A Critical Review of Bottom-Up Proteomics: The Good, the Bad, and the Future of This Field

Abstract

<jats:p>Proteomics is the field of study that includes the analysis of proteins, from either a basic science prospective or a clinical one. Proteins can be investigated for their abundance, variety of proteoforms due to post-translational modifications (PTMs), and their stable or transient protein–protein interactions. This can be especially beneficial in the clinical setting when studying proteins involved in different diseases and conditions. Here, we aim to describe a bottom-up proteomics workflow from sample preparation to data analysis, including all of its benefits and pitfalls. We also describe potential improvements in this type of proteomics workflow for the future.</jats:p>

Tailoring the Health-Promoting Potential of Protein Hydrolysate Derived from Fish Wastes and Flavonoids from Yellow Onion Skins: From Binding Mechanisms to Microencapsulated Functional Ingredients

“…Low molecular weight peptides from fish waste were obtained by enzymatic treatment followed by centrifugal ultrafiltration, resulting a yield of 7.08%. Furthermore, the results were correlated with MALDI-TOF experiment within database searches using bioinformatic tools aiming to identify the peptides that correspond to the spectrum [ 20 ]. Therefore, in order to simulate the complete papain-assisted hydrolysis of the main proteins found in fish bones, the amino acids sequence of collagen type I α1 and collagen type I α2 was taken from UniProtKB database.…”

Top-Down and Bottom-Up Proteomics Methods to Study RNA Virus Biology

“…In addition to top-down and structural approaches, bottom-up proteomics methods have also seen remarkable progresses in the past few years, reaching unprecedented analytical depth, sensitivity, and throughput (reviewed in [ 99 ]). For these reasons, in the last few years, bottom-up proteomics have been increasingly employed to study human pathogenic RNA viruses, shedding unprecedented light on virus-induced host perturbations at multiple levels.…”

Protocol for the Bottom-Up Proteomic Analysis of Mouse Spleen

“…Prior to designing a specific proteomic workflow, it is crucial to initially investigate which biochemical screening approaches and protein identification methodologies are the most suitable way for the efficient analysis of the particular type of cell, tissue, or organ under investigation ( Kang et al., 2020 ). In analytical protein biochemistry, the main high-throughput approaches can be divided into top-down proteomics versus bottom-up proteomics ( Dupree et al., 2020 ). Besides antibody-based screening methods, the most frequently employed detection method for protein identification is mass spectrometry.…”

“…Proteomics has developed into a key biochemical methodology for the systematic cataloging of protein populations in biofluids, cells, tissues, and organs ( Uzozie and Aebersold, 2018 ), whereby the development of highly sensitive mass spectrometric methods plays a central role in the unequivocal identification of individual proteoforms ( Kang et al., 2020 ). This protocol describes a typical bottom-up proteomic approach ( Dupree et al., 2020 ) that has combined tissue homogenization, protein concentration measurements, controlled protein digestion by trypsination, the removal of interfering chemicals, peptide sequencing with the help of an Orbitrap Fusion Tribrid mass spectrometer and bioinformatic analysis of proteomic data ( Dowling et al., 2020 ). The expected outcome of a typical proteomic survey of crude tissue samples is the identification of several thousand individual proteins.…”