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Methods in Molecular Biology

Azad Eshghi, Christoph H Borchers
Multiple reaction monitoring (MRM) is a technique used in tandem mass spectrometry where the first mass analyzer preselects parent ions for fragmentation and the second mass analyzer transmits selected product ions to the detector. This targeted technique has found widespread application in bottom-up proteomics for monitoring target peptides in a complex enzymatic digest. Quantitative MRM can be performed on enzymatically digested samples using spiked-in synthetic peptide standards, providing unsurpassed quantitative accuracy and a dynamic range of four orders of magnitude, often eliminating the need for prior depletion of high-abundance proteins...
December 19, 2017: Methods in Molecular Biology
Feng Guo, Nathan Wlodarchak, Patrick Menden, Yongna Xing
Proteomics is a powerful approach for systematic identification and quantification of the entire proteome of a biological system (cell, tissue, organ, biological fluid, or organism) at specific time points ( ). Extracting and purifying target proteins from native tissues are essential steps for many aspects of proteomic studies. In this chapter, we will introduce the experimental procedures to obtain soluble proteins from two different tissues: (1) the CCT (cpn-containing TCP-1) complex from bovine testes and (2) the protein phosphatase 2A (PP2A) catalytic subunit (PP2Ac or C) from porcine brains...
December 17, 2017: Methods in Molecular Biology
Yi Chen, David Britton, Elizabeth R Wood, Stephen Brantley, Michelle Fournier, Marek Wloch, Vonetta L Williams, Joseph Johnson, Anthony Magliocco, Ian Pike, John M Koomen
Liquid chromatography-selected reaction monitoring (LC-SRM) mass spectrometry has developed into a versatile tool for quantification of proteins with a wide range of applications in basic science, translational research, and clinical patient assessment. This strategy uniquely complements traditional pathology approaches, like hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC). The multiplexing capabilities offered by mass spectrometry are currently unmatched by other techniques. However, quantification of biomarkers in tissue specimens without the other data obtained from H&E-stained slides or IHC, including tumor cellularity or percentage of positively stained cells inter alia, may not provide as much information that is needed to fully understand tumor biology or properly assess the patient...
December 15, 2017: Methods in Molecular Biology
Rémi Longuespée, Deborah Alberts, Dominique Baiwir, Gabriel Mazzucchelli, Nicolas Smargiasso, Edwin De Pauw
Matrix-assisted laser desorption ionization (MALDI) imaging is widely used for in situ proteomic mapping and finds multiple applications in pathology. However, low fragmentation yields in MALDI avoid an optimal identification of peptides from tissues. On the other hand, LMD-based microproteomic analyses allow for the identification of hundreds to thousands of proteins from small tissue regions. Herein, we present the combination of MALDI imaging and LMD-based microproteomic approaches for parallel identification...
December 10, 2017: Methods in Molecular Biology
Judita Koreivienė
The staining of lipids in algae cells with BODIPY dyes is much less studied compared to Nile red; therefore, a complex of issues concerning staining details and fluorescence measurements still should be clarified for the species that vary in cell wall complexity. Nevertheless, some general guidelines could be given, and a preliminary protocol of the method is provided based on the existing data. The semiquantification of lipid could be reliable if the staining protocol will be developed and adapted for particular microalgae species...
December 8, 2017: Methods in Molecular Biology
S Van Wychen, L M L Laurens
The carbohydrate fraction of microalgal biomass is complex and consists of a combination of neutral, acidic, amino sugars, and sugar alcohols. These carbohydrate amalgamations can be difficult to accurately quantify with the most analytical carbohydrate determination techniques. The method described here provides a straightforward two-step sulfuric acid hydrolysis followed by soluble carbohydrate quantification by either a spectrophotometric method (based on aldehyde functional group derivatization), for an overall combined monomeric sugar concentration determination, or a liquid chromatography method, for a more specific monomeric sugar profile...
December 5, 2017: Methods in Molecular Biology
S Van Wychen, L M L Laurens
The aliphatic chains of fatty acids are the most prominent and potentially the highest value precursor constituents of algal biomass, and thus accurately quantifying the algal biomass total fatty acid content is a prerequisite for comparing algal strains, growth conditions, and processes. Direct, acid-catalyzed transesterification of whole microalgal biomass is a simple, effective, and widely used method to determine the fatty acid content in whole algal biomass. Such a direct transesterification procedure typically covers the following steps: first, solubilizing the lipids in the biomass matrix and then liberating the fatty acids to make these available for catalytic upgrading to fatty acid methyl esters (FAMEs), subsequent extraction into hexane, and then quantification by gas chromatography...
December 5, 2017: Methods in Molecular Biology
Parhom Towfighi, Jacob Shaw, Tara Sigdel
In order to simplify a complex mixture of soluble proteins from tissues, a protocol to fractionate samples prior to two-dimensional (2D) gel electrophoresis has been developed. These methods involve the use of DEAE-Sepharose, SP-Sepharose, and phenyl Sepharose chromatographic columns and the fractionation of the protein mixtures based on differential anionic, cationic, and hydrophobic properties of the proteins, respectively. Fractionation of the soluble proteins with DEAE-Sepharose can result in an increase in the number of detectable 2D gel spots...
December 2, 2017: Methods in Molecular Biology
Kristian Spilling, Jukka Seppälä
Measuring fluorescence is a noninvasive, inexpensive, and quick way of determining biomass concentration and health of the algae. Fluorescence is generally positively correlated with chlorophyll a and can as such be used as a proxy for biomass. In addition, the proportion variable fluorescence of maximal fluorescence is a measure of photochemical efficiency, which is affected by stress in a very early stage and can as such be used as a proxy for algal health.
December 2, 2017: Methods in Molecular Biology
Iva Procházková, Juraj Lenčo, Pavel Bouchal
Breast cancer is the most common and molecularly well-characterized malignant cancer in women; however, its progression to metastatic cancer remains lethal for 78% of patients within 5 years of diagnosis. Identifying novel markers in high risk patients using quantitative methods is essential to overcome genetic, inter-tumor, and intra-tumor variability, and to translate novel findings into cancer diagnosis and treatment. Using untargeted proteomics, we recently identified 13 proteins associated with some key factors of breast cancer aggressiveness: estrogen receptors, tumor grade, and lymph node status...
December 2, 2017: Methods in Molecular Biology
Kristian Spilling
In this chapter, I go through the very basic methods for isolating and setting up a microalgae culture. Isolation can be done from local algal strains with a micropipette and microscope. Determining algal growth is important and can be done by counting cells under a microscope or by determining the change in dry weight (DW).
December 2, 2017: Methods in Molecular Biology
Tuulikki Seppänen-Laakso, Heli Nygren, Heiko Rischer
Constituents of microalgae and sample preparation for UPLC-ELSD and GC-MS analyses are described. Bound fatty acids from acylglycerols, alkylacylglycerols, galactosyldiacylglycerols, glycerophospholipids, and sterol esters are derivatized by using transesterification with sodium methoxide to form fatty acid methyl esters. Compounds containing free hydroxyl groups, either present originally or formed during previous step, like free fatty acids, sterols, α-tocopherol, phytol, and nonesterified alkoxyglycerols, are trimethylsilylated...
November 21, 2017: Methods in Molecular Biology
Jaana Koistinen, Mervi Sjöblom, Kristian Spilling
Carbon is the element which makes up the major fraction of lipids and carbohydrates, which could be used for making biofuel. It is therefore important to provide enough carbon and also follow the flow into particulate organic carbon and potential loss to dissolved organic forms of carbon. Here we present methods for determining dissolved inorganic carbon, dissolved organic carbon, and particulate organic carbon.
November 21, 2017: Methods in Molecular Biology
Andrea Peloso, Antonio Citro, Valeria Corradetti, Szandra Brambilla, Graziano Oldani, Francesco Calabrese, Tommaso Dominioni, Marcello Maestri, Lorenzo Cobianchi
Whole-organ decellularization is recently gaining interest in the transplantation field as strategy to obtain acellular scaffold only composed by extracellular matrix. These structures, that still remain organ-specific in terms of biological cues and tridimensional morphology could be then recellularized with patient's autologous cells. The final result should be a nwe transplantable autologous organ that should by-pass, at the same time, the problem of organ shortage and secondly the consequences related to the immunosuppression need...
November 21, 2017: Methods in Molecular Biology
Jaana Koistinen, Mervi Sjöblom, Kristian Spilling
Phosphorus (P) is a macronutrient for all microalgal species, and the main form of uptake is orthophosphate (PO4). In this chapter we present a colorimetric method for determining the PO4 concentration and dissolved organic phosphorus (DOP) based on total phosphorus (TP) measurements. We also describe a method for determining particulate organic phosphorus (POP) based on the same principles.
November 21, 2017: Methods in Molecular Biology
Heli Nygren, Tuulikki Seppänen-Laakso, Heiko Rischer
In this paper, an analytical method for the analysis of molecular lipids in algae samples is reported. The sample preparation is based on a modified Folch extraction, and the analysis is carried out with ultrahigh performance liquid chromatography combined with mass spectrometry (UPLC-MS). For further characterization of lipids, MS/MS analyses are carried out utilizing either a separate instrument (e.g., LTQ-Orbitrap mass spectrometer) or simultaneous fragmentation with the same instrument. Throughput of the method is over 100 samples/d...
November 21, 2017: 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
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