Profiling of Histone Post-translational Modifications in Mouse Brain with High Resolution Top Down Mass Spectrometry

Transcription

1 Profiling of Histone Post-translational Modifications in Mouse Brain with High Resolution Top Down Mass Spectrometry Mowei Zhou, Ljiljana Paša-Tolić, and David L. Stenoien Supporting Information: Figure S1. Experimentally observed histone H3 with glutathione modification on the cysteine. Figure S2. Experimentally observed hemoglobin alpha chain with glutathione modification on the cysteine. Figure S3. Experimentally observed hemoglobin beta chain with glutathione modification on the cysteine. Figure S4. Annotated HCD spectrum for the bromine containing H4. Figure S5. MS1 spectra for the major H2A.X species containing one acetylation. Figure S6. Annotated ETD and HCD spectra for the novel H2A2A proteoform containing acetylation at the two termini. Figure S7. Annotated (a) ETD and (b) HCD spectra for the H2A2A proteoform containing phosphorylation at S16. Figure S8. Zoom-in of the annotated ETD spectra for the phosphorylated H2A proteoform. Table S1. Summary for the identified proteoforms with high scores. S-1

2 Figure S1. (a) Experimentally observed histone H3 with glutathione modification on the cysteine. (b) ETD and (c) HCD fragmentation spectra confirming the sequence, with corresponding fragment mass error map in (d) and (e), respectively. The glutathionylation coexist with other modifications near the N- terminus of H3, and multiple modified forms of H3 can be detected (data not shown). Because these H3 species (the H3+Glutathione region highlighted in Figure 1d) elute at similar retention time as the gultathionylated hemoglobin beta chain and have distinct masses from the major H3 species, we postulate that they are all glutathionylated H3 despite the fact that most of them only had limited coverage in MS 2 spectra due to the low signal intensities. The modified cysteine 110 is highlighted in the error map with the vertical bar. S-2

3 Figure S2. (a) Experimentally observed hemoglobin alpha chain with glutathione modification on the cysteine. (b) ETD and (c) HCD fragmentation spectra confirming the sequence, both of which showed good coverage and uniform mass accuracy as shown by the fragment mass error map in (d) and (e), respectively. The modified cysteine 104 is highlighted in the error map with the vertical bar. S-3

4 Figure S3. (a) Experimentally observed hemoglobin beta chain with glutathione modification on the cysteine. (b) ETD and (c) HCD fragmentation spectra confirming the sequence, both of which showed good coverage and uniform mass accuracy as shown by the fragment mass error map in (d) and (e), respectively. The modified cysteine 93 is highlighted in the error map with the vertical bar. S-4

5 Figure S4. (a) Annotated HCD spectrum for the bromine containing H4 and (b) the fragment mass error map. The insert in (a) is the zoom-in of the y15/y16 ions which bear the unique isotope pattern from bromine. The red peaks are matched to the y ions containing the 2nd isotope of bromine, while the unlabeled peaks pointed by the orange arrows are from the 1st isotope of bromine. S-5

6 (a) Major H2A.X species: C 664 H 1116 N 208 O 190 (+1Ac) (b) Feature Map of H2A.X Region (c) +80Da species: C 664 H 1116 N 208 O HPO (e) +160Da species: C 664 H 1116 N 208 O (HPO 3 ) (g) +240Da species: C 664 H 1116 N 208 O (HPO 3 ) (d) +80Da species: C 664 H 1116 N 208 O Br - H (f) +160Da species: C 664 H 1116 N 208 O Br -2 H (h) +240Da species: C 664 H 1116 N 208 O Br -3 H Figure S5. (a) MS1 spectra for the major H2A.X species containing one acetylation. The experimental isotope distribution in red matches to the theoretical distribution (formula C664H1116N208O190) shown in blue. (b) Feature map showing of the H2A.X region. There are a series of species with mass difference around 80 Da between min. The H2A.X+1Ac+80Da species near the retention time of min was identified to be H2A.X S1Ac Y57Br (data not shown). Matching the ETD fragments with bromination gave lower mass error than with phosphorylation, similar to the spectra shown in Figure 2. (c, d) The high resolution MS1 spectrum of the +80 Da species helps confirm the presence of bromine instead of phosphorylation. (e-h) Precursor isotope distributions suggest the +160 Da and +240 Da species are from multiple Br addition instead of phosphorylation. S

7 Figure S6. Annotated ETD and HCD spectra for the novel H2A2A proteoform containing acetylation at the two termini. S-7

8 Figure S7. Annotated (a) ETD and (b) HCD spectra for the H2A2A proteoform containing phosphorylation at S16. The insert shows the precursor ion in the preceding MS1 spectrum. The highlighted region indicates the precursor isolation window (0.6 Da as set up in the method). HCD spectrum showed strong b ions corresponding to the loss of phosphate group. S-8

9 Figure S8. Zoom-in of the annotated ETD spectra for the phosphorylated H2A proteoform with the phosphate group assigned at (a) S1, (b) S16, and (c) S18. Major signature c ions are highlighted with either open diamond (not containing phosphate group) or filled diamond (containing phosphate group). The spectrum can match to all three proteoforms, implying the precursor is a mixture of multiple species. The proteoform with S16Phos can match to several of the c ions with the highest abundance. It is noted that most of these signature fragments are at comparable intensities, thus indicating the isoforms coexist in comparable relative abundances in the precursor. Direct quantitation of the isoforms based on phosphorylated/non-phosphorylated fragment intensities was not successful and generated conflicting numbers (data not shown), presumably because the different fragmentation efficiencies for different phosphorylated forms and varying transmission efficiencies across the mass range. S-9

10 Protein Modifications Mass Spectral Counts sp P62806 H4_MOUSE Histone H4 full length Acetyl 0,Acetyl 16,DiMethyl Acetyl 0,DiMethyl Acetyl 0,DiMethyl 20,Oxidation Acetyl 0,Acetyl 16,DiMethyl 20,Oxidation Acetyl 0,Acetyl 12,Acetyl 16,DiMethyl Acetyl 0,Acetyl 8,Acetyl 16,DiMethyl Acetyl 0,Phospho 1,Acetyl 16,DiMethyl Acetyl 0,Acetyl 16,TriMethyl Acetyl 0,TriMethyl Acetyl 0,TriMethyl 20,Oxidation Acetyl 5,Acetyl 12,Acetyl 16,DiMethyl Acetyl 0,Acetyl 16,TriMethyl ,Oxidation 84 sp Q64475 H2B1B_MOUSE Histone H2B type 1-B full length unmodified Oxidation 59 or Oxidation 59,Oxidation Acetyl 12,Acetyl 15,Acetyl sp Q6ZWY9 H2B1C_MOUSE Histone H2B type 1-C/E/G full length unmodified Oxidation 59 or Oxidation 59,Oxidation Acetyl Acetyl Acetyl 15 or 16,Oxidation 59 or Acetyl 15,Oxidation 59,Oxidation Acetyl truncation unmodified sp P10853 H2B1F_MOUSE Histone H2B type 1-F/J/L full length unmodified Acetyl Acetyl 12,Acetyl 15,Acetyl Acetyl Oxidation 59 or Acetyl 12,Acetyl Acetyl Acetyl 12,Acetyl 15,Acetyl 16,Acetyl S-10

11 Acetyl 15,Oxidation Acetyl 16,Acetyl Oxidation 59,Oxidation Acetyl 15,Oxidation 59,Oxidation Acetyl 12,Acetyl Acetyl 15,Acetyl Acetyl 15,Oxidation Acetyl 15,Acetyl Acetyl Acetyl 16,Oxidation 59,Oxidation Acetyl 11,Acetyl 15,Acetyl 16,Acetyl Acetyl 12,Acetyl 16,Acetyl BR 40,Oxidation 59,Oxidation Acetyl 12,Acetyl 15,Oxidation Acetyl 12,Acetyl Acetyl 11,Acetyl Acetyl 16,Oxidation Acetyl 12,Acetyl 15,Acetyl Acetyl 12,Acetyl 15,Oxidation sp P10854 H2B1M_MOUSE Histone H2B type 1-M full length unmodified Oxidation 59 or sp Q64525 H2B2B_MOUSE Histone H2B type 2-B full length unmodified Acetyl Oxidation 59 or Acetyl Acetyl Acetyl 16,Oxidation Oxidation 59,Oxidation Acetyl 20,Oxidation sp Q64524 H2B2E_MOUSE Histone H2B type 2-E full length unmodified Oxidation sp Q9D2U9 H2B3A_MOUSE Histone H2B type 3-A full length unmodified Phospho Oxidation Acetyl Phospho 7,Oxidation Oxidation 59,Oxidation Oxidation Acetyl S-11

12 Acetyl 12,Acetyl 15,Acetyl Acetyl 12,Acetyl Phospho Phospho 7,Oxidation 59,Oxidation Acetyl 12,Acetyl Acetyl 12,Phospho Acetyl 12,Phospho Phospho 7,Acetyl sp Q8CGP0 H2B3B_MOUSE Histone H2B type 3-B full length unmodified Oxidation 59 or Oxidation 59,Oxidation sp P22752 H2A1_MOUSE Histone H2A type 1 full length Acetyl Acetyl 0,Phospho Acetyl 0,Acetyl Acetyl 0,Acetyl Acetyl 0,Phospho Acetyl 0,BR 39,BR 50,BR 57* (primarily in one sample, mono-/di-br observed as well in the same sample) Acetyl 0,Acetyl Acetyl 0,Acetyl Acetyl 0,Acetyl 15,Phospho Acetyl 0,Acetyl Acetyl 0,Acetyl Phospho 1,Acetyl truncation Acetyl truncation Acetyl sp Q8CGP5 H2A1F_MOUSE Histone H2A type 1-F full length Acetyl sp Q8CGP6 H2A1H_MOUSE Histone H2A type 1-H full length Acetyl Acetyl 0,Phospho sp Q6GSS7 H2A2A_MOUSE Histone H2A type 2-A full length Acetyl Acetyl 0,Phospho Acetyl 0,Acetyl Acetyl 0,Acetyl Acetyl 0,Oxidation Phospho 1,Acetyl Acetyl 0,Acetyl 5,Acetyl Acetyl 0,Phospho 1,Oxidation Acetyl Acetyl 0,Acetyl S-12

13 Acetyl 0,Acetyl Acetyl 0,Acetyl 15,Phospho Acetyl 0,Phospho 1,Acetyl Acetyl 0,Acetyl Acetyl 0,Acetyl 5,Oxidation Acetyl 0,Phospho 1,Acetyl truncation Acetyl truncation Acetyl truncation Acetyl truncation Acetyl truncation Acetyl truncation Acetyl truncation Acetyl sp Q64522 H2A2B_MOUSE Histone H2A type 2-B full length Acetyl Acetyl 0,Acetyl Acetyl 0,Phospho sp Q64523 H2A2C_MOUSE Histone H2A type 2-C full length Acetyl Acetyl 0,Phospho Acetyl 0,Acetyl Acetyl 0,Acetyl Acetyl 0,Oxidation Acetyl 0,Acetyl Acetyl Phospho 1,Acetyl sp Q8BFU2 H2A3_MOUSE Histone H2A type 3 full length Acetyl Acetyl 0,Acetyl Acetyl 0,Acetyl Acetyl 0,Phospho 1,Methyl Acetyl 0,Acetyl truncation Acetyl truncation unmodified sp Q8R1M2 H2AJ_MOUSE Histone H2A.J full length Acetyl Acetyl 0,Phospho Acetyl 0,Acetyl Acetyl 0,Acetyl truncation Acetyl sp Q3THW5 H2AV_MOUSE Histone H2A.V full length unmodified sp P27661 H2AX_MOUSE Histone H2AX full length Acetyl S-13

14 Acetyl 0,Phospho Acetyl 0,Acetyl Acetyl 0,Acetyl 5,Acetyl Acetyl 0,Phospho Acetyl 0,Acetyl Phospho 1,Acetyl truncation Acetyl truncation Acetyl truncation Acetyl sp P0C0S6 H2AZ_MOUSE Histone H2A.Z full length unmodified Acetyl Acetyl sp P68433 H31_MOUSE Histone H3.1 full length DiMethyl 9,Acetyl 14,DiMethyl 27,DiMethyl DiMethyl 9,Acetyl 18,DiMethyl 27,DiMethyl sp P84228 H32_MOUSE Histone H3.2 full length DiMethyl 9,Acetyl 14,DiMethyl 27,DiMethyl TriMethyl 9,DiMethyl TriMethyl 9,Methyl 27,Methyl DiMethyl 9,Acetyl 14,Methyl 27,DiMethyl DiMethyl 9,DiMethyl 27,DiMethyl DiMethyl 17,DiMethyl DiMethyl 9,Acetyl 18,DiMethyl 27,DiMethyl DiMethyl 9,Methyl 17,DiMethyl TriMethyl 9,Methyl sp P84244 H33_MOUSE Histone H3.3 full length DiMethyl 27,DiMethyl tr A8DUK4 A8DUK4_MOUSE Beta-globin full length unmodified Oxidation Nitrosylation Oxidation 93,Oxidation Beta-methylthiolation Persulfide/Dioxidation GSH Trioxidation tr Q91VB8 Q91VB8_MOUSE Alpha globin 1 full length unmodified Oxidation S-14

15 Nitrosylation GSH truncation unmodified Nitrosylation truncation unmodified Table S1. Summary for the identified proteoforms with high scores. The identifications are categorized by protein name. All residue counts start from the 2 nd residue in the full length sequence in UniProt with the initiator methionine excised. The modification column lists the identified modifications with the number of the residue modified annotated after the modification name. The abbreviations for the modification names are: Acetyl acetylation, BR bromination, Deami deamidation, Methyl monomethylation, DiMethyl demethylation, TriMethyl trimethylation, Phospho phosphorylation, GSH glutathionation. Some of the modifications listed for alpha and beta globins in the table are not commonly reported to the best of our knowledge. They are based on the masses of the precursors and fragments in top down mass spectrometry data only and may need further validation from other techniques. The masses of the proteoforms and the counts of the matched spectra after filtering are listed on the last two columns on the right. S-15