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Biochemical Markers in Ophthalmology
Published in Ching-Yu Cheng, Tien Yin Wong, Ophthalmic Epidemiology, 2022
Abdus Samad Ansari, Pirro G. Hysi
High-throughput methods include protein microarrays [145]. This process involves the application of small amounts of sample to a “chip” for analysis. Antibodies are subsequently fixated to the chip surface and used to capture target proteins in a complex model. This process is often referred to as analytical protein microarray [145]. Functional microarrays allow for the characterization of protein functions, including enzyme substrate turnover and protein–RNA interactions [146]. Reverse-phase protein microarray involves the process of using both healthy and diseased tissue bound to a chip, which is subsequently probed with antibodies against target proteins. MS-based proteomics is also a form of complex gel-free methods of separating proteins. This includes isotope-coded affinity tag, stable isotope labeling with amino acids in culture, and isobaric tags [147].
Understanding the Proteomics of Medicinal Plants under Environmental Pollution
Published in Azamal Husen, Environmental Pollution and Medicinal Plants, 2022
Pooja Singh, V.K. Mishra, Rohit Kashyap, Rahul Rawat
Two-dimensional gel electrophoresis is now a mature and well-established technique; however, it suffers from some ongoing concerns regarding quantitative reproducibility and limitations on the ability to study certain classes of proteins (Abdallah et al. 2012). Alternatives to 2-DE gel-based quantification of proteins are gel-free MS-based proteomics. Currently, label-based proteomic approaches, such as isotope-coded affinity tags (ICAT), isotope-coded protein labelling (ICPL) isobaric tags for relative and absolute quantification (iTRAQ), tandem mass tag (TMT), stable isotopic labelling with amino acids in cell culture (SILAC), and label-free LC/MS represent attractive alternatives. The workflow in proteomics is depicted in Figure 12.3.
Omics Technology: Novel Approach for Screening of Plant-Based Traditional Medicines
Published in Megh R. Goyal, Hafiz Ansar Rasul Suleria, Ademola Olabode Ayeleso, T. Jesse Joel, Sujogya Kumar Panda, The Therapeutic Properties of Medicinal Plants, 2019
Rojita Mishra, Satpal Singh Bisht, Mahendra Rana
Proteomics mainly describes about the function, interaction, modification, and targeting as well as regulation of proteome expressed by the cell [20]. The simplest way of analyzing protein expression, function localization is by 2D-gel electrophoresis; subsequently, the cellular fractions of the gel are studied using fluorescent dyes and compared with wild type homologs and analogs for comparative study regarding expression level and post-transcriptional modifications [21–23]. Proteomics includes isolation of sub proteomes based on the cellular localization of isoelectric point [23]; mass spectrometric techniques like isotope-coded affinity tags (ICAT) [24] and multidimensional protein identification technology (MudPIT) [25]. Protein function and interactions are being addressed on a genome-wide scale through the development of protein arrays and protein interaction maps [26].
Advances in phosphoproteomics and its application to COPD
Published in Expert Review of Proteomics, 2022
Xiaoyin Zeng, Yanting Lan, Jing Xiao, Longbo Hu, Long Tan, Mengdi Liang, Xufei Wang, Shaohua Lu, Tao Peng, Fei Long
Stable isotope labeling using amino acids (SILAC) is a metabolic labeling method that involves binding isotopes to samples for cell culture, relative quantification by comparing isotope peak shape area size in primary mass spectra, and identifying peptides in secondary mass spectrometry maps. Zanivan et al. [97] used the SILAC technique in a mouse model to study phosphorylated proteins during skin cancer development. Stepath et al. [98] compared SILAC, TMT, and label-free quantification of phosphorylation sites in the epidermal growth factor receptor (EGFR) signaling pathway in colorectal cancer. They found that SILAC was more accurate than TMT and nonlabeled quantification for the quantification of phosphorylation sites. In addition to SILAC, ICAT and 18O isotope labeling were also quantified based on primary spectrograms. Isotope-coded affinity tag (ICAT) is a chemical labeling method, and ICAT is a chemical reagent labeled with an isotopically encoded chemical reagent on specific amino acid residues of a peptide segment chemical reaction for quantification [99]. In contrast, 18O isotope labeling belongs to enzymatic reaction labeling, quantified by adding 18O isotope labels to the enzymatic reaction [100]. However, these two methods are not widely used at present.
Could a blood test for PTSD and depression be on the horizon?
Published in Expert Review of Proteomics, 2018
Another valuable proteomic method, which allows to simultaneously measuring multiple proteins in a small volume of sample, is the Luminex bead-based approach. This technique has been successfully used for biomarker discovery and is more common in several clinical studies such as in studies of autoimmune disorders [262], or Alzheimer’s disease [263]. Several other techniques involve the use of stable isotopes to label peptides. The most widely known is the Isotope Coded Affinity Tags, which permits to distinguish between different protein in one sample based on their isotopic composition, separating the peptide fragments of interest and quantifying them by MS [264]. By labeling only cysteine residues, this approach only analyzes proteins containing this amino-acid residue, hence excluding many other relevant proteins.
Protein amino-termini and how to identify them
Published in Expert Review of Proteomics, 2020
Annelies Bogaert, Kris Gevaert
To correct for the slow scan speed of mass spectrometers used at the time, in the late 1990s, scientists introduced novel proteomic methods. Pioneers were the Yates and Aebersold labs, with the former implementing two consecutive and orthogonal stages of peptide separation (based on peptide charge and peptide hydrophobicity) prior to analysis [25] and the latter enriching for cysteine-containing peptides [26], by which both methods decomplexed the peptide mixture prior to analysis, thus increasing the overall proteome coverage. Instead of introducing an affinity tag on specific amino acid side-chains as is the case for Aebersold’s isotope-coded affinity tags to modify cysteines [26], our lab explored the concept of diagonal chromatography for enriching specific classes of peptides. In essence, peptide diagonal chromatography involves two consecutive, identical chromatographic separations of peptides, interrupted by a chemical or enzymatic reaction that targets a functionality present in a selected class of peptides. Methionine-containing peptides were first on the radar as the methionine side-chain can be readily and quite specifically oxidized by hydrogen peroxide to introduce a sulfoxide. This renders methionine-containing peptides less hydrophobic, thus resulting in a hydrophilic shift during reverse-phase chromatography, based on which such peptides were collected for further analysis. Further, as the extent of this hydrophilic shift can be measured for individual synthetic peptides containing methionine, following the first peptide separation, several fractions could be combined, explaining the COFRADIC acronym as combined fractional diagonal chromatography [27].