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Methods in Enzymology

Arun K Shukla
No abstract text is available yet for this article.
2017: Methods in Enzymology
A Hashiguchi, S Komatsu
Posttranslational modifications (PTMs) of proteins such as phosphorylation and ubiquitination are crucial for controlling protein stability, localization, and conformation. Genetic information encoded in DNA is transcribed, translated, and increases its complexity by multiple PTMs. Conformational change introduced by PTMs affects interacting partners of each proteins and their downstream signaling; therefore, PTMs are the major level of modulations of total outcome of living cells. Plants are living in harsh environment that requires unremitting physiological modulation to survive, and the plant response to various environment stresses is regulated by PTMs of proteins...
2017: Methods in Enzymology
M Miyagi
Acetylation of ɛ-amino group of lysine is one of the most common protein posttranslational modifications. The modification is reversible and catalyzed by lysine acetyltransferases in one direction and lysine deacetylases in the other direction. Although numerous lysine acetylation sites have been identified in many proteins involved in a diverse range of cellular processes, little has been revealed about the roles of this modification at the level of individual sites. To understand better the site-specific roles of this modification, it is important to investigate what fraction of each modified site is actually acetylated (stoichiometry of acetylation) in vivo in different physiological conditions...
2017: Methods in Enzymology
A Tsirigotaki, M Papanastasiou, M B Trelle, T J D Jørgensen, A Economou
Protein folding is an intricate and precise process in living cells. Most exported proteins evade cytoplasmic folding, become targeted to the membrane, and then trafficked into/across membranes. Their targeting and translocation-competent states are nonnatively folded. However, once they reach the appropriate cellular compartment, they can fold to their native states. The nonnative states of preproteins remain structurally poorly characterized since increased disorder, protein sizes, aggregation propensity, and the observation timescale are often limiting factors for typical structural approaches such as X-ray crystallography and NMR...
2017: Methods in Enzymology
J V Arrington, C-C Hsu, W A Tao
Dissection of direct kinase-substrate relationships provides invaluable information about phosphorylation pathways and can highlight both pathogenic mechanisms and possible drug targets for diseases in which abnormal kinase activity is linked to onset and progression. Here, we describe a mass spectrometry-based strategy to define the direct substrates of a kinase of interest. The kinase assay-linked phosphoproteomics approach examines putative kinase substrates both in vitro and in vivo to produce a list of highly confident substrates...
2017: Methods in Enzymology
J Svozil, K Baerenfaller
Mass spectrometry-based proteomics allows in principle the identification of unknown target proteins of posttranslational modifications and the sites of attachment. Including a variety of posttranslational modifications in database-dependent searches of high-throughput mass spectrometry data holds the promise to gain spectrum assignments to modified peptides, thereby increasing the number of assigned spectra, and to identify potentially interesting modification events. However, these potential benefits come for the price of an increased search space, which can lead to reduced scores, increased score thresholds, and erroneous peptide spectrum matches...
2017: Methods in Enzymology
J B Shabb, W W Muhonen, A A Mehus
Metallothioneins (MTs) are a family of small, highly conserved, cysteine-rich metal-binding proteins that are important for zinc and copper homeostasis, protection against oxidative stress, and buffering against toxic heavy metals. Individual human MT isoforms are candidate biomarkers for heavy metal toxicity, and selected cancers and neurodegenerative diseases. The similar antigenicity of human MT-1 and MT-2 isoforms precludes development of antibody-based assays for their individual quantitation. Metal-based MT quantitation methods do not directly measure MT isoforms...
2017: Methods in Enzymology
C L Yu, S Brooks, Y Li, M Subramanian, R Summers, M Pope
Understanding the pathophysiology of genes and enzymes involved in caffeine metabolism can have extracurricular benefits, such as providing distinct methylxanthines as intermediates for pharmaceutical synthesis, and also improve environmental waste remediation. The strains Pseudomonas putida CBB5 and CES may provide insights into these applications because they may both be induced to degrade caffeine, yet the latter thrives in concentrations >8.0gL(-1); threefold higher than any other bacteria. We took a novel approach toward identifying the enzymatic pathways in both Pseudomonas sp...
2017: Methods in Enzymology
I Trbojević-Akmačić, I Ugrina, G Lauc
Large-scale glycomics studies enable identification of aberrant glycosylation patterns in disease and provide information about functional relevance of individual glycans through genome-wide association studies. Developed high-throughput methodologies have to be sensitive, robust, and stable during long periods of time (few months) to be able to reliably detect small biological variations in glycosylation. Here, we describe a simple, robust, and affordable protocol for immunoglobulin G N-glycan analysis by hydrophilic interaction liquid chromatography-ultra-performance liquid chromatography (HILIC-UPLC), as well as useful strategies for method optimization: Plackett-Burman screening design and analysis of source of variation...
2017: Methods in Enzymology
K A Janssen, S Sidoli, B A Garcia
Functional epigenetic regulation occurs by dynamic modification of chromatin, including genetic material (i.e., DNA methylation), histone proteins, and other nuclear proteins. Due to the highly complex nature of the histone code, mass spectrometry (MS) has become the leading technique in identification of single and combinatorial histone modifications. MS has now overcome antibody-based strategies due to its automation, high resolution, and accurate quantitation. Moreover, multiple approaches to analysis have been developed for global quantitation of posttranslational modifications (PTMs), including large-scale characterization of modification coexistence (middle-down and top-down proteomics), which is not currently possible with any other biochemical strategy...
2017: Methods in Enzymology
J Mehla, J H Caufield, N Sakhawalkar, P Uetz
Two-hybrid systems are one of the most popular, preferred, cost effective, and scalable in vivo genetic approaches for screening protein-protein interactions. A number of variants of yeast and bacterial two-hybrid systems exist, rendering them ideal for modern, flexible proteomics-driven studies. For mapping protein interactions at genome scales (that is, constructing an interactome), the yeast two-hybrid system has been extensively tested and is preferred over bacterial two-hybrid systems, given that users have created more resources such as a variety of vectors and other modifications...
2017: Methods in Enzymology
R Noberini, T Bonaldi
Histone posttranslational modifications (hPTMs) generate a complex combinatorial code that plays a critical role in the regulation of gene activity and nuclear architecture during physiological and pathological processes. Mass spectrometry (MS) offers an unbiased, comprehensive, and quantitative view on hPTM patterns, and has emerged as a powerful tool in epigenetic research. Stable isotope labeling by amino acid in cell culture (SILAC) is a MS-based quantitative method that relies on the metabolic labeling of cell populations, which has been widely applied in global proteomic studies and can also be exploited for the accurate quantitation of hPTM changes among distinct functional states...
2017: Methods in Enzymology
J Wang, Y K Wong, J Zhang, Y-M Lee, Z-C Hua, H-M Shen, Q Lin
Identifying the cellular binding targets of drugs and other bioactive small molecules is a crucial step for understanding their molecular mechanisms of action as well as potential off-target effects. The field of chemical proteomics is an emerging discipline in chemical biology using synthetic chemistry and high-throughput detection techniques to study small molecule-protein interactions. In this chapter, we describe a quantitative chemical proteomics protocol combining bioorthogonal click chemistry and quantitation by isobaric tags for relative and absolute quantification (iTRAQ) to identify the specific binding targets of drugs and bioactive small molecules such as natural products...
2017: Methods in Enzymology
A G Nuccio, M Bui, Y Dalal, A Nita-Lazar
Histone posttranslational modifications (PTMs) are key epigenetic marks involved in gene silencing or activation. Histone modifications impact chromatin organization and transcriptional processes through the changes in charge density between histones and DNA. They also serve as recognition and binding sites for specific binding proteins. Histone tails and globular cores contain many basic amino acid residues, which are subject to various dynamic modifications, making the modification repertoire extremely diverse...
2017: Methods in Enzymology
C S Ang, M S Baker, E C Nice
Colorectal cancer (CRC) is the third leading cause of cancer mortality for both men and women, and the second leading cause of cancer death for men and women combined. If detected early, before metastasis has occurred, survival following surgical resection of the tumor is >90%. Early detection is therefore critical for effective disease surveillance. Unfortunately, current biomarker assays lack the necessary sensitivity and specificity for reliable early disease detection. Development of new robust, non- or minimally invasive specific and sensitive biomarkers or panels with improved compliance and performance is therefore urgently required...
2017: Methods in Enzymology
Y Yakkioui, Y Temel, E Chevet, L Negroni
Quantitative proteomics represents a powerful approach for the comprehensive analysis of proteins expressed under defined conditions. These properties have been used to investigate the proteome of disease states, including cancer. It has become a major subject of studies to apply proteomics for biomarker and therapeutic target identification. In the last decades, technical advances in mass spectrometry have increased the capacity of protein identification and quantification. Moreover, the analysis of posttranslational modification (PTM), especially phosphorylation, has allowed large-scale identification of biological mechanisms...
2017: Methods in Enzymology
J Armengaud, C Duport
Pathogens are known to release in their environment a large range of toxins and other virulence factors. Their pathogenicity relies on this arsenal of exoproteins and their orchestrated release upon changing environmental conditions. Exoproteomics aims at describing and quantifying the proteins found outside of the cells, thus takes advantage of the most recent methodologies of next-generation proteomics. This approach has been applied with great success to a variety of pathogens increasing the fundamental knowledge on pathogenicity...
2017: Methods in Enzymology
X Zhao, Y Xie, J Liu
Exosomes are small membrane vesicles that are produced by cells and excreted into extracellular space. Contents of exosomes generally include lipid, membrane, and soluble proteins, and various types of coding and noncoding RNAs. Over the past decades, it has become clear that exosomes constitute an important vector for intercellular transport and communication with significant functional relevance. Evaluating exosome contents and their changes are vital for understanding its role in different physiological and pathological processes...
2017: Methods in Enzymology
C Zhang, Y Liu
Posttranslational modifications (PTMs) of histones are one of the main research interests in the rapidly growing field of epigenetics. Accurate and precise quantification of these highly complex histone PTMs is critical for understanding the histone code and the biological significance behind it. It nonetheless remains a major analytical challenge. Mass spectrometry (MS) has been proven as a robust tool in retrieving quantitative information of histone PTMs, and a variety of MS-based quantitative strategies have been successfully developed and employed in basic research as well as clinical studies...
2017: Methods in Enzymology
K C Tsolis, A Economou
Due to their physicochemical properties, membrane protein proteomics analyses often require extensive sample preparation protocols resulting in sample loss and introducing technical variation. Several methods for membrane proteomics have been described, designed to meet the needs of specific sample types and experimental designs. Here, we present a complete membrane proteomics pipeline starting from the membrane sample preparation to the protein identification/quantification and also discuss about annotation of proteomics data...
2017: Methods in Enzymology
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