Special Focus on Top‐down Proteomics

Abstract

Without doubt, so-called “shotgun” or “bottom-up” proteomics dealing with peptides obtained upon chemical or enzymatic breakdown of proteins has been the driving force behind the tremendous progress and possibilities that we have seen and are still seeing in proteome analysis. Nevertheless, as the term indicates, “shooting” at pieces of proteins with a shotgun leaves one ambiguous about the actual target. Moreover, the real objects of desire in proteomic analyses are the intact proteins, their function, location, modifications, concentration, interactions, and relevance in biological processes. Many of these latter aspects may get lost or overseen when breaking down proteins to their corresponding peptide fragments. In due consequence, it is not surprising that from the very beginning of intact protein mass spectrometry, which has been primarily facilitated by the introduction of electrospray ionization and matrix-assisted laser desorption/ionization, researchers have been trying to investigate proteins at the intact molecule level, including all posttranslational modifications and sequence variations. This strategy of approaching proteomes via intact protein analysis has been termed “top-down” proteomics, striving to directly derive sequences and identification information from fragment spectra of intact proteins. While shotgun approaches have the advantage of dealing with peptides having relatively homogenous chemical and physical properties, which facilitates the application of quite generic separation and mass spectrometric identification routines, top-down technologies struggle with the immense structural diversity and the corresponding broad range of physical and chemical properties of proteins. Therefore, a significantly larger, multifaceted, and more sophisticated toolbox is required in order to handle intact proteins efficiently on a global scale. This toolbox includes one- and multidimensional separation methods, elaborate fragmentation techniques, technologies for high-resolution and highly accurate mass analysis, and finally, algorithms for automated interpretation of mass spectrometric and chromatographic data. We are indebted to researchers in the field, kindly following our invitation to contribute to this Focus Issue devoted to top-down proteomics in order to demonstrate the current status of the technology. The articles collected in this issue cover a significant portion of the major challenges of top-down protein analysis, including technical aspects such as gas-phase ion chemistry of intact proteins, automated software platforms for large-scale top-down proteomics, and the high-resolution single- and multidimensional separation and detection of intact proteins. Applications of the top-down technologies presented in this issue range from the investigation of ribosomal proteins and histones, the phyloproteomic classification of bacterial proteins, the characterization of hemoglobin variants, to the characterization of integral plant membrane protein complexes. Some of the contributions to this Focus Issue also shed light on the possibilities and limitations both of shotgun and top-down approaches, revealing both methods as highly complementary and thereby enabling more comprehensive investigations into biological problems. We thank all contributing authors for investing their time and sharing with us valuable insights into the world of top-down proteome analysis. We hope that the workflows and data presented in this issue will stimulate you own new ideas and approaches to problem solving in biological, biochemical, or biomedical analysis involving intact proteins. Enjoy reading, Sincerely, Christian Huber University of Salzburg Lukas Huber Innsbruck Medical University