on 05-01-201210:54 AM - edited on 11-09-202101:18 AM by usermigration2
Hao Z, Jiang L, Zhang T, Hühmer AFR. Scientific Poster Conventional methodology of protein mass spectrometry using a bottom-up approach has limitations with respect to characterization of protein isoforms. In the bottom-up approach, proteins are enzymatically digested into a collection of peptides of relatively short size making the post-analysis assembly of protein isoform information challenging. The analysis of large peptides or intact proteins would reduce or even overcome this problem, thus, provide more complete sequence coverage, including site-specific modifications or mutations. Electron transfer dissociation (ETD), compared to collisional activation, is relatively insensible to the size of peptides, therefore offers a great opportunity for large peptide sequencing. From multiply charged precursors of large peptides, ETD generates information rich spectra containing full series of c and z. types of product ions. With the increase of precursor ion charge state, fragment ions are often obtained carrying charge of +5 or even higher which are not well resolved in unit resolution instruments. Data interpretation of such complex, information rich, yet not well resolved ETD spectra would be difficult. High resolution and accurate mass would greatly facilitate analysis of complex ETD spectra. ETD was recently implemented with a hybrid linear ion trap – LTQ Orbitrap XL ETD mass spectrometer. An orbitrap mass analyzer with ETD capabilities was applied here for direct, unambiguous sequencing of large peptide or small intact proteins. It has been observed that ETD fragmentation efficiency decreases with the increase of precursor m/z, regardless of precursor charge or peptide molecular mass. A supplemental activation approach can enhance ETD performance by converting the non-dissociative electron transfer product into c and z type of ions. Automated implementation of supplemental activation on precursor ions of charge +2 through +6 is now available under instrument control software. In this study, the automated supplemental activation was evaluated using standard peptides and applied to large peptide identification from protein digest.