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Current Protocols in Protein Science Feb 2019Stable isotope labeling by amino acids in cell culture (SILAC) has become very popular as a quantitative proteomic method since it was firstly introduced by Matthias... (Review)
Review
Stable isotope labeling by amino acids in cell culture (SILAC) has become very popular as a quantitative proteomic method since it was firstly introduced by Matthias Mann's group in 2002. It is a metabolic labeling strategy in which isotope-labeled amino acids are metabolically incorporated in vivo into proteins during translation. After natural (light) or heavy amino acid incorporation, differentially labeled samples are mixed immediately after cell lysis and before any further processing, which minimizes quantitative errors caused by handling different samples in parallel. In this unit, we describe protocols for basic duplex SILAC, triplex SILAC for use in nondividing cells such as neurons, and for measuring amounts of newly synthesized proteins. © 2018 by John Wiley & Sons, Inc.
Topics: Animals; Humans; Isotope Labeling; Nerve Tissue Proteins; Neurons; Proteome; Proteomics
PubMed: 30238645
DOI: 10.1002/cpps.74 -
Proteomics Feb 2015The highly complex and species-selective mechanism of fertilization is a central theme of developmental biology. Gametogenesis, sperm activation, and egg-sperm... (Review)
Review
The highly complex and species-selective mechanism of fertilization is a central theme of developmental biology. Gametogenesis, sperm activation, and egg-sperm recognition are fundamental biological processes, warranting detailed studies into the molecular composition of gametes. Biological MS has been instrumental for the comprehensive itemizing of gamete proteomes. The protein constellation of sperm cells and its subcellular structures has been established for a variety of animal species. Spermatogenesis and the crucial activation of sperm cells as a prerequisite of successful fertilization and physiological adaptations to external stressors was investigated using proteomics, as well as the underlying mechanisms of male infertility with respect to proteome-wide alterations. This review outlines recent achievements of sperm proteomics and exemplifies the usefulness of gel-based surveys by outlining the comparative analysis of abnormal spermatozoa in globozoospermia. Besides label-free MS techniques and cell-based labeling methodology, high-resolution fluorescence 2DE has been shown to be highly suitable as a proteomic biomarker discovery tool in sperm protein research. The appropriateness of novel protein markers for improving our understanding of normal spermatogenesis and sperm activation versus the molecular pathogenesis of male infertility will be discussed. New biomarker candidates might be useful to improve diagnostic, prognostic, and therapeutic aspects of infertility.
Topics: Animals; Humans; Male; Proteome; Proteomics; Spermatogenesis; Spermatozoa
PubMed: 24909132
DOI: 10.1002/pmic.201400032 -
Nucleic Acids Research Jan 2022Proteome-pI 2.0 is an update of an online database containing predicted isoelectric points and pKa dissociation constants of proteins and peptides. The isoelectric...
Proteome-pI 2.0 is an update of an online database containing predicted isoelectric points and pKa dissociation constants of proteins and peptides. The isoelectric point-the pH at which a particular molecule carries no net electrical charge-is an important parameter for many analytical biochemistry and proteomics techniques. Additionally, it can be obtained directly from the pKa values of individual charged residues of the protein. The Proteome-pI 2.0 database includes data for over 61 million protein sequences from 20 115 proteomes (three to four times more than the previous release). The isoelectric point for proteins is predicted by 21 methods, whereas pKa values are inferred by one method. To facilitate bottom-up proteomics analysis, individual proteomes were digested in silico with the five most commonly used proteases (trypsin, chymotrypsin, trypsin + LysC, LysN, ArgC), and the peptides' isoelectric point and molecular weights were calculated. The database enables the retrieval of virtual 2D-PAGE plots and customized fractions of a proteome based on the isoelectric point and molecular weight. In addition, isoelectric points for proteins in NCBI non-redundant (nr), UniProt, SwissProt, and Protein Data Bank are available in both CSV and FASTA formats. The database can be accessed at http://isoelectricpointdb2.org.
Topics: Amino Acid Sequence; Computational Biology; Databases, Protein; Electrophoresis, Gel, Two-Dimensional; Isoelectric Point; Molecular Weight; Peptides; Proteome; Proteomics
PubMed: 34718696
DOI: 10.1093/nar/gkab944 -
Proteomics Feb 2015There is increasing realisation that human health status in adulthood depends critically upon environmental conditions pertaining around the time of conception and... (Review)
Review
There is increasing realisation that human health status in adulthood depends critically upon environmental conditions pertaining around the time of conception and during pregnancy. Poor maternal diet or adverse environmental conditions around the periconception period somehow induces the resultant embryo to adapt predictively in order to survive this level of stress for the whole of its life. However, if there is a mismatch between expectation and reality, where the conditions during later life are better than expected, things go wrong and the adult suffers a range of illnesses, including diabetes, heart disease, hypertension and stroke. Understanding the molecular signals that direct the early embryo to adopt appropriate adaptations to suit its future life would be extremely valuable. However, although it appears to be an ideal task for proteomic applications, there are technical, ethical and practical limitations to what can be achieved with the current framework of proteomic technology. Here, we review what has been achieved to date, explain some of the experimental problems and suggest some strategies for taking this field forward.
Topics: Animals; Female; Fertilization; Humans; Mice; Pregnancy; Proteome; Proteomics
PubMed: 25404351
DOI: 10.1002/pmic.201400362 -
Advances in Experimental Medicine and... 2015As noninvasive and easily available biological fluid, urine is becoming an ideal sample for proteomic study. In recent years, researchers endeavored in profiling urinary... (Review)
Review
As noninvasive and easily available biological fluid, urine is becoming an ideal sample for proteomic study. In recent years, researchers endeavored in profiling urinary proteome and discovering potential disease biomarkers. However, there are still many challenges in the studies of urinary proteome for the complexity of urine. In this article, we review current status of urinary sample preparation, including collection, storage, and extraction of urinary proteins, and the overall urinary proteome analysis so far, which may be helpful for urinary proteome analysis.
Topics: Biomarkers; Female; Humans; Male; Mass Spectrometry; Proteome; Proteomics; Specimen Handling
PubMed: 25355567
DOI: 10.1007/978-94-017-9523-4_4 -
Proteomics Jan 2016Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells that are defined by their myeloid origin, immature state, and ability to potently... (Review)
Review
Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells that are defined by their myeloid origin, immature state, and ability to potently suppress T-cell responses. They regulate immune responses and the population significantly increases in the tumor microenvironment of patients with glioma and other malignant tumors. For their study, MDSCs are usually isolated from the spleen or directly of tumors from a large number of tumor-bearing mice although promising ex vivo differentiated MDSC production systems have been recently developed. During the last years, proteomics has emerged as a powerful approach to analyze MDSCs proteomes using shotgun-based mass spectrometry (MS), providing functional information about cellular homeostasis and metabolic state at a global level. Here, we will revise recent proteome profiling studies performed in MDSCs from different origins. Moreover, we will perform an integrative functional analysis of the protein compilation derived from these large-scale proteomic studies in order to obtain a comprehensive view of MDSCs biology. Finally, we will also discuss the potential application of high-throughput proteomic approaches to study global proteome dynamics and post-translational modifications (PTMs) during the differentiation process of MDSCs that will greatly boost the identification of novel MDSC-specific therapeutic targets to apply in cancer immunotherapy.
Topics: Animals; Humans; Immunotherapy; Myeloid Cells; Neoplasms; Proteome; Proteomics; Tumor Microenvironment
PubMed: 26403437
DOI: 10.1002/pmic.201500229 -
Briefings in Bioinformatics Jan 2021Empowered by the advancement of high-throughput bio technologies, recent research on body-fluid proteomes has led to the discoveries of numerous novel disease biomarkers... (Review)
Review
Empowered by the advancement of high-throughput bio technologies, recent research on body-fluid proteomes has led to the discoveries of numerous novel disease biomarkers and therapeutic drugs. In the meantime, a tremendous progress in disclosing the body-fluid proteomes was made, resulting in a collection of over 15 000 different proteins detected in major human body fluids. However, common challenges remain with current proteomics technologies about how to effectively handle the large variety of protein modifications in those fluids. To this end, computational effort utilizing statistical and machine-learning approaches has shown early successes in identifying biomarker proteins in specific human diseases. In this article, we first summarized the experimental progresses using a combination of conventional and high-throughput technologies, along with the major discoveries, and focused on current research status of 16 types of body-fluid proteins. Next, the emerging computational work on protein prediction based on support vector machine, ranking algorithm, and protein-protein interaction network were also surveyed, followed by algorithm and application discussion. At last, we discuss additional critical concerns about these topics and close the review by providing future perspectives especially toward the realization of clinical disease biomarker discovery.
Topics: Biomarkers; Body Fluids; Humans; Proteome; Proteomics
PubMed: 32020158
DOI: 10.1093/bib/bbz160 -
Journal of Proteome Research Mar 2023Accurate protein quantification is key to identifying protein markers, regulatory relationships between proteins, and pathophysiological mechanisms. Realizing this... (Review)
Review
Accurate protein quantification is key to identifying protein markers, regulatory relationships between proteins, and pathophysiological mechanisms. Realizing this potential requires sensitive and deep protein analysis of a large number of samples. Toward this goal, proteomics throughput can be increased by parallelizing the analysis of both precursors and samples using multiplexed data independent acquisition (DIA) implemented by the plexDIA framework: https://plexDIA.slavovlab.net. Here we demonstrate the improved precisions of retention time estimates within plexDIA and how this enables more accurate protein quantification. plexDIA has demonstrated multiplicative gains in throughput, and these gains may be substantially amplified by improving the multiplexing reagents, data acquisition, and interpretation. We discuss future directions for advancing plexDIA, which include engineering optimized mass-tags for high-plexDIA, introducing isotopologous carriers, and developing algorithms that utilize the regular structures of plexDIA data to improve sensitivity, proteome coverage, and quantitative accuracy. These advances in plexDIA will increase the throughput of functional proteomic assays, including quantifying protein conformations, turnover dynamics, modifications states and activities. The sensitivity of these assays will extend to single-cell analysis, thus enabling functional single-cell protein analysis.
Topics: Mass Spectrometry; Proteomics; Algorithms; Proteome
PubMed: 36735898
DOI: 10.1021/acs.jproteome.2c00721 -
Journal of Proteome Research Jan 2021Protein -acylation (commonly known as palmitoylation) is a widespread reversible lipid modification, which plays critical roles in regulating protein localization,... (Review)
Review
Protein -acylation (commonly known as palmitoylation) is a widespread reversible lipid modification, which plays critical roles in regulating protein localization, activity, stability, and complex formation. The deregulation of protein -acylation contributes to many diseases such as cancer and neurodegenerative disorders. The past decade has witnessed substantial progress in proteomic analysis of protein -acylation, which significantly advanced our understanding of -acylation biology. In this review, we summarized the techniques for the enrichment of -acylated proteins or peptides, critically reviewed proteomic studies of protein -acylation at eight different levels, and proposed major challenges for the -acylproteomics field. In summary, proteome-scale analysis of protein -acylation comes of age and will play increasingly important roles in discovering new disease mechanisms, biomarkers, and therapeutic targets.
Topics: Acylation; Lipoylation; Protein S; Proteome; Proteomics
PubMed: 33253586
DOI: 10.1021/acs.jproteome.0c00409 -
Current Opinion in Biotechnology Apr 2016Environmental biotechnology relies on the exploration of novel biological systems and a thorough understanding of the underlying molecular mechanisms. Next-generation... (Review)
Review
Environmental biotechnology relies on the exploration of novel biological systems and a thorough understanding of the underlying molecular mechanisms. Next-generation proteomics based on the latest generation of mass analyzers currently allows the recording of complete proteomes from any microorganism. Interpreting these data can be straightforward if the genome of the organism is established, or relatively easy to perform through proteogenomics approaches if a draft sequence can be obtained. However, next-generation proteomics faces new, interesting challenges when the organism is distantly related to previously characterized organisms or when mixtures of organisms have to be analyzed. New mass spectrometers and innovative bioinformatics tools are reshaping the possibilities of homology-based proteomics, proteogenomics, and metaproteomics for the characterization of biological systems. Novel time- and cost-effective screening strategies are also possible with this methodology, as exemplified by whole proteome thermal profiling and subpopulation proteomics. The complexity of environmental samples allows for unique developments of approaches and concepts.
Topics: Animals; Biotechnology; Genome; Proteome; Proteomics
PubMed: 26950175
DOI: 10.1016/j.copbio.2016.02.025