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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].
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].
Protein amino-termini and how to identify them
Published in Expert Review of Proteomics, 2020
Annelies Bogaert, Kris Gevaert
In general, enriching proteins and/or peptides carrying a modification of interest is essential to reach sufficient coverage of the proteins affected by this modification. Such enrichment occurs through affinity (e.g. antibodies recognizing specific modification motifs) or is based on the physicochemical parameters of the modified peptides that can be different from those of non-modified peptides. Bottom-up proteomics still is the most popular tool in mass spectrometry-based proteomics [18]. Here, proteins get digested into peptides and as N-terminal peptides only account for a fraction of the generated peptide mixture, their enrichment prior to analysis is necessary. Several enrichment methods were developed, which can be classified as negative or positive selection methods. The former indirectly enrich N-terminal peptides by depleting internal peptides, whereas the latter directly enrich N-terminal peptides (Figure 1).
Approaching complexity: systems biology and ms-based techniques to address immune signaling
Published in Expert Review of Proteomics, 2020
Joseph Gillen, Caleb Bridgwater, Aleksandra Nita-Lazar
Proteomics offers more and more broad strategies for approaching the study of proteins (Figure 1). Bottom-up proteomics is by far the most common approach and entails the digestion of protein mixtures with proteolytic enzymes, peptide separation via chromatography, and the creation of a mass spectra in tandem mass spectrometry through peptide fragmentation [9]. This approach is carried out on protein mixtures, and is the most common variety of bottom-up proteomic analysis, known as shotgun proteomics [10]. Fast and robust, shotgun proteomics does have drawbacks including the loss of some PTMs and structure. In contrast, top-down proteomics is the analysis of intact proteins and protein complexes [11]. Analysis of difficult post-translational modifications and structural artifacts of proteins is possible using top-down proteomics, but the increased complexity of the resulting signals and difficulty in pre-MS protein separation of complex mixtures for analysis are still barriers. Lastly, middle-down proteomics bridge the two approaches by using incomplete or perturbed proteolytic digestion to prepare mixtures of larger peptide fragments than in bottom-up approaches [9]. When properly utilized, proteomic methods can add important insights to larger studies and open new routes to understand the complex nature of immune signaling.