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Integrated Omics Technology for Basic and Clinical Research
Published in Jyoti Ranjan Rout, Rout George Kerry, Abinash Dutta, Biotechnological Advances for Microbiology, Molecular Biology, and Nanotechnology, 2022
Kuldeep Giri, Vinod Singh Bisht, Sudipa Maity, Kiran Ambatipudi
In today’s date, widely used methodologies to characterize peptides/ proteins are bottom-up and top-down approaches. In the bottom-up approach, which is more specific and sensitive, includes liquid chromatographic-based separation of peptides derived from enzymatic digestion (i.e., trypsin) of selected protein and their subsequent identification through MS. However, posttranslation modification such as glycosylation and phosphorylation is identified by performing a top-down approach that involves intact protein identification through the MS (Barbosa et al., 2012). Similarly, for labeled quantification of proteins, Isotope Coded Affinity Tag, is a sensitive technique used for relative quantitative analysis by tagging cysteine residues. Similarly, isobaric tags for relative and absolute quantification (iTRAQ) in protein extract and stable isotope labeling with amino acid in cell culture (SILAC) is used for quantitative comparison of experimentally tagged proteins to discriminate their abundances by MS in different samples (healthy vs. disease state) (Ong et al., 2002; Ross et al., 2004). High throughput MS technique such as MudPIT which is a two dimensional chromatography technique, using high-performance liquid chromatography (incorporate strong cation exchange and reversed-phase columns) coupled with tandem MS identification and quantifies protein from a complex mixture and determination of protein-protein interaction (Washburn et al., 2001). The label-free and labeled (SILAC and iTRAQ) approach for proteomes’ quantitation and subsequent analysis is represented in Figure 14.8.
Omics to address the opportunities and challenges of nanotechnology in agriculture
Published in Critical Reviews in Environmental Science and Technology, 2021
Sanghamitra Majumdar, Arturo A. Keller
Electrospray ionization (ESI) and Matrix-Assisted Laser Desorption Ionization (MALDI) are the most common platforms used to ionize peptides and proteins. The precise molecular mass of the resulting ions is then analyzed using mass analyzers, such as ion trap, quadrupole, Orbitrap, time-of-flight (TOF), and Fourier transform ion cyclotron resonance (FTICR), which are often used in tandem to achieve higher degrees of ion separation (Lai et al., 2012). In untargeted proteomics using LC-MS/MS, data dependent acquisition is performed, where the highest abundance peptide ions from full MS scans are selected for MS/MS. However, this may generate datasets skewed toward the identification of relatively high abundance proteins, thereby masking and excluding the low abundance proteins from quantification (Hart-Smith et al., 2017). Several labeling techniques such as isobaric Tags for Relative and Absolute Quantitation (iTRAQ) and Stable Isotope Labeling by Amino acids in Cell culture (SILAC) are also available which can help to reduce errors introduced during measurement conditions. However, untargeted proteomic analysis requires extensive data processing and is currently challenged by incomplete and limited nature of plant genomic and proteomic databases. In recent times, the use of label-free shotgun proteomic techniques have become increasingly popular, as they do not restrict the number of proteins identified compared to gel-based methods (Majumdar et al., 2015, 2019; Mirzajani et al., 2014; Vannini et al., 2014; Verano-Braga et al., 2014). However, gel-free methods have several drawbacks, such as masking of low abundant peptides and unavailability of protein database for all species.