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The Precision Medicine Approach in Oncology
Published in David E. Thurston, Ilona Pysz, Chemistry and Pharmacology of Anticancer Drugs, 2021
A second group of techniques referred to as “shotgun proteomics” is based on liquid chromatography combined with tandem mass spectrometry (LC-MS-MS), such as ESI-LC-MS (Electrospray Ionization Liquid Chromatography Tandem Mass Spectroscopy). Peptides from trypsin-digestion are separated by LC and then fragmented on-line into amino acid patterns by MS-MS. A prerequisite for the successful application of this method is a reduction in complexity of the protein content (i.e., a “clean-up” process) in the biological matrices being studied. This may be achieved either by coupling different chromatographic separations upfront or by selectively enriching a subset of specific peptides through chemical reactions.
Detection of Food Allergen Residues by Immunoassays and Mass Spectrometry
Published in Andreas L. Lopata, Food Allergy, 2017
Sridevi Muralidharan, Yiqing Zhao, Steve L. Taylor, Nanju A. Lee
The shotgun proteomics workflow is suited for discovery studies and typically adopts the bottom-up approach, where proteins are pre-fractionated, subjected to proteolytic digestion, followed by nanoflow-liquid chromatography- tandem mass spectrometry (nLC-MS/MS). This workflow is becoming ideal for identifying proteins and characterizing post translational modifications. Alternatively, top-down approach is more suited for characterizing intact proteins typically analysed by high resolution mass analysers, followed by MS/MS based on collision induced dissociation of ions. Gene ontology annotations and analysis have been possible from using databases such as UniProt, Interpro, KEGG, and new tools such as WEGO (Ye et al. 2006), MaxQuant (Cox and Mann 2008), STRAP (Bhatia et al. 2009), DAVID or MAPMAN. This approach is predominantly used for biomarker discovery across different areas of research but has recently been successfully demonstrated for discovery of potential allergens in uncharacterised novel allergenic foods (in house unpublished). As an alternative, data acquired using data-independent acquisition mode based on simultaneous fragmentation activation of all ions (all co-eluting peptides) could exponentially enhance the depth of data available for analysis, and has the potential for multiple retrospective analyses from a single experiment. This approach has been exploited in certain other areas of research, however it requires the generation of high quality spectral libraries for peptide identification and quantification and its application in food allergen research is still at infancy.
“Omics” Technologies in Vaccine Research
Published in Mesut Karahan, Synthetic Peptide Vaccine Models, 2021
Proteomics is the high-throughput analysis of the complete set of proteins of an organism, a cell, or the part of a cell. In this approach, the protein sample is isolated from the cells or the specific subcellular compartment according to the intended type of proteome such as secretome or surfome. Following the protein isolation, gel-based or gel-free techniques are used for the identification of each protein in the sample. In gel-based approaches, the proteins are separated by either conventional one-dimensional gel electrophoresis (1-DE) or two-dimensional gel electrophoresis (2-DE) for a higher resolution. In 2-DE, the proteins are separated according to their isoelectric point (pI) values in the first dimension, called isoelectric focusing (IEF). In the second dimension, proteins on the IEF strips are separated according to their molecular weight (MW) using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and following the staining of the gel using a dye such as Coomassie brilliant blue, the proteins are visualized as fine spots (Figure 16.3). The protein samples can be labeled prior to 2-DE using spectrally resolvable fluorophores for given masses and charges comigrating with proteins on 2D-gel, a technique called 2D difference in-gel electrophoresis (2D-DIGE). Desired bands in 1-DE or spots in 2-DE are isolated from the gel, and mass spectrometric techniques such as matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) and tandem mass spectrometry (MS/MS) are used for the peptide identification. Gel-based techniques are well-established and used widely. However, they have some limitations such as application problems for certain classes of proteins, and quantitative reproducibility. Gel-free approaches are used to overcome these problems. In shotgun proteomics, proteins are digested using proteases to obtain complex peptide fractions, and they are resolved via different high-throughput fractionation approaches such as ion-exchange chromatography, reversed-phase chromatography, 2D liquid chromatography, or OFFGEL electrophoresis (Kaushik and Sehgal 2008; Walters and Mobley 2010; Scherp et al. 2010; Abdallah et al. 2012; Baiwir et al. 2018).
LC-MS/MS: A sensitive and selective analytical technique to detect COVID-19 protein biomarkers in the early disease stage
Published in Expert Review of Proteomics, 2023
Siva Nageswara Rao Gajula, Ankita Sahebrao Khairnar, Pallavi Jock, Nikita Kumari, Kendre Pratima, Vijay Munjal, Pavan Kalan, Rajesh Sonti
Untargeted biochemical profiling is the preliminary step in identifying biomarkers and comprehensive analysis of all the measurable analytes in the sample, including unknown chemicals; this offers novel target discovery. In contrast, targeted profiling measures defined chemically characterized and biochemically annotated metabolites [46,47]. Shotgun proteomics using mass spectrometry is an effective method for identifying protein biomarkers to study the global abundance of protein changes in response to biological perturbations. However, sample enrichment and stronger separation techniques are often required to increase the sensitivity of identification and the accuracy of quantifying low-abundant proteins. This shotgun proteomics using mass spectrometry method has two quantification strategies for differentiating proteins in biological systems, such as stable isotope labeling and label-free methods. Shotgun proteomics-compatible isotope-labeled techniques offer flexible tools to examine protein changes in complex biological matrices with good accuracy and precision. Label-free quantification approaches are simple, and cost-effective displaying good reproducibility with linearity for both peptide and protein quantifications. It should be noted that not all the LC MS/MS based methods are equally accurate or reproducible, and that this depends on both the method and the standards used [48].
Peptidomics and proteogenomics: background, challenges and future needs
Published in Expert Review of Proteomics, 2021
Rui Vitorino, Manisha Choudhury, Sofia Guedes, Rita Ferreira, Visith Thongboonkerd, Lakshya Sharma, Francisco Amado, Sanjeeva Srivastava
The proteogenomics and peptidomics involve identification of peptides, usually based on advances in basic techniques of gel electrophoresis, mass spectrometry, ionization, or chromatography. These technologies, in conjunction with advanced sequencing techniques, form the basis of modern peptidomics and proteogenomics technology. 2-DE polyacrylamide gel electrophoresis (PAGE) was the first and most suitable technique for protein purification [14]. In this technique, proteins are first separated according to their isoelectric point (pI) and then according to their molecular weight using a sodium dodecyl sulfate gel PAGE (SDS-PAGE). The ability to analyze complete proteins in terms of their post-translational modifications (PTMs) is of great theoretical and practical interest in proteomics. To maximize the biological relevance of the method, proteomics should ideally be able to analyze complete, native, folded proteins with their PTMs and prosthetic groups [15,16,17]. However, gel-based proteomics has low sensitivity, low throughput, and poor predictive power. Further, protein spots/bands identified by gel-based methods are subjected to mass spectrometric techniques for protein identification [18]. To overcome several challenges associated with gel-based proteomics now there is more emphasis on mass spectrometry-based shotgun proteomics.
A proteogenomic approach to target neoantigens in solid tumors
Published in Expert Review of Proteomics, 2020
Ayushi Verma, Ankit Halder, Soumitra Marathe, Rahul Purwar, Sanjeeva Srivastava
The shotgun proteomics strategy includes the steps of protein digestion and separation through a liquid chromatography column, tandem mass spectrometry, peptide sequence mapping to MS/MS spectra data, and sequence database searching [6]. The conventional MS-based proteomics also suffered from low sequence coverage. The advancement of the mass spectrometers redefined the field of proteomics by providing high-resolution and high-accuracy MS data. It decreases the search time and reduces the detection of false-positive peptides when searched against six-frame translation databases. The major concern for the sequence coverage of mass spectrometers arises from the fact that extreme hydrophobic or hydrophilic peptides are difficult to elute out of the Liquid Chromatography (LC) column. Also, the proteins undergoing trypsin digestion having peptides with Lysine, Arginine residues that are less than 6 amino acids or greater than 30 amino acids tend to remain undetected by mass spectrometry [1]. Post-translational modifications such as glycosylation and disulfide bridges can interfere with the trypsin digestion. Incomplete digestion (usually with trypsin) can lead to false negative identification of some peptides. Recently in one study, Darie et al. have shown that disulfide bridges prevent the fragmentation of ions that results in poor MS/MS spectra [7].