Peptide-mass fingerprinting (PMF) for the characterization of histone variants

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1 BioScience Trends. 26; (5): Supporting Information Supplementary Materials and Methods Peptide-mass fingerprinting (PMF) for the characterization of histone variants For the digestion, each fraction containing histone proteins was incubated with buffer (2mmol Tris-HCl, ph 8.) and trypsin (. µg/µl) at 37 C for 2-5 hours. After the incubation, these samples were mixed at a ratio of.5: (v/v) with the matrix (α-cyano-4-hydroxycinnamic acid in 3% ACN/.% TFA). The characterization of histone variants were performed by UltrafleXtreme MALDI-TOF/TOF mass spectrometer (Bruker Daltonics, Billerica, MA, USA) equipped with a Smartbeam II laser. MS data was conducted in the reflectron mode at the range of 4-5, m/z with the 25. kv acceleration voltage. The lens voltage was set to 8. kv and kv was used for the reflector voltage. We used LIFT mode to acquire MS/MS data from precursor ions of MS spectra. Ions were accelerated at 7.5 kv and the lens voltage was 3.5 kv. A 29.5 kv was used as the reflector voltage and the LIFT voltage was set to 9. kv. H3 variants were characterized using LC-MS. The sample was analyzed by a Q-Exactive mass spectrometer (Thermo-Fisher Scientific, Bremen, Germany) using a linear gradient of -% solvent B over 3 min (solvent A:.% TFA; solvent B: 8% acetonitrile with.% TFA). MS was directed using Xcalibur software version (Thermo-Fisher Scientific). A nanoelectrospray (Thermo-Fisher Scientific) was applied with a.9 kv spray voltage and a capillary temperature of 275 C. Mass spectra (at the range of 3-2, m/z) were acquired with a resolution of 7, at m/z 4 Da and MS/MS spectra were obtained in a data-dependent manner. Spectra of histone peptides were searched against a protein database (NCBInr and SwissProt) using the MASCOT program (version 2.5, Matrix Science, UK).

2 BioScience Trends. 26; (5): Supplementary Table. List of histone peptides from mouse testis and epididymis # Testis Retention Peptides (Observed masses) Miss Histone Score Time (min) Cleavage KKPAGPSVSELIVQAVSSSK (22.268) H ASGPPVSELITK (98.784) KASGPPVSELITK ( ) SGVSLAALK ( ) SGVSLAALKK ( ) ALAAAGYDVEK (7.695) ALAAAGYDVEKNNSR ( ) IKLGLK ( ) KPAGAAKKPK ( ) KASGPPVSELITK ( ) ASGPPVSELITK ( ) SGVSLAALKK ( ) ALAAAGYDVEK (7.5624) ALAAAGYDVEKNNSR ( ) IKLGLK ( ) GILVQTK ( ) H.4 6 H LIPEALSTSQER ( ) Ht KESYSVYVYK ( ) ESYSVYVYK (37.662) QVHPDTGISSK (68.724) AMGIMNSFVNDIFER ( ) LAHYNKR (9.5938) STITSR ( ) EIQTAVR ( ) LLLPGELAK ( ) HKQGGK ( ) AGLQFPVGR (944.55) H2B 7 H2A 69

3 BioScience Trends. 26; (5): VGAGAPVYMAAVLEYLTAEILELAGNAAR ( ) HLQLAIR (85.556) HLQLAIRNDEELNK ( ) VTIAQGGVLPNIQAVLLPK (93.228) TESHHK ( ) IGAGAPVYLAAVLEYLTAEVLELAGNAAR ( ) TH2A AGLQFPVGR ( ) VGAGAPVYLAAVLEYLTAEILELAGNAAR ( ) HLQLAIR( ) HLQLAIRNDEELNK ( ) NDEELNKLLGR (3.74) VTIAQGGVLPNIQAVLLPK (93.926) TESHHK ( ) H2A AMSIMNSFVTDIFER ( ) TH2B 82 VGAGAPVYLAAVLEYLTAEILELAGNAAR ( ) H2A DNIQGITKPAIR ( ) RISGLIYEETR ( ) ISGLIYEETR (8.627) ISGLIYEETRGVLK ( ) VFLENVIR ( ) VFLENVIRDAVTYTEHAK (25.54) TVTAMDVVYALKR ( ) TLYGFGG ( ) LPFQR ( , +2) VTIMPK ( , +2; ) DIQLAR ( , +2; 75.4) SAPATGGVK ( ) KLPFQR ( ,+2; ) STELLIR (46.253,+2; ) RVTIMPK ( , +2; ) EIAQDFK ( , +2; ) KSAPATGGVK ( , +2; ) YRPGTVALR ( , +3; 56.85, +2; ) H4 68 H3.2 34

4 BioScience Trends. 26; (5): FQSSAVMALQEASEAYLVGLFEDTNLCAIHAK (52.952, +3) LPFQR ( , +2) VTIMPK ( , +2; ) DIQLAR (358.28, +2; ) KLPFQR ( , +2; ) SAPSTGGVK (42.265, +2; ) STELLIR (46.254,+2; ) RVTIMPK ( , +2; ) EIAQDFK ( , +2; 85.43) KSAPSTGGVK ( , +2; ) YRPGTVALR ( , +3; 56.83, +2; ) EIAQDFKTDLR ( , +2) FQSAAIGALQEASEAYLVGLFEDTNLCAIHAK ( , +3; , +4) H Q LATK (56.347) Cluster 68 IRGER ( , +2; ) 3,H3 LPFQR ( , +2) (Rattus VTIMPK ( ) norvegicus) KQLATK (344.72, +2; ) DIQLAR ( , +2; ) SAPATGGVK (394.29, +2; ) KLPFQR ( , +2; ) STELLIR (46.254, +2; ) RVTIMPK ( , +2; ) EIAQDFK (425.79, +2; ) KSAPATGGVK ( , +3; , +2; ) YHPGTVALR ( , +3; , +2; ) EIAQDFKTDLR (445.95, +3; , +2) FQSSAVMALQEACESYLVGLFEDTNLCAIHAK ( , +3) LPFQR (33.695, +2) VTIMPK ( , +2; ) DIQLAR ( , +2; 75.42) SAPATGGVK (394.29, +2; ) KLPFQR ( ,+2; ) H3. 459

5 BioScience Trends. 26; (5): STELLIR (46.254,+2; ) RVTIMPK ( , +2; ) EIAQDFK ( , +2; ) KSAPATGGVK ( , +2; ) YRPGTVALR (344.87, +3; 56.8, +2; ) EIAQDFKTDLR (445.96, +3; , +2) FQSSAVMALQEACEAYLVGLFEDTNLCAIHAK (58.225, +3) #Epididymis Retention Peptides (Observed masses) Miss Histone Score Time (min) Cleavage KTSGPPVSELITK ( ) TSGPPVSELITK ( ) SGVSLAALKK ( ) ALAAAGYDVEKNNSR ( ) IKLGLK (67.538) RAGAAK ( ) KPAGVRR( ) KASGPPVSELITK ( ) ASGPPVSELITK ( ) SGVSLAALKK( ) ALAAAGYDVEKNNSR ( ) IKLGLK (67.494) GILVQTK ( ) KESYSVYVYK ( ) ESYSVYVYK (37.468) QVHPDTGISSK (68.574) AMGIMNSFVNDIFER ( ) LAHYNKR (9.444) EIQTAVR ( ) LLLPGELAK ( ) LLLPGELAKHAVSEGTK ( ) H.4 56 H.2 4 H2B AGLQFPVGR ( ) H2A 94

6 BioScience Trends. 26; (5): VGAGAPVYMAAVLEYLTAEILELAGNAAR ( ) HLQLAIR (85.532) HLQLAIRNDEELNK ( ) VTIAQGGVLPNIQAVLLPK (93.927) TESHHK ( ) AGLQFPVGR ( ) VGAGAPVYLAAVLEYLTAEILELAGNAAR ( ) HLQLAIR(85.597) HLQLAIRNDEELNK ( ) NDEELNKLLGR (3.694) VTIAQGGVLPNIQAVLLPK (93.588) 72.9 DNIQGITKPAIR ( ) RISGLIYEETR ( ) ISGLIYEETR (8.6582) ISGLIYEETRGVLK ( ) VFLENVIR ( ) VFLENVIRDAVTYTEHAK (25.524) TVTAMDVVYALKR ( ) QLATK (56.343) IRGER ( , +2; ) LPFQR ( , +2; ) VTIMPK ( , +2; ) KQLATK (344.72, +2; ) DIQLAR ( , +2; ) SAPATGGVK ( , +2; ) KLPFQR ( ,+2; ) STELLIR (46.255,+2; ) RVTIMPK ( , +2; ) EIAQDFK ( , +2; 85.43) KSAPATGGVK ( , +2; ) STELLIRK ( , +2) YRPGTVALR ( , +3; 56.83, +2; ) SAPATGGVKKPHR ( , +3) H2A 99 H4 68 H3.2 44

7 BioScience Trends. 26; (5): EIAQDFKTDLR (445.92, +3; , +2) FQSSAVMALQEASEAYLVGLFEDTNLCAIHAK (52.926, +3) LPFQR ( , +2) VTIMPK ( ) DIQLAR ( , +2; 75.42) KLPFQR ( , +2; ) SAPSTGGVK (42.265, +2; ) STELLIR (46.25,+2; ) RVTIMPK ( , +2; ) EIAQDFK ( , +2; ) DIQLARR ( ) KSAPSTGGVK ( , +2; 93.52) YRPGTVALR ( , +3; 56.84, +2; ) YQKSTELLIR ( , +3; , +2) EIAQDFKTDLR (445.98, +2; , +2) VTIMPKDIQLAR ( , +3) FQSAAIGALQEASEAYLVGLFEDTNLCAIHAK ( , +3; , +4) 97.9 IRGER ( , +2) LPFQR ( , +2) VTIMPK ( , +2; ) KQLATK (344.72, +2; ) DIQLAR ( , +2; 75.4) SAPATGGVK ( , +2; ) KLPFQR ( ,+2; ) STELLIR (46.253,+2; ) RVTIMPK ( , +2; ) EIAQDFK ( , +2; 85.43) KSAPATGGVK ( , +2; ) YRPGTVALR ( , +3; 56.83, +2; ) EIAQDFKTDLR (445.99, +3; , +2) FQSSAVMALQEACEAYLVGLFEDTNLCAIHAK (58.266, +3; , +4) H H3. 75

8 BioScience Trends. 26; (5): Note: Histone variants were digested with trypsin. Peptides of histone variants were identified by PMT using MALDI-TOF-TOF/MS and LC-MS and then mass spectra were matched with the database in MASCOT (version 2.5, Matrix Science, UK).