Superior Dissociation of Intact Proteins with High Capacity Electron Transfer Dissociation

SUPPLEMENTAL MATERIAL

Superior Dissociation of Intact Proteins with High Capacity Electron Transfer Dissociation

Nicholas M. Riley, Christopher Mullen, Chad R. Weisbrod, Seema Sharma, Michael W. Senko, Vlad Zabrouskov,Michael S. Westphall, John E.P. Syka, and Joshua J. Coon

Journal of the AmericanSociety for Mass Spectrometry

Supplemental Table 1. ETD reaction times for myoglobin.

Supplemental Figure 1. The number of fragments produced by high capacity ETD for higher AGC targets outmatches the number of fragments generated in standard ETD, regardless of the standard or elevated reactions times used. The difference between fragments matched in high capacity ETD and standard ETD reacted for the same duration (red and green, respectively) is most likely due to overreaction in the standard ETD condition, further confirming that the number of precursors stored in high capacity ETD is larger. Data here is shown for the z = +15 precursor of myoglobin.

Supplemental Figure 2. Larger precursor populations in high capacity ETD improve the rate of protein sequence coverage achieved per second of acquisition time over standard ETD. For each line, the five points represent 1-5 transients averaged for fragmentation of the z = + 18 precursor.

Supplemental Figure 3. The ability to perform ETD on larger precursor populations also benefits the hybrid fragmentation method EThcD, shown here with a normalized collision energy of 10 and 10 transients averaged for activation of the z = +15 precursor of myoglobin.

Supplemental Figure 4. Determination of the best reaction times to use for standard and high capacity ETD on carbonic anhydrase, z = +34. Each data point is the result of 10 averaged scans at 120K resolving power, using an AGC of 3e5 for standard ETD and 1e6 for high capacity ETD. The open symbols (4 ms and 7 ms, respectively) indicate the reaction times that were used for further analyses.

Supplemental Figure 5. Discussion of S/N of fragments matched for standard and high capacity ETD.Panel (a) shows the overlap in matched fragments from standard ETD and high capacity ETD when analyzing carbonic anhydrase at 240K resolving power and 64 averaged transients. The percentage of peaks explained from all peaks in the deconvoluted spectrum, i.e., the number of peaks that matched as fragments, were 20% and 17% for standard ETD and high capacity ETD, respectively.Panel (b) shows a density plot of signal for fragments that matched to sequence for standard ETD and high capacity ETD, as well as only the fragments that are unique to high capacity ETD. This trend matches the expected results: many of the fragments gained from high capacity ETD are relatively low signal because they were already too low signal to be detected with the standard ETD condition, but are boosted to a detectable level with high capacity ETD.