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Figure S1. General experimental procedure

Figure S1. General experimental procedure. WT. Nuclei prep/Acid Extraction. H2B. Intentisity. Step 1. Propionylation Step 2. Trypsin digestion Step 3. Propionylation. Intentisity. ΔEZL2. H4. H2A. H3. ΔTXR1. RT. M/Z. Equal mixture of L&H histones. N15 WT.

cynthia-nichols
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Figure S1. General experimental procedure

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  1. Figure S1. General experimental procedure WT Nuclei prep/Acid Extraction H2B Intentisity Step 1. Propionylation Step 2. Trypsin digestion Step 3. Propionylation Intentisity ΔEZL2 H4 H2A H3 ΔTXR1 RT M/Z Equal mixture of L&H histones N15 WT RP-18 HPLC separation NanoLC-MS/MS analysis

  2. Figure S2. Histone variants, sequence coverage, and their PTMs a. H2A variants H2A   --------STTGKGGKAKG-KTASSKQVSRSARAGLQFPVGRISRFLKNGRYS-ERIGTG 50 H2A.X  --------STTGKGGKAKG-KTASSKQVSRSARAGLQFPVGRISRFLKHGRYS-ERVGTG 50 H2A.Z  AGGKGGKGGKGGKGGKVGGAKNKKTPQ-SRSYKAGLQFPVGRIHRFLKGRVSAKNRVGAT 59 H2A APVYLAAVLEYLAAEVLELAGNAAKDNKKTRIVPRHILLAIRNDEELNKLMANTTIADGG 110 H2A.X APVYLAAVLEYLAAEVLELAGNAAKDNKKTRIVPRHILLAIRNDEELNKLMANTTIADGG 110 H2A.Z AAVYAAAILEYLTAEVLELAGNASKDFKVRRITPRHLLLAIRGDEEL-DILIKATIAGGG 118 H2A   VLPNINPMLLPSKTKKS-----TEPEH  132 H2A.X  VLPNINPMLLPSKSKKTESRGQASQDL  137 H2A.Z  VIPHIHKALLGKHSTKNRSSAKTAEPR  145 Sequence coverage: 60% # of PTMs identified (17 PTMs): H2A.X/H2A.1:K5Ac, K8Ac, K10Ac, K12Ac, S1/T2/T3Phos*, R33Me1, R36Me2, K39Form, K42Me1, R92Me1 H2A.Z:K4Ac,K7Ac,K10Ac,K13Ac,K16Ac,K21Ac,K140Ac # of PTMs quantified (13 PTMs): H2A.X/H2A.1:K5Ac, K8Ac, K10Ac, K12Ac, K39Form, R92Me1 H2A.Z:K4Ac,K7Ac,K10Ac,K13Ac,K16Ac,K21Ac,K140Ac *Localization is difficult due to incomplete fragment ion information

  3. b. H2B variants H2B1 APKKAPAAAAEKKVKKAPTTEKKNKKKRSETFAIYIFKVLKQVHPDVGISKKAMNIMNSF 60 H2B2 APKKAPAATTEKKVKKAPTTEKKNKKKRSETFAIYIFKVLKQVHPDVGISKKAMNIMNSF 60 H2B1 INDSFERIALESSKLVRFNKRRTLSSREVQTAVKLLLPGELARHAISEGTKAVTKFSSST 120 H2B2 INDSFERIALESSKLVRFNKRRTLSSREVQTAVKLLLPGELARHAISEGTKAVTKFSSSS 120 H2B1 N 121 H2B2 N 121 Sequence coverage: 68% # of PTMs identified (12 PTMs): H2B-1: A1Me1, K3Me1/Me2/Me3, K4Ac, K115Ac, (S117-T120)Phos* H2B-2: A1Me1, K3Me1/Me2/Me3, K4Ac # of PTMs quantified (10 PTMs): H2B-1: A1Me1, K3Me1/Me2/Me3, K4Ac, K115Ac, (S117-T120)Phos* H2B-2: K3Me2/Me3, K4Ac *Localization is difficult due to incomplete fragment ion information

  4. c. H3 variants H3 H3.3 H3.4 H3 H3.3 H3.4 H3 H3.3 H3.4 ARTKQTARKSTGAKAPRKQLASKAARKSAPATGGIKKPHRFRPGTVALREIRKYQKSTDL 60 ARTKQTARKSTGVKAPRKQLATKAARKSAPVSGGVKKPHKFRPGTVALREIRKYQKTTDL 60 ARTKQTARKSTSIKAPRKQLAAKAARKSAPISGGIKKPHKFRPGTVALREIRKYQKTTDL 60 LIRKLPFQRLVRDIAHEFKAELRFQSSAVLALQEAAEAYLVGLFEDTNLCAIHARRVTIM 120 LIRKLPFQRLVRDIAMEMKSDIRFQSQAILALQEAAEAYLVGLFEDTNLCAIHARRVTIM 120 LIRKLPFQRLVRDIAMEMKSDIRFQSQAILALQEAAEAYLVGLFEDTNLCAIHARRVTIM 120 TKDMQLARRIRGERF 135 TKDLHLARRIRGERF 135 TKDLHLARRIRGERF 135 Sequence coverage: 76% # of PTMs identified (32 PTMs): H3: K4Me1/Me2/Me3, K9Ac, K14Ac, K14Me1, K18Ac, K23Ac, K23Me1/Me2/Me3, K23Form, K27Me1/Me2/Me3, K27Ac, K36Me1/Me2, R40Me1, R49Me1, K56Me1, K79Me1, R83Me1, R128Me1 H3.3: K9Ac, K14Ac, K14Form, K18Ac, K23Ac, K23Form, K56Ac, K79Me1 # of PTMs quantified (25 PTMs): H3: K4Me1/Me3, K9Ac, K14Ac, K18Ac, K23Ac, K23Me1/Me2/Me3, K23Form, K27Me1/Me2/Me3, K27Ac, K36Me1, K56Me1, R83Me1, R128Me1 H3.3: K9Ac, K14Ac, K14Form, K18Ac, K23Ac, K23Form, K56Ac

  5. d. H4 AGGKGGKGMGKVGAKRHSRKSNKASIEGITKPAIRRLARRGGVKRISSFIYDDSRQVLKS 60 FLENVVRDAVTYTEHARRKTVTAMDVVYALKRQGRTLYGFGG 102 Sequence coverage: 97% # of PTMs identified (11 PTMs): H4: K4Ac, K7Ac, K7Ub, K11Ac, K15Ac, R35Me1, R55Me1, R67Me1, R77Me1, K91Me1, R92Me1 # of PTMs quantified (7 PTMs): H4: K4Ac, K7Ac, K11Ac, K15Ac, R55Me1, R77Me1, K91Me1

  6. Figure S3. All PTMs identified by tandem MS H2A.X-K5Ac 1STTGKGGKAK10 [M-2H2O+2H]2+ b8 b9 y2 y1 y3 b6 y4 b7 y5 M/Z: 572.8141, Charge: 2+, Mass Error: 0ppm, Ion Score: 23

  7. H2A.X-K5AcK8AcK10AcK12Ac 1STTGKGGKAKGKTASSKQVSR21 [M-2H2O+2H]2+ y7 b14-H2O y11 y9 y8 y6 b17-H2O y4 y5 y3 b13 b15-H2O b18 b16 b16-H2O y13 y12 M/Z: 1172.628, Charge: 2+, Mass Error: 1ppm, Ion Score: 54

  8. H2A.X-K5AcK8AcK10Ac 1STTGKGGKAKGKTASSKQVSR21 [M-2H2O+2H]2+ y19-NH3 y7 b20 y4 y6 b7-NH3 y8-H2O y2 y5 y3 y9 y8 y10 b8 b12 M/Z: 786.7592, Charge: 3+, Mass Error: 0ppm, Ion Score: 34

  9. H2A.X-K5AcK8AcK12Ac 1STTGKGGKAKGKTASSKQVSR21 [M-2H2O+2H]2+ y11 y7 b12 b14-H2O y9 y6 y8 b17-H2O b18-H2O y12 b15 y4 y13 b16-H2O y3 y5 b18 M/Z: 1179.6361, Charge: 2+, Mass Error: 1ppm, Ion Score: 69

  10. H2A.X-K5AcK8Ac 1STTGKGGKAKGKTASSKQVSR21 [M-2H2O+2H]2+ y9 b12 y7 y11 b14-H2O b10 b15-H2O y6 y8 y4 b13 y5 y16 y12 y13 b15 b14 M/Z: 1186.6435, Charge: 2+, Mass Error: 1ppm, Ion Score: 101

  11. H2A.X-STT(1-3)Phos 1STTGKGGKAKGKTASSKQVSR21 [M-H2O+2H]2+ b10-NH3 b17 b6 y9 y7 b9 y11 b12 b13 b14 b16 b5 y6 b15 M/Z: 1212.6277, Charge: 2+, Mass Error: 1ppm, Ion Score: 43

  12. H2A.X-K10AcK12Ac 6GGKAKGKTASSKQVSR21 y11 y9 y7 y8 b14 y6 b7 y4 b5 b14-H2O b12-H2O y3 y5 y10 b13 b15 b12 y2 b10 b11 b15-H2O b6 M/Z: 893.4959, Charge: 2+, Mass Error: 2ppm, Ion Score: 72

  13. H2A.X-K10Ac 6GGKAKGKTASSKQVSR21 [M-2H2O+2H]2+ y7 y9 b14 y4 y11 y6 y5 b15 b7 y10 b13 b14-H2O b6 y2 y3 b10 b12 y12 M/Z: 900.504, Charge: 2+, Mass Error: 2ppm, Ion Score: 79

  14. H2A.X-K12Ac 6GGKAKGKTASSKQVSR21 [M-H2O+2H]2+ y7 y9 y4 b6-NH3 b14 y11 b15 y5 b10 b12 b6 b14-NH3 b5 y6 b12-NH3 b11 b13 y10 y13 M/Z: 912.4688, Charge: 2+, Mass Error: 2ppm, Ion Score: 58

  15. H2A.X-R33Me1 25AGLQFPVGR33 [M-H2O+2H]2+ y5 y6 y4 b5 b3 y2 b4 b7 y1 b6 y3 y8 M/Z: 514.2709, Charge: 2+, Mass Error: 0ppm, Ion Score: 30

  16. H2A.X-R36Me2 34ISRFLKHGR42 [M-H2O+2H]2+ y8,2+ y3 y4 y6 y5 y8 y7,2+ b7 b3-H2O y2 b6 b5 y7 b8 M/Z: 627.3735, Charge: 2+, Mass Error: -6ppm, Ion Score: 43

  17. H2A.X-R42Me1 37FLKHGR42 y3 y5,2+ y4 y5 y2 [M-H2O+2H]2+ b1 b4 b3 b2 b5 M/Z: 448.2434, Charge: 2+, Mass Error: 0ppm, Ion Score: 39

  18. H2A.X-K39Form 37FLKHGR42 y5,2+ y4 [M-2H2O+2H]2+ y5 y3 y2 y1 b5-NH3 b4,2+ b4-NH3 b1 b2 b5 M/Z: 421.2374, Charge: 2+, Mass Error: 0ppm, Ion Score: 36

  19. H2A.X-R92Me1 86HILLAIR92 b5 b4 b3 b2 [M-H2O+2H]2+ b6 y4 y5 y3 y6 M/Z: 917.5589, Charge: 1+, Mass Error: 2ppm, Ion Score: 53

  20. H2A.Z-K7AcK10Ac 1AGGKGGKGGKGGK13 y9 y11,2+ y10,2+ y8 b7 b10 b3 y3 b11 y11 b5 y7 b4 y5 b9 b12 y4 M/Z: 571.8189, Charge: 2+, Mass Error: 3ppm, Ion Score: 58

  21. H2A.Z-K4AcK7AcK10Ac 1AGGKGGKGGKGGK13 y9 y11,2+ y8 y6 y10,2+ b10 y3 b7 y7 b4 b5 b11 b12 y4 b6 y5 y11 b9 b8 M/Z: 592.8227, Charge: 2+, Mass Error: 0ppm, Ion Score: 68

  22. H2A.Z-K4AcK7AcK10AcK13Ac 1AGGKGGKGGKGGKGGK16 y9 y12 y6 y14,2+ b13 b10 y11 b7 y8 y10 y5 y3 b14 b4 b15 y4 b11 b12 y14 b5 b9 M/Z: 734.897, Charge: 2+, Mass Error: 0ppm, Ion Score: 77

  23. H2A.Z-K4AcK7AcK10AcK13AcK16Ac 1AGGKGGKGGKGGKGGKVGGAK21 y14 y14,2+ y11 b17 y8 y19,2+ b16 y17 y4 b13 y5 b7 y17,2+ y13 b18 y10 y9 y15 y12 y7 b11 b20 b12 b5 b4 b19 b15 M/Z: 962.0239, Charge: 2+, Mass Error: 0ppm, Ion Score: 97

  24. H2A.Z-K4AcK7AcK10AcK13AcK16AcK21Ac 1AGGKGGKGGKGGKGGKVGGAKNK23 b17,2+ y6 b16,2+ y7 y16,2+ b13 b10 y10 b18,2+ b12 y5 y18,2+ y19,2+ b4 b7 b14 b16 b17 b6 y2 y3 b15 y11 y13 b18 M/Z: 736.4026, Charge: 3+, Mass Error: 2ppm, Ion Score: 91

  25. H2A.Z-K7AcK10AcK13Ac 5GGKGGKGGKGGK16 y14,2+ y9 y6 y10,2+ b3 b9 b6 y8 y3 y7 y5 b4 b11 b5 b10 b7 y4 y10 M/Z: 557.3039, Charge: 2+, Mass Error: 0ppm, Ion Score: 67

  26. H2A.Z-K7AcK10AcK13AcK16Ac 5GGKGGKGGKGGKVGGAK21 y11 y8 b13 y14 y4 b6 y15,2+ b9 b16 y14,2+ y10 y5 b3 y13 y7 y9 b12 y12 b14 b5 b15 y3 M/Z: 784.4312, Charge: 2+, Mass Error: 0ppm, Ion Score: 78

  27. H2A.Z-K7AcK10AcK13AcK16AcK21Ac 5GGKGGKGGKGGKVGGAKNK23 b6 y7 y13,2+ b9 y16,2+ y5 b12 b14,2+ y2 y3 b10 b17,2+ b3 b13 y10,2+ y7,2+ y14,2+ y10 y8 b5 y12 M/Z: 618.005, Charge: 3+, Mass Error: -2ppm, Ion Score: 61

  28. H2A.Z-K13AcK16Ac 8GGKGGKGGKVGGAK21 y11 y8 [M-H2O+2H]2+ b6 y12,2+ b10 y10 y4 b13 b9 y5 y7 y9 b12 b4 b5 b11 y3 M/Z: 621.3515, Charge: 2+, Mass Error: 0ppm, Ion Score: 55

  29. H2A.Z-K13AcK16AcK21Ac 8GGKGGKGGKVGGAKNK23 y6 [M-H2O+2H]2+ b10 y10 y14,2+ y13 b6 b14 b13 b9 y9 y8 y3 y13,2+ b15 b11 y11,2+ y12 y2 y11 b12 b4 b5 y7 M/Z: 774.3933, Charge: 2+, Mass Error: 0ppm, Ion Score: 102

  30. H2A.Z-K10AcK13AcK16AcK21Ac 8GGKGGKGGKVGGAKNK23 y10 y7 y13 y9 b6 b9 [M-H2O+2H]2+ y3 b13 b15 y13,2+ y8 b3 y12 y12,2+ y6 y11 b10 y14,2+ b11 b4 b14 b5 y4 y5 M/Z: 784.4318, Charge: 2+, Mass Error: 1ppm, Ion Score: 81

  31. H2A.Z-K16AcK21Ac 11GGKGGKVGGAKNK23 y10 y7 [M-H2O+2H]2+ b6 y11,2+ y9 b12 y3 b10 y8 b11 y2 b5 y5 b3 y4 b9 y11 M/Z: 621.351, Charge: 2+, Mass Error: -1ppm, Ion Score: 63

  32. H2A.Z-K16Ac 14GGKVGGAK21 b7-H2O y4 y5 b3 b7 y6 b4 y3 y2 M/Z: 725.3801, Charge: 1+, Mass Error: 0ppm, Ion Score: 31

  33. H2A.Z-K140Ac 137SSAKTAEPR145 [M-H2O+2H]2+ y2 y5 b7 y3 b7-H2O y7,2+ y6 y4 b4 b8 y7 y1 b3 M/Z: 494.7563, Charge: 2+, Mass Error: 0ppm, Ion Score: 66

  34. H2B-1-A1Me1K3Me1 1APKKAPAAAAEK12 b11 b9 b10 b8 y4 [M-2H2O+2H]2+ b7 [M-H2O+2H]2+ y2 y3 b11,2+ y2-NH3 y1 b6,2+ y5 y8 y7 y9 M/Z: 682.373, Charge: 2+, Mass Error: 0ppm, Ion Score: 52

  35. H2B-1-K3Me2 1APKKAPAAAAEK12 b8,2+ b9 y3 b9,2+ b5 b7 y8 b10 b11 y2-H2O b8 b3-NH3 y9 y11 b2 M/Z: 646.8825, Charge: 2+, Mass Error: 0ppm, Ion Score: 34

  36. H2B-1-K3Me3 1APKKAPAAAAEK12 b7,2+ b8,2+ b9 b10 b8 b11 b7 b9,2+ b4 [M-H2O+2H]2+ y2-H2O y3 y5 y7 b6,2+ y2 y8 M/Z: 653.8902, Charge: 2+, Mass Error: 0ppm, Ion Score: 56

  37. H2B-1-A1Me1 1APKKAPAAAAEKK13 y12,2+ y8 y9 y11,2+ b4 b5 y10 b11 y2 b9,2+ [M-H2O+2H]2+ b9 y5 b7 y11 y4 b10 y12 b3 y3 M/Z: 731.9352, Charge: 2+, Mass Error: 0ppm, Ion Score: 47

  38. H2B-1-K3Me3K4Ac 1APKKAPAAAAEKK13 b11 b9 b10 b8 b8,2+ y2 y12-H2O [M-2H2O+2H]2+ b7 y6 y3-H2O y4 b7,2+ y5 y8 b6,2+ y9 y10 M/Z: 738.9436, Charge: 2+, Mass Error: 1ppm, Ion Score: 53

  39. H2B-1-K115Ac 104HAISEGTKAVTKFSSSTN121 b10 [M-2H2O+2H]2+ y14-NH3 y10 y15-NH3 b11 b8 y15 y8 y13 y16,2+ y11 y17 y7 y14 b7 y12 b6 M/Z: 1010.0059, Charge: 2+, Mass Error: 1ppm, Ion Score: 40

  40. H2B-1-SSST(117-120)Phos 104HAISEGTKAVTKFSSSTN121 [M-H2O+2H]2+ b10 y13 y15 b3 y8 y14 y17,+2 b11 y7,+2 y10 y11 y13,+2 b3-H2O y16 y17 M/Z: 1028.9841, Charge: 2+, Mass Error: 1ppm, Ion Score: 38

  41. H2B-2-K3Me3 1APKKAPAATTEK12 [M-2H2O+2H]2+ b11 b9 b5 b10 b8 y5 b7 y4 y7 y2 y3 y1 M/Z: 719.3925, Charge: 2+, Mass Error: 1ppm, Ion Score: 44

  42. H2B-2-A1Me1 1APKKAPAATTEKK13 b6 y8 y9 b5 b4 y10 y2 b11 y7 y5 b3 b7 b8 b9 b12 b10 M/Z: 761.9465, Charge: 2+, Mass Error: 1ppm, Ion Score: 54

  43. H2B-2-K3Me2 1APKKAPAATTEKK13 b11 [M-2H2O+2H]2+ [M-H2O+2H]2+ b8 y2 b9 b5 b12 b10 y7 y4 y8 b7,2+ y3 y12 y5 y9 b2 M/Z: 768.9542, Charge: 2+, Mass Error: 1ppm, Ion Score: 49

  44. H2B-2-K3Me3 1APKKAPAATTEKK13 b11 b6 b8 y2 b9 b12 b5 b10 b7 y8 b3 y4 y2-NH3 y9 y3-NH3 b11-NH3 b8,2+ M/Z: 784.4372, Charge: 2+, Mass Error: 1ppm, Ion Score: 58

  45. H2B-2-K3Me3K4Ac 1APKKAPAATTEKK13 b11 [M-H2O+2H]2+ [M-2H2O+2H]2+ y2 b8 b10 b9 b12 b5 y7,2+ y2-NH3 y4 y7 y8 y5 b8,2+ y6,2+ y9 y3-NH3 y10 b11-NH3 y3 M/Z: 777.4291, Charge: 2+, Mass Error: 1ppm, Ion Score: 51

  46. H3-K4Me1 3TKQTAR8 [M-H2O+2H]2+ y4 [M-2H2O+2H]2+ y5,2+ y1 b2 y3 b3 y5-NH3 y4-H2O y5 b1 y2 M/Z: 387.7271, Charge: 2+, Mass Error: 0ppm, Ion Score: 33

  47. H3-K4Me2 3TKQTAR8 [M-H2O+2H]2+ [M-2H2O+2H]2+ b5 y1 y5,2+ y4 b4 b5-H2O b2 y5 y3 b3 y2 M/Z: 400.2186, Charge: 2+, Mass Error: 0ppm, Ion Score: 23

  48. H3-K4Me3 3TKQTAR8 y5-NH3 [M-H2O+2H]2+ y1 [M-2H2O+2H]2+ b4 b1 b3 y5 b2 y2 y4 y3 M/Z: 401.743, Charge: 2+, Mass Error: 1ppm, Ion Score: 19

  49. H3-K9Ac 9KSTGAKAPR17 [M-H2O+2H]2+ [M-2H2O+2H]2+ y8 y2 y3 b1 y7 y6 b4 y4 b7 b2 b5 y5 y1 b3 b6 b8 M/Z: 535.3029, Charge: 2+, Mass Error: -1ppm, Ion Score: 61

  50. H3-K14Ac 9KSTGAKAPR17 [M-2H2O+2H]2+ [M-H2O+2H]2+ y2 y7 y3 y7-H2O y6 y8 y1 y5 b3 b6 b8 y4 M/Z: 507.2902, Charge: 2+, Mass Error: -1ppm, Ion Score: 35

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