Abstract
Theoretically, the gas-phase interrogation of whole proteoforms via mass spectrometry, known as top-down proteomics, bypasses the protein inference problem that afflicts peptide-centric proteomic approaches. Despite this obvious advantage, the application of top-down proteomics remains rare, mainly due to limited throughput and difficulty of analyzing proteins >30 kDa. Here we will discuss some of the problems encountered during the characterization of large proteoforms, and guided by a combination of theoretical background and experimental evidence we will describe some innovative data acquisition strategies and novel mass spectrometry technologies that can at least partially overcome such limitations.
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