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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.
The Precision Medicine Approach in Oncology
Published in David E. Thurston, Ilona Pysz, Chemistry and Pharmacology of Anticancer Drugs, 2021
MALDI-MSI (Matrix-Assisted Laser Desorption Ionization MS Imaging) and LCM-MS (Laser Capture Microdissection MS) are two new MS-based technologies that are being developed for use in the proteomics area. These techniques, which are still experimental, attempt to use mass spectrometry to image proteins and peptides in cells and tissues. Gel-free isotopic labeling methods such as SILAC (Stable Isotope Labeling with Amino Acids in Cell Culture), iTRAQ (Isobaric Tags for Relative and Absolute Quantitation) and I-TMTs (Isobaric Tandem Mass Tags) are currently capable of quantifying up to thousands of proteins in a single analysis with high reproducibility.
Emerging Biomedical Analysis
Published in Lawrence S. Chan, William C. Tang, Engineering-Medicine, 2019
DDA and DIA acquisitions are widely used in bottom-up proteomics for both qualitative and quantitative purposes. Peptides are identified by comparing the experimental tandem mass spectra with theoretical tandem mass spectra generated from in silico fragmentation or a spectral library collected in previous experiments. Without any derivatizations, proteins can be quantitatively compared using precursor ion intensities, peak areas, spectral counts or the transition ion intensities (SRM/MRM only) of peptides. Several labeling techniques including stable isotopic labeling of amino acids in cell culture (SILAC), 18O, demethylation, isobaric tags for relative and absolute quantitation (iTRAQ) and tandem mass tags (TMT) are also available for relative and absolute quantitative analysis of peptides and proteins (Zhang et al. 2013).
Identification of Rab7 as an autophagy marker: potential therapeutic approaches and the effect of Qi Teng Xiao Zhuo granule in chronic glomerulonephritis
Published in Pharmaceutical Biology, 2023
Xiujuan Qin, Huiyu Chen, Xiaoli Zhu, Xianjin Xu, Jiarong Gao
For extraction of proteins, renal cortex tissues were homogenized with SDT buffer containing a protease inhibitor cocktail (150 mM Tris-HCl, pH 8.0, 4% SDS, 100 mM dithiothreitol). The homogenate was then boiled for 5 min and centrifuged at 40,000 g for 30 min. The supernatant was collected to measure protein concentrations by a bicinchoninic acid assay (BCA, Pierce, USA). For protein digestion, trypsin solution with triethylamine borane (150 µL, 50 mM) was added to the proteins at 1:50 (M:M) overnight at 37 °C. Peptides from each sample were labeled by tandem mass tags. Peptides were fractionated using the Agilent 1200 LC system (Agilent Technologies, Santa Clara, CA, USA). They were reconstituted with buffer A (20 mM ammonium acetate in H2O, pH 10) and loaded onto a C18 column. Elution was performed via 5-35% buffer B (20 mM ammonium acetate, 90% ACN/H2O, pH 10) for 35 min, 35-90% buffer B for 5 min, and 90% buffer B for 5 min. Elution was monitored by measuring the absorbance at 214 nm, and fractions were collected every min. The eluted peptides were pooled into 10 fractions and vacuum dried. A Q Exactive Mass Spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) was run in positive ion mode in a data-dependent manner: full MS scan from 350 to 2000 m/z; full scan resolution at 70,000; MS/MS scan resolution at 17,500; normalized collision energy 28% and stepped 20%.
New advances in quantitative proteomics research and current applications in asthma
Published in Expert Review of Proteomics, 2021
Yanting Lan, Xiaoyin Zeng, Jing Xiao, Longbo Hu, Long Tan, Mengdi Liang, Xufei Wang, Shaohua Lu, Fei Long, Tao Peng
Tandem mass tag (TMT) is one of the isobaric labeling used for relative quantification in LC-MS/MS analysis. The general structure of a TMT consists of an amine reactive group, a mass-normalized group, and a reporter ion group [32]. The overall mass of the amine reactive group is the same for each tag, but the distribution of heavy isotopes between the reporter ion group and the mass normalization group is different. Peptides labeled with different TMT tags have the same molecular weight and mass-to-charge ratio. Upon high-energy collisions, these peptides are dissociated and produce characteristic reporter ions that provide relative quantitative information (Figure 2c). Thompson et al. first used the synthetic TMT-tagged peptides for LC-MS/MS analysis in 2003. They proposed that quantification based on MS2 spectra greatly improved the signal-to-noise ratio compared to MS1 spectra [36].
TMT-Based proteomics analysis of LPS-induced acute lung injury
Published in Experimental Lung Research, 2021
Shengsong Chen, Yi Zhang, Qingyuan Zhan
Tandem mass tag (TMT) for relative quantification combined with liquid chromatography/tandem mass spectrometry (LC-MS/MS) is widely used in qualitative and quantitative proteomics research of a variety of disease markers.5–7 TMT technology has been widely used in the medical research field by virtue of its advantages; this method is high throughput, shows good repeatability, enables accurate quantification at a high resolution, provides a wealth of data and involves a high degree of automation.8 In this study, TMT technology combined with LC-MS/MS was used to identify proteins involved in LPS-induced ALI, and the functions of the differentially expressed proteins (DEPs) were preliminarily explored by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. Moreover, parallel reaction monitoring (PRM) was further used to identify key protein targets for ALI/ARDS.