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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
The ultimate downstream MS workflow comprises several common steps, including ionization of peptides and their separation according to their mass charge ratio. Top-down proteomics separates proteins in a sample prior to them being ionized and bottom-up proteomics digests proteins into a complex mixture of peptides first [148, 149].
Emerging Biomedical Analysis
Published in Lawrence S. Chan, William C. Tang, Engineering-Medicine, 2019
The development of tandem mass spectrometry technologies dramatically improved the depth of proteomics studies by providing highly sensitive and specific analytical approaches. Coupled with a separation front end, usually LC or CE, tandem mass spectrometry is exclusively used as the analytical platform for different proteomic strategies. There are two major classes of proteomic strategies: bottom-up proteomics and top-down proteomics (Fig. 8). In bottom-up strategies, the characterization of proteins is achieved by the analysis of peptides released from proteins through proteolysis. In contrast, top-down strategies mainly focus on the analysis of intact proteins. Compared with intact proteins, peptides are more easily fractionated, ionized and fragmented. Therefore, bottom-up proteomics is more universally used in practice.
Proteomics Approaches to Uncover the Drug Resistance Mechanisms of Microbial Biofilms
Published in Chaminda Jayampath Seneviratne, Microbial Biofilms, 2017
Chaminda Jayampath Seneviratne, Tanujaa Suriyanarayanan, Lin Qingsong, Juan Antonio Vizcaíno
Classically there are two major approaches in gel-free proteomics: bottom-up and top-down [98,108]. Top-down proteomics involves the analysis of intact proteins, whereas bottom-up proteomics approach represents the analysis of a complex peptide mixture after proteolysis with an enzyme, most commonly trypsin. Currently, the bottom-up (also known as shotgun) proteomics approach is more popular and more commonly employed because of its wider spectrum of applications and existing instrumentation. In the bottom-up approach, proteins are first digested to obtain peptides, which are subsequently subjected to MS-based analysis. Some studies have combined top-down and bottom-up proteomics approaches for better interpretation of results [109].
How can platelet proteomics best be used to interrogate disease?
Published in Platelets, 2023
However, two fundamentally different technologies are available to perform these analyses, top-down and bottom-up proteomics. The main difference between these technologies is that for bottom-up proteomics, also called shotgun analysis, the proteins of a biological sample have to be digested into peptides to enable the analysis process. This strange step of preparing biological samples using enzymatic digestion is necessary because an adequate liquid chromatographic separation and subsequent mass spectrometric measurement of complex protein mixtures are only possible with their peptides than with the corresponding intact proteins. On the other hand, top-down proteomic methods involve the analysis of intact proteins from a biological sample. The term “top-down proteomics” was first coined in the early 2000s to define mass spectrometry-based method developments that should also enable the analysis of intact proteins. However, the intact proteome can currently only be separated most efficiently using two-dimensional gel electrophoresis.5–7 Different staining methods directly visualize the two-dimensional gel electrophoresis separated protein profiles in a nice analogue image of a proteomic spot map. In this process step the intact proteins can be quantified and biological differences can be identified. It is important to note that only after the intact proteins have been fractionated, and their identity is still unknown, are they digested into peptides for identification by MS.
An overview of technologies for MS-based proteomics-centric multi-omics
Published in Expert Review of Proteomics, 2022
Andrew T. Rajczewski, Pratik D. Jagtap, Timothy J. Griffin
In contrast to the bottom-up approach, ‘top-down’ proteomics performs LC-MS/MS analyses on whole, undigested proteins using high-resolution mass spectrometers. Top-down mass spectrometry is performed with the goal of detecting the chemical modifications and post-translational modifications that distinguishes individual proteins encoded by the same gene, from one another in vivo[13]. ‘Top-down proteomics performs LC-MS analyses on whole, undigested proteins using high-resolution mass spectrometers. Top-down mass spectrometry is performed with the goal of detecting the chemical modifications and post-translational modifications (PTMs) that distinguish individual proteins, even chemically distinct proteoforms [14] encoded by the same gene, from one another in vivo[13]. Top-down has proven valuable in many contexts, although limitations in characterizing larger massed proteins and proteins that are not easily solubilized have limited their ability to comprehensively characterize complex proteomes.
Proteogenomic interrogation of cancer cell lines: an overview of the field
Published in Expert Review of Proteomics, 2021
While the vast majority of proteomics studies are carried out using bottom-up approaches, top-down proteomics is an alternative proteome analysis approach that can yield complementary information. In top-down proteomics, intact proteins are directly resolved by LC and analyzed by MS and MS/MS. The advantage of top-down proteomics is that it can identify specific proteoforms that cannot be resolved through bottom-up proteomics. Furthermore, it is particularly suitable for analyzing small proteins that lack sufficient proteolytic sites for identification by bottom-up approaches. Nevertheless, compared to bottom-up proteomics, the top-down approach lacks sensitivity as intact proteins are difficult to be resolved well by LC, and large proteins can be challenging to ionize and fragment. Top-down proteomics has been applied to profile the proteome of the H1299 lung cancer cell line, identifying more than 2,000 proteoforms [52]. It has also been used more specifically to identify proteoforms of the RAS gene in colorectal cancer cell lines [53].