Top-Down Characterization of Histones using 193 nm Ultraviolet Photodissociation (UVPD) Mass Spectrometry. Sylvester Greer, Jennifer Brodbelt*

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1 Top-Down Characterization of Histones using 193 nm Ultraviolet Photodissociation (UVPD) Mass Spectrometry Sylvester Greer, Jennifer Brodbelt* Department of Chemistry University of Texas at Austin Austin, TX Correspondence to: Jenny Brodbelt, Supporting Information Figure S1. Examples of nano LC traces for HCD, EThcD and UVPD experiments illustrating the level of reproducibility. Figure S2. Examples of MS2 spectra for histone H3K9me2K27me (23+), sequence maps and score metrics for: A) HCD, B) EThcD and C) UVPD. Figure S3. A) Number of proteoforms identified using one laser pulse at different energies. B) Examples of sequence coverage and proteoform metrics at various laser energies for H2A proteoform (19+). Figure S4. A) Results obtained for different numbers of laser pulses showing the number of identified proteoforms and average C-scores using one or two laser pulses at 1.7 mj. B) Examples of sequence coverage and proteoform metrics obtained using one or two laser pulses (1.7 mj). Figure S5. Typical liquid chromatogram of a mixture of intact calf histones separated on a 30 cm x 75 um column packed with PLRP-S stationary phase. Figure S6. Distribution of fragment ion types generated by UVPD, EThcD and HCD of histone ach2a (19+). Figure S7. Histogram showing the percentage of all PSMs identified by HCD, EThcD or UVPD as a function of the number of matched diagnostic ions for all identified proteoforms. Figure S8. Difference in sequence coverage for the 177 proteoforms identified in common by HCD and UVPD from the Venn diagram in Figure 1. Each point represents the difference in sequence coverage between UVPD and HCD. Results are ordered based on the caliber of results for UVPD relative to HCD. All data points above the x-axis indicate better performance for UVPD. Figure S9. Box and whisker plot showing the sequence coverage of proteoforms common to UVPD and EThcD from Figure 1b. Results were sorted by mass to show impact of protein size. Figure S10. C-score distribution for all proteoforms identified by UVPD and EThcD with 1,2,3 or 4 modifications. The n numbers in each legend indicate the number of proteoforms included for each MS/MS method. S 1

2 Figure S11. C-score distribution for proteoforms identified in common by UVPD and EThcD with 1,2,3 or 4 modifications. The n numbers next to each sub-title indicate the number of proteoforms in common for UVPD and EThcD. Table S1. Lists of proteoforms from Prosight for HCD, UVPD and EThcD. All spectra are archived and available at: The accession numbers are PXD for ProteomeXchange and JPST for jpost. S 2

3 Figure S1. Examples of nano LC traces for HCD, EThcD and UVPD experiments illustrating the level of reproducibility. S 3

4 Figure S2. Examples of MS2 spectra for histone H3K9me2K27me (23+), sequence maps and score metrics for: A) HCD, B) EThcD and C) UVPD. S 4

5 Figure S3. A) Number of proteoforms identified using one laser pulse at different energies. B) Examples of sequence coverage and proteoform metrics at various laser energies for H2A proteoform (19+). S 5

6 Figure S4. A) Results obtained for different numbers of laser pulses showing the number of identified proteoforms and average C-scores using one or two laser pulses at 1.7 mj. B) Examples of sequence coverage and proteoform metrics obtained using one or two laser pulses (1.7 mj).. S 6

7 Figure S5. Typical liquid chromatogram of a mixture of intact calf histones separated on a 30 cm x 75 um column packed with PLRP-S stationary phase. S 7

8 Figure S6. Typical distribution of fragment ion types generated by UVPD, EThcD and HCD of histone ach2a (19+). S 8

9 Figure S7. Histogram showing the percentage of all PSMs identified by HCD, EThcD or UVPD as a function of the number of matched diagnostic ions for all identified proteoforms. S 9

10 Fig. S8. Difference in sequence coverage for the 177 proteoforms identified in common by HCD and UVPD from the Venn diagram in Figure 1. Each point represents the difference in sequence coverage between UVPD and HCD. Results are ordered based on the caliber of results for UVPD relative to HCD. All data points above the x-axis indicate better performance for UVPD. S 10

11 Fig. S9. Box and whisker plot showing the sequence coverage of proteoforms common to UVPD and EThcD from Figure 1b. Results were sorted by mass to show impact of protein size. S 11

12 Figure S10. C-score distribution for all proteoforms identified by UVPD and EThcD with 1,2,3 or 4 modifications. The n numbers in each legend indicate the number of proteoforms included for each MS/MS method. S 12

13 Figure S11. C-score distribution for proteoforms identified in common by UVPD and EThcD with 1,2,3 or 4 modifications. The n numbers next to each sub-title indicate the number of proteoforms in common for UVPD and EThcD. S 13

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