Top or Middle? Up or Down? Toward a Standard Lexicon for Protein Top-Down and Allied Mass Spectrometry Approaches

Abstract

In recent years, there has been increasing interest in top-down mass spectrometry (TDMS) approaches for protein analysis, driven both by technological advancements and efforts such as those by the multinational Consortium for Top-Down Proteomics (CTDP). Today, diverse sample preparation and ionization methods are employed to facilitate TDMS analysis of denatured and native proteins and their complexes. The goals of these studies vary, ranging from protein and proteoform identification, to determination of the binding site of a (non)covalently-bound ligand, and in some cases even with the aim to study the higher order structure of proteins and complexes. Currently, however, no widely accepted terminology exists to precisely and unambiguously distinguish between the different types of TDMS experiments that can be performed. Instead, ad hoc developed terminology is often used, which potentially complicates communication of top-down and allied methods and their results. In this communication, we consider the different types of top-down (or top-down-related) MS experiments that have been performed and reported, and define distinct categories based on the protocol used and type(s) of information that can be obtained. We also consider the different possible conventions for distinguishing between middle- and top-down MS, based on both sample preparation and precursor ion mass. We believe that the proposed framework presented here will prove helpful for researchers to communicate about TDMS and will be an important step toward harmonizing and standardizing this growing field. Graphical Abstract.

Keywords: Native mass spectrometry; Proteoform; Top-down mass spectrometry; Top-down proteomics.

Graphical Abstract

Figure 1

Various (non-)native top-down and allied methods as defined in this work

Figure 2

How the methods described in Figure 1 relate to one another, as well as to (extended) bottom-up approaches. The “true” top-down methods are given in bolded text

Figure 3

(Center) general structure of a monoclonal antibody (IgG1), surrounded by simulated (using Bruker DataAnalysis 4.1) isotope distributions for (top-left) the 49 kDa heavy chain, (top-right) the 23 kDa light chain, (bottom-left) an example 1.9 kDa enzymatically derived peptide consisting of the first 18 N-terminal light chain amino acid residues, and (bottom-right) an example 4.4 kDa enzymatically derived peptide consisting of the first 39 light chain N-terminal residues. The former two represent typical precursor masses for middle-up (MU) and middle-down (MD) MS, whereas the latter two fall within the bottom-up (BU) and extended bottom-up (eBU) mass range, respectively. Isotope distributions were simulated using the sequence of adalimumab, and the monoisotopic peak for each species (too low in intensity to be detectable for the heavy and light chain) is indicated in blue. The isotopic envelope is outlined using a solid red line, simulating the signal obtained at a resolution insufficient to resolve the individual isotope peaks