Top‐down tandem mass spectrometry on RNase A and B using a Qh/FT‐ICR hybrid mass spectrometer

Abstract

Protein characterization using top-down approaches emerged with advances in high-resolution mass spectrometers and increased diversity of available activation modes: collision induced dissociation (CID), infrared multiphoton dissociation (IRMPD) electron capture dissociation (ECD) and electron transfer dissociation (ETD). Nevertheless, top-down approaches are still rarely used for glycoproteins. Hence, this work summarized the capacity of top-down approaches to improve sequence coverage and glycosylation site assignment on the glycoprotein Ribonuclease B (RNase B). The glycan effect on the protein fragmentation pattern was also investigated by comparing the fragmentation patterns of RNase B and its non-glycosylated analog RNase A. The experiments were performed on a Bruker 12-T-Qh/FTICR SolariX mass spectrometer using vibrational (CID/IRMPD) and radical activation (ECD/ETD) with/without pre- or post-activation (IRMPD or CID, respectively). The several activation modes yielded complementary sequence information. The radical activation modes yielded the most extensive sequence coverage that was slightly improved after a CID pre-dissociation-activation event. The combination of the data made it possible to obtain 90% final sequence coverage for RNase A and 86% for RNase B. Vibrational and radical activation modes showed high retention of the complete glycan moiety (>98% for ETD and ECD) facilitating unambiguous assignment of the high-mannose glycosylation site. Moreover, the presence of the high-mannose glycan enhanced fragmentation around the glycosylation site.

Phosphoproteomics with Activated Ion Electron Transfer Dissociation

“…AI-ETD improves fragmentation and sequence coverage for top-down proteomics, 95–97 but it has yet to be applied to intact phosphoproteins. To investigate the utility of AI-ETD for top-down phosphoproteomics, we fragmented five charge states of α-casein spanning the charge state envelope of the protein (z = +22, +20, +18, +16, and +14) and compared performance to HCD, ETD, and EThcD.…”

Ion Activation Methods for Peptides and Proteins

“…194 Sequence coverage obtained by ETD and ECD was two to three times more extensive than that obtained from CID and IRMPD data, and the glycan was retained which allowed facile assignment of the glycosylation site. 194 …”

“…Reproduced from Frese, C.K., Altelaar, A.F.M.,van den Toorn, H., Nolting, D., Griep-Raming, J., Heck, A.J.R., Mohammed, S., Anal. Chem ., 2012 , 84, 9668–9673 (ref 194). Copyright 2012 American Chemical Society.…”

Benchmarking Multiple Fragmentation Methods on an Orbitrap Fusion for Top-down Phospho-Proteoform Characterization

“…A more widely used approach to increase fragmentation efficiency for peptides is the combination of ETD with supplemental activation (ETciD, sometimes also called ETcaD), which targets the chargereduced precursors 10 . Moreover, although not commonly applied in top-down proteomics, the collisional activation applied during ETciD could disrupt the tertiary structure of larger proteins 11,12 . Another alternative to increase ETD fragmentation efficiency is combining ETD and HCD sequentially in a single event, a fragmentation method termed EThcD 13,14 .…”