various existing protein extraction buffers with zebrafish liver tumor samples and found that sodium deoxycholate- (DOC-) based extraction buffer with heat denaturation was the most effective approach for highly efficient extraction of proteins from complex tissues such as the zebrafish liver tumor. A total of 4,790 proteins have been identified using shotgun proteomics approach with 2D LC, which to our knowledge is the most comprehensive study for zebrafish liver tumor proteome.

scholarly journals Comparison of different digestion methods for proteomic analysis of isolated cells and FFPE tissue samples

Talanta ◽  
2021 ◽  
pp. 122568
Author(s):  
Artur Pirog ◽  
Jakub Faktor ◽  
Zuzanna Urban-Wojciuk ◽  
Sachin Kote ◽  
Elżbieta Chruściel ◽  
...  
Keyword(s):  
Proteomic Analysis ◽  
Ffpe Tissue ◽  
Isolated Cells ◽  
Tissue Samples ◽  
Digestion Methods

scholarly journals Proteomic analysis-based discovery of a novel biomarker that differentiates intestinal Behçet’s disease from Crohn’s disease

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jihye Park ◽  
Daeun Jeong ◽  
Youn Wook Chung ◽  
Seunghan Han ◽  
Da Hye Kim ◽  
...  
Keyword(s):  
Crohn’S Disease ◽  
Crohn's Disease ◽  
Proteomic Analysis ◽  
Behcet’S Disease ◽  
Differentially Expressed ◽  
Tandem Mass ◽  
Intestinal Behçet’S Disease ◽  
Tissue Samples ◽  
Intestinal Behcet’S Disease ◽  
Intestinal Behçet's Disease

AbstractIntestinal Behçet’s disease (BD) and Crohn’s disease (CD) present similar manifestations, but there are no specific diagnostic tests to differentiate them. We used a proteomic approach to discover novel diagnostic biomarkers specific to intestinal BD. Colon mucosa tissue samples were obtained from patients with intestinal BD or CD using colonoscopy-guided biopsy of the affected bowel. Peptides from seven intestinal BD and seven CD patients were extracted and labeled using tandem mass tag (TMT) reagents. The labeled peptides were identified and quantified using liquid chromatography-tandem mass spectrometry (LC–MS/MS). The proteins were further validated using immunohistochemical (IHC) analysis with tissue samples and an ELISA test with serum samples from 20 intestinal BD and 20 CD patients. Using TMT/LC–MS/MS-based proteomic quantification, we identified 39 proteins differentially expressed between intestinal BD and CD. Beta-2 glycoprotein 1 (APOH) and maltase-glucoamylase (MGAM) showed higher intensity in the IHC staining of intestinal BD tissues than in CD tissues. The serum MGAM level was higher in intestinal BD patients. Proteomic analysis revealed that some proteins were differentially expressed in patients with intestinal BD compared with those with CD. Differential MGAM expression in intestinal BD suggests its role as a potential novel diagnostic biomarker.

scholarly journals P058 Proteomic analysis-based discovery of a novel biomarker that differentiates intestinal Behçet’s disease from Crohn’s disease

2021 ◽  
Vol 15 (Supplement_1) ◽  
pp. S164-S165
Author(s):  
J Park ◽  
J Daeun ◽  
C Youn Wook ◽  
C Jae Hee ◽  
R Ji-Hwan
Keyword(s):  
Crohn’S Disease ◽  
Crohn's Disease ◽  
Proteomic Analysis ◽  
Differentially Expressed ◽  
Tandem Mass ◽  
Diagnostic Biomarker ◽  
Intestinal Behçet’S Disease ◽  
Tissue Samples ◽  
Intestinal Behcet’S Disease ◽  
Intestinal Behçet's Disease

Abstract Background Intestinal Behçet’s disease (BD) and Crohn’s disease (CD) present similar manifestations, but there are no specific pathognomonic clinical, laboratory, or histological diagnostic tests to differentiate intestinal BD from CD. We used a proteomic approach to discover a novel diagnostic biomarker specific to intestinal BD. Methods The colon mucosa tissue samples were obtained using colonoscopy-guided biopsy of the affected bowel from patients with intestinal BD or CD at the Inflammatory Bowel Disease Clinic of Severance Hospital, Seoul, Korea. Peptides from seven intestinal BD and seven CD patients were extracted and labeled using tandem mass tag (TMT) reagents. The labeled peptides were identified and quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The differentially expressed proteins were further validated using immunohistochemical (IHC) analysis with tissue samples and an ELISA test with serum samples from 20 intestinal BD and 20 CD patients. Results A total of 3,266 proteins were identified using TMT/LC-MS/MS-based proteomic quantification, including 39 candidate proteins differentially expressed between intestinal BD and CD. Beta-2 glycoprotein 1 (APOH) and maltase-glucoamylase (MGAM) showed significantly higher intensity in the IHC staining of the intestinal BD tissues than that of CD tissues. Furthermore, the serum MGAM level was significantly higher in patients with intestinal BD than in patients with CD. Conclusion Our proteomic analysis revealed that some proteins were differentially expressed in intestinal BD patients compared to CD patients. Differential MGAM expression in intestinal BD suggests its role as a potential novel diagnostic biomarker in the differentiation of intestinal BD from CD.

scholarly journals High‐throughput proteomic analysis of FFPE tissue samples facilitates tumor stratification

2019 ◽  
Vol 13 (11) ◽  
pp. 2305-2328 ◽  
Author(s):  
Yi Zhu ◽  
Tobias Weiss ◽  
Qiushi Zhang ◽  
Rui Sun ◽  
Bo Wang ◽  
...  
Keyword(s):  
High Throughput ◽  
Proteomic Analysis ◽  
Ffpe Tissue ◽  
Tissue Samples

scholarly journals FACS-Based Proteomics Enables Profiling of Proteins in Rare Cell Populations

2020 ◽  
Vol 21 (18) ◽  
pp. 6557
Author(s):  
Evelyne Maes ◽  
Nathalie Cools ◽  
Hanny Willems ◽  
Geert Baggerman
Keyword(s):  
Proteomic Analysis ◽  
Mononuclear Cells ◽  
Protein Extraction ◽  
Cell Types ◽  
Clinical Samples ◽  
Cell Populations ◽  
Peripheral Blood Mononuclear ◽  
Limited Protein ◽  
Instrument Sensitivity ◽  
Acquisition Method

Understanding disease pathology often does not require an overall proteomic analysis of clinical samples but rather the analysis of different, often rare, subpopulations of cells in a heterogeneous mixture of cell types. For the isolation of pre-specified cellular subtypes, fluorescence activated cell sorting (FACS) is commonly used for its ability to isolate the required cell populations with high purity, even of scarce cell types. The proteomic analysis of a limited number of FACS-sorted cells, however, is very challenging as both sample preparation inefficiencies and limits in terms of instrument sensitivity are present. In this study, we used CD14+CD15+ immune cells sorted out of peripheral blood mononuclear cells isolated from whole blood to improve and evaluate FACS-based proteomics. To optimize both the protein extraction protocol and the mass spectrometry (MS) data acquisition method, PBMCs as well as commercialized HeLa digest were used. To reflect the limited number of sorted cells in some clinical samples, different numbers of sorted cells (1000, 5000, 10,000, or 50,000) were used. This allowed comparing protein profiles across samples with limited protein material and provided further insights in the benefits and limitations of using a very limited numbers of cells.

scholarly journals Optimization of protein isolation by proteomic qualification from Cutaneotrichosporon oleaginosus

2019 ◽  
Vol 412 (2) ◽  
pp. 449-462 ◽  
Author(s):  
Dania Awad ◽  
Thomas Brueck
Keyword(s):  
Protein Extraction ◽  
Feedback Regulation ◽  
Genetic Optimization ◽  
Physiochemical Properties ◽  
High Lipid ◽  
Extraction Buffer ◽  
Optimized Protocol ◽  
Significant Step ◽  
One Step ◽  
Purification Methods

AbstractIn the last decades, microbial oils have been extensively investigated as a renewable platform for biofuel and oleochemical production. Offering a potent alternative to plant-based oils, oleaginous microorganisms have been the target of ongoing metabolic engineering aimed at increasing growth and lipid yields, in addition to specialty fatty acids. Discovery proteomics is an attractive tool for elucidating lipogenesis and identifying metabolic bottlenecks, feedback regulation, and competing biosynthetic pathways. One prominent microbial oil producer is Cutaneotrichosporon oleaginosus, due to its broad feedstock catabolism and high lipid yield. However, this yeast has a recalcitrant cell wall and high cell lipid content, which complicates efficient and unbiased protein extraction for downstream proteomic analysis. Optimization efforts of protein sample preparation from C. oleaginosus in the present study encompasses the comparison of 8 lysis methods, 13 extraction buffers, and 17 purification methods with respect to protein abundance, proteome coverage, applicability, and physiochemical properties (pI, MW, hydrophobicity in addition to COG, and GO analysis). The optimized protocol presented in this work entails a one-step extraction method utilizing an optimal lysis method (liquid homogenization), which is augmented with a superior extraction buffer (50 mM Tris, 8/2 M Urea/Thiourea, and 1% C7BzO), followed by either of 2 advantageous purification methods (hexane/ethanol or TCA/acetone), depending on subsequent applications and target studies. This work presents a significant step forward towards implementation of efficient C. oleaginosus proteome mining for the identification of potential targets for genetic optimization of this yeast to improve lipogenesis and production of specialty lipids.

scholarly journals Comparison of Different Protein Extraction Methods for Gel-Based Proteomic Analysis of Ganoderma spp.

2016 ◽  
Vol 35 (2) ◽  
pp. 100-106 ◽  
Author(s):  
Jameel R. Al-Obaidi ◽  
Noor Baity Saidi ◽  
Siti Rokhiyah Ahmad Usuldin ◽  
Siti Nahdatul Isnaini Said Hussin ◽  
Noornabeela Md Yusoff ◽  
...  
Keyword(s):  
Proteomic Analysis ◽  
Protein Extraction ◽  
Extraction Methods

Proteomic features of colorectal cancer independently predict relapse-free survival.

2015 ◽  
Vol 33 (3_suppl) ◽  
pp. 616-616
Author(s):  
Callisia Clarke ◽  
Michael Sangmin Lee ◽  
Ganiraju C. Manyam ◽  
Zhi-Qin Jiang ◽  
Feng Tian ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Energy Balance ◽  
Proteomic Analysis ◽  
Tumor Recurrence ◽  
Cox Regression ◽  
Protein Extraction ◽  
Univariate Analysis ◽  
The Cancer Genome Atlas ◽  
Cancer Center ◽  
Bootstrap Validation

616 Background: Proteomic analysis continues to provide major insight into the pathophysiology of colorectal cancer (CRC). Recently, the Cancer Genome Atlas Project and others have utilized reverse-phase protein arrays (RPPAs) to identify critical protein markers and signaling pathways in a variety of tumor types. However, the prognostic relevance of many of these proteins remains unclear. We utilized RPPA to analyze stage 2 and 3 CRC to investigate the prognostic implications of the functional proteome. Methods: Protein extraction was performed on 232 snap frozen stage 2/3 samples from the MD Anderson Cancer Center with a median 5 year follow up. 163 validated proteins and phospho-proteins were analyzed by RPPA (with dichotomization by the median value), and used to identify protein predictors of tumor recurrence. Cox regression was used for univariate analysis with bootstrap validation, followed by inclusion of proteins with corrected p≤ 0.05 into multivariate model with clinical and pathologic variables, including stage, grade, and microsatellite status. Results: 12 proteins were found to be significant predictors of tumor recurrence on univariate analysis after bootstrap validation, which are notable for components of the energy balance/MTOR signaling pathway including AMPK, mTOR, PI3 Kinase p85, FoxO3a. On multivariate analysis, inclusive of known prognostic clinical and pathology variables, phospho-Bad (Ser112), FoxO3a, HER3, and phospho-S6 (Ser240-244) remained significant. Conclusions: Functional proteomic analysis has identified key proteomic features with prognostic importance independent of known clinical/pathological variables. Confirmation studies are ongoing to explore regulators of energy balance and apoptosis in colorectal cancer. [Table: see text]

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