| Summary: | Mass spectrometry has emerged as a high accuracy, high sensitivity approach for the analysis of proteins. A conventional scheme for protein analysis by mass spectrometry involves reduction, alkylation, proteolytic digestion, followed by analysis either by electrospray ionization mass spectrometry (ESI-MS) directly or with a prior separation by liquid chromatography (LC). This approach, termed bottom-up proteomics, offers the advantage of high sensitivity and allows for the analysis of complex mixtures of tens, hundred, and even thousands of proteins. However, it is well-known that proteins arising from the same gene may be chemically-different species as a result of sequence truncation, alternative splicing variants, and/or different post-translation modifications (PTMs). Such species are defined as proteoforms. A fundamental problem with the bottom-up approach is thus the inability to identify proteoforms, which may even possess multiple PTMs at once. In the course of proteolytic digestion, as a protein is converted into a mixture of peptides, associations between different PTMs are lost. In essence, what was once a single proteoform composed of numerous PTMs has become a set of smaller peptides scrambled within a mixture of other peptides from other proteoforms. Moreover, peptide fragments with bound PTMs may not contain basic amino acid residues and would not exhibit protonation to be detected by MS, resulting in losses and underrepresentation of such PTMs. An alternative approach to protein analysis by mass spectrometry is therefore top-down proteomics, in which proteins are analyzed in their intact forms. A further extension of top-down proteomics native mass spectrometry (native MS) involves the analysis of proteins at near-physiological conditions, where all-levels of protein structure, including any non-covalent associations, are maintained. The ability to perform top-down proteomics, and especially native MS, is a consequence of the numerous advancements in analytical instrumentation that have occurred over the past several decades.
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