Top-down analysis of 30–80 kDa proteins by electron transfer dissociation time-of-flight mass spectrometry

Electron transfer dissociation (ETD)-based top-down mass spectrometry (MS) is the method of choice for in-depth structure characterization of large peptides, small- and medium-sized proteins, and non-covalent protein complexes. Here, we describe the performance of this approach for structural analysis of intact proteins as large as the 80 kDa serotransferrin. Current time-of-flight (TOF) MS technologies ensure adequate resolution and mass accuracy to simultaneously analyze intact 30–80 kDa protein ions and the complex mixture of their ETD product ions. Here, we show that ETD TOF MS is efficient and may provide extensive sequence information for unfolded and highly charged (around 1 charge/kDa) proteins of ∼30 kDa and structural motifs embedded in larger proteins. Sequence regions protected by disulfide bonds within intact non-reduced proteins oftentimes remain uncharacterized due to the low efficiency of their fragmentation by ETD. For serotransferrin, reduction of S–S bonds leads to significantly varied ETD fragmentation pattern with higher sequence coverage of N- and C-terminal regions, providing a complementary structural information to top-down analysis of its oxidized form. Figure ETD TOF MS provides extensive sequence information for unfolded and highly charged proteins of ~30 kDa and above. In addition to charge number and distribution along the protein, disulfide bonds direct ETD fragmentation. For intact non-reduced 80 kDa serotransferrin, sequence regions protected by disulfide bonds oftentimes remain uncharacterized. Reduction of disulfide bonds of serotransferrin increases ETD sequence coverage of its N- and C-terminal regions, providing a complementary structural information to the top-down analysis of its oxidized form