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Methods in Molecular Biology

Nathan Salomonis
The analysis of proteomics data can be significantly challenging. Beyond the technical challenges of accurately identifying and quantifying peptides, identifying the most biologically coherent set of biomarkers can be a particularly daunting step. In this chapter, we will review a series of methods implemented in the software AltAnalyze that can be used to normalize proteomics peptide counts, identify a minimal set of the most distinguishing morbidity-associated biomarkers, and connect up these results to known pathways and interacting protein and regulatory networks...
November 18, 2017: Methods in Molecular Biology
Guixue Hou, Yang Wang, Xiaomin Lou, Siqi Liu
Quantitative proteome analysis using iTRAQ is an important technique to find disease-related proteins. As an important component of tumor microenvironment, tissue interstitial fluid (TIF) has drawn a great attention for its potential as a source for exploration of the solid tumor biomarkers. On the basis of a mouse model of colorectal cancer (CRC) that was induced by the carcinogenetic reagents, we adopted a quantitative proteome analysis with iTRAQ to discover the CRC-related proteins in the TIFs and with MRM to evaluate the corresponding abundance changes in the individual mouse TIF and serum samples...
November 15, 2017: Methods in Molecular Biology
Emily T Kostas, Stuart J Wilkinson, Daniel A White, David J Cook
Accurate quantification of the carbohydrate content of biomass is crucial for many bio-refining processes. The most commonly followed protocol is typically a modification of the NREL-based assay (specifically designed for carbohydrate analysis from lignocellulosic biomass). However, this NREL protocol was revealed to be excessively thermochemically harsh for seaweed biomass. This can result in erroneously low total sugar quantification as the reaction severity can degrade a proportion of the liberated sugars to decomposition products such as furans...
November 14, 2017: Methods in Molecular Biology
Eva Tomaskovic-Crook, Jeremy M Crook
Human brain organoids provide opportunities to produce three-dimensional (3D) brain-like tissues for biomedical research and translational drug discovery, toxicology, and tissue replacement. Here we describe a protocol for rapid and defined induction of brain organoids from human induced pluripotent stem cells (iPSCs), using commercially available culture and differentiation media and a cheap, easy to handle and clinically approved semisynthetic hydrogel. Importantly, the methodology is uncomplicated, well-defined, and reliable for reproducible and scalable organoid generation, and amendable to principles of current good laboratory practice (cGLP), with the potential for prospective adaptation to current good manufacturing practice (cGMP) toward clinical compliance...
November 9, 2017: Methods in Molecular Biology
Yuqian Gao, Hui Wang, Carrie D Nicora, Tujin Shi, Richard D Smith, Tara K Sigdel, Minnie M Sarwal, David G Camp, Wei-Jun Qian
Liquid chromatography (LC)-selected reaction monitoring (SRM) is a powerful protein quantification technique in terms of sensitivity, reproducibility, and multiplexing capability. LC-SRM can accurately measure the concentrations of surrogate proteotypic peptides for targeted proteins in complex biological samples by using their stable heavy isotope-labeled counterparts as internal standards. Herein, we describe a step-by-step protocol of the application of LC-SRM to quantify candidate protein biomarkers in human urine...
November 8, 2017: Methods in Molecular Biology
Tara K Sigdel, Minnie M Sarwal
Because of our access to human genome data and ever-improving genome sequencing and proteome analysis methods, we are much better in terms of our understanding of biological processes. In addition to genomics, proteomics, and other "omics" methods, availability of more sophisticated molecular assaying methods has augmented our knowledge about immune processes toward auto- and allogeneic targets. High-density protein arrays are developed to analyze protein-small molecule interactions, enzyme-substrate profiling, protein-protein interaction, and immune monitoring by assessing antibodies in the serum...
November 8, 2017: Methods in Molecular Biology
Matteo Ghiringhelli, Alessandro Zenobi, Stefano Brizzola, Fulvio Gandolfi, Valentino Bontempo, Sandro Rossi, Tiziana A L Brevini, Fabio Acocella
The development of new approaches for organ transplantation has become crucial in the last years. In particular, organ engineering, involving the preparation of acellular matrices that provide a natural habitat for reseeding with an appropriate population of cells, is an attractive although technically demanding approach. We here describe a method that allows for the derivation of functional in vitro hepatic organoids and that does not require a previous selection of all the parenchymal hepatocytes and non-parenchymal cells, namely, Kupffer cells, liver endothelial cells, and hepatic stellate cells...
November 4, 2017: Methods in Molecular Biology
Iris Pla-Palacín, Pilar Sainz-Arnal, Sara Morini, Manuel Almeida, Pedro M Baptista
Currently, due to the progress made in the field of regenerative medicine, whole-organ bioengineering is becoming a valid alternative to cope with the shortages of organs for transplantation. In this chapter, we describe the main techniques carried out for pig liver bioengineering, which serves as an essential model for future human liver bioengineering. These include porcine whole-liver decellularization, endothelial and mesenchymal stem cell isolation, porcine ES-derived hepatoblasts, and scaffold recellularization using a bioreactor perfusion system...
November 4, 2017: Methods in Molecular Biology
Javad Alizadeh, Shahla Shojaei, Adel Sepanjnia, Mohammad Hashemi, Eftekhar Eftekharpour, Saeid Ghavami
Autophagy is increasingly identified as a central player in many cellular activities from cell proliferation to cell division, migration, and differentiation. However, it is also considered as a double-edged sword in cancer biology which either promotes oncogenesis/invasion or sensitizes the tumor cells to chemotherapy induced apoptosis. Recent investigations have provided direct evidence for regulation of cellular phenotype via autophagy pathway. One of the most important types of phenotype conversion is Epithelial-Mesenchymal-Transition (EMT), resulting in alteration of epithelial cell properties to a more mesenchymal form...
November 4, 2017: Methods in Molecular Biology
Dejan Dobi, Zoltan G Laszik
Quantitative metrics on the tissue distribution of different cell phenotypes, extracellular matrix components, and signaling/cell cycle markers hold the promise for the advent of new-generation tissue-based predictive/prognostic biomarkers in clinical diagnostics. The workflow of this approach is composed of three major phases: (1) detection of multiple molecular targets on a single histologic section, (2) image acquisition, and (3) digital image processing and analysis. Here, we present the most prevalent current alternatives for step (1) and describe a three-plex staining and image acquisition platform that captures the spatial distribution of macromolecules from two different species...
October 27, 2017: Methods in Molecular Biology
Yi Zhu, Tiannan Guo
In the era of precision medicine, there is an increasing need to measure several thousand proteins expressed in minimal amount of fresh-frozen biopsy tissue samples from clinical cohorts. Here we present the detailed protocol for high-throughput proteomic analysis of less than 1 mg fresh-frozen tissue sample using pressure cycling technology (PCT), SWATH mass spectrometry, and OpenSWATH workflow. The PCT procedures enable simultaneous analysis of 16 biopsy tissue samples per batch, followed by SWATH analysis of about 15 samples per mass spectrometer per working day...
October 26, 2017: Methods in Molecular Biology
Timothy D Cummins, Gopal P Sapkota
Identification and characterization of large protein complexes is a mainstay of biochemical toolboxes. Utilization of cross-linking chemicals can facilitate the capture and identification of transient or weak interactions of a transient nature (Huang and Kim, PloS One 8:e61430, 2013; Gao et al., J Vis Exp doi: 10.3791/51387, 2014). Here we describe a detailed methodology for a cell culture-based proteomic approach. We describe the generation of cells stably expressing green fluorescent protein (GFP)-tagged proteins under the tetracycline-inducible promoter and subsequent proteomic analysis of GFP-interacting proteins...
October 25, 2017: Methods in Molecular Biology
Peggi M Angel, Anand Mehta, Kim Norris-Caneda, Richard R Drake
Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is a unique and well developed tool for probing the protein content of formalin-fixed, paraffin-embedded tissue (FFPE). Integral to this approach is the application of trypsin, and more recently peptide N-glycosidase F, to release tryptic peptides or N-glycans from tissue and report localization of distinct species. This is typically done on serial or adjacent tissue sections, and there is an emerging need to understand the colocalized protein population linked to the exact same regions of N-glycans...
October 21, 2017: Methods in Molecular Biology
François-Ludovic Sauvage, Sébastien Passeron, Pierre Marquet
In renal transplantation, the discovery of early urine biomarkers of graft lesions would be useful in helping physicians to improve patient care and minimize the use of invasive techniques such as biopsies. Over the last years, high-resolution mass spectrometry has been used extensively for the search of biomarkers in various biological fluids. Here we describe a procedure based on reverse-phase nano-HPLC, offline plate spotting, and MALDI-TOF and TOF/TOF applied in our laboratory for the search of natural peptides in urine samples from renal transplant patients...
October 17, 2017: Methods in Molecular Biology
Deniz Gulfem Ozturk, Muhammed Kocak, Devrim Gozuacik
Autophagy is a cellular survival pathway that is necessary for the degradation of cellular constituents such as long-lived proteins and damaged organelles. Conditions resulting in cellular stress such as starvation or hypoxia might activate autophagy. Being at the crossroads of various cellular response pathways, dysregulation of autophagy might result in pathological states including cancer and neurodegenerative diseases. Autophagy has also been shown to participate in stemness. MicroRNAs were introduced as novel regulators of autophagy, and accumulating results underlined the fact that they constituted an important layer of biological control mechanism on the autophagic activity...
October 12, 2017: Methods in Molecular Biology
Nicole Valenzuela, Nwe Nwe Soe, Fang Li, Xiaohai Zhang, Yi-Ping Jin, Elaine F Reed
The signaling capacity of HLA molecules in vascular cells has been well established. Intracellular signaling and association with the coreceptor integrin β4 has been well-studied for HLA class I. However, little is known regarding HLA class II intracellular signaling in human endothelial cells. Investigation of HLA class II has been challenging due to the loss of HLA class II expression in cultured primary cells. Herein, we describe methods for inducing expression of endogenous alleles and loci of HLA class II molecules, as well as for studying intracellular signaling...
October 10, 2017: Methods in Molecular Biology
Kelly J Shields, Changgong Wu
Differential proteomic analysis (comparative quantitative proteomics) is a robust quantitative technique used to detect and identify the proteome of selected tissues. The expression levels (upregulated vs. downregulated) of proteins in tissue samples that differ by experimental design or anatomic location are determined by a series of assays including (1) 2D difference gel electrophoresis (2D-DiGE), (2) protein spot picking based on a priori thresholds, (3) Mass Spectrometry, and (4) follow-up Western Blot for antibody validation (Chen et al...
October 10, 2017: Methods in Molecular Biology
Manousos Makridakis, Antonia Vlahou
Application of various proteomics methodologies have been implemented for the global and targeted proteome analysis of many different types of biological samples such as tissue, urine, plasma, serum, blood, and cell lines. Among the aforementioned biological samples, tissue has an exceptional role into clinical research and practice. Disease initiation and progression is usually located at the tissue level of different organs, making the analysis of this material very important for the understanding of the disease pathophysiology...
October 10, 2017: Methods in Molecular Biology
Chih-Wei Liu, Qibin Zhang
The pancreas is an organ with both endocrine and exocrine functions, and various pathologies, such as pancreatic cancer and diabetes are associated with this organ. Owing to the limited pancreatic biopsy samples available for research, it is critical to make the best use of cadaveric pancreatic tissue for biomarker studies and mechanistic understanding of pancreas-related pathologies. Discovery-phase quantitative proteomics has attracted a lot of attention for its capabilities in large-scale protein identification and accurate protein quantification...
October 7, 2017: Methods in Molecular Biology
Yvonne Pasing, Armin Schniers, Terkel Hansen
After conducting systematic and quantitative comparisons of different sample preparation techniques regarding their capability to efficiently and reproducibly recover proteins from biopsies, we present here our superior protocol for extracting proteins from low amounts of adipose tissue. Adipose tissue as a matrix in bottom-up proteomics is challenging due to the extremely high lipid content.The lysis buffer utilized contains the detergent sodium deoxycholate, which does not impair the activity of trypsin and therefore enables direct digestion without detergent removal steps...
October 5, 2017: Methods in Molecular Biology
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