Black et al. 10.1073/pnas.0700390104. |
Fig. 5. Side-by-side analysis of raw data for the H4 peptide from Fig. 2B (peptide retention time was in the range of 19-21 min in all cases) either from canonical nucleosomes (Left) or centromeric nucleosomes (Right). Dashed red and green lines are guideposts to highlight the differences in shifts generated by H/D exchange. Red arrows mark the location of the centroid value of the peptide after H/D exchange at the indicated timepoints determined using DXMS software.
Fig. 6. Slowing in H/D exchange measured for a peptide overlapping a large portion of the a3 helix from H4 (diagrammed in A; peptide retention time was in the range of 15-17 min in all cases) is observed in the centromeric nucleosome as compared to the canonical nucleosome (B). (C) Side-by-side analysis of raw data from B using the same labeling scheme as in SI Fig. 5 for this peptide (amino acids 85-102).
Fig. 7. H/D exchange profile of a CENP-A-containing nucleosome assembled with non-centromeric DNA (from sea urchin 5S rDNA). Multiple charge states were detected for a subset of peptides, and in these cases each charge state is represented by its own horizontal color-coded block.
Fig. 8. H/D exchange profile of a H3-containing nucleosome assembled with non-centromeric DNA (from sea urchin 5S rDNA). Multiple charge states were detected for a subset of peptides, and in these cases each charge state is represented by its own horizontal color-coded block.
Fig. 9. Side-by-side analysis of raw data from Fig. 3B (using the same labeling scheme as in SI Fig. 5).
Fig. 10. Comparison of H/D exchange from an identical peptide from CENP-A (peptide retention time was in the range of 16-18 min in all cases) from nucleosomes assembled with centromeric DNA (Left) or non-centromeric DNA (Right). Note that measurable exchange on this peptide occurs only at the final timepoint (1 ´ 106 s), regardless of which DNA is used to assemble the nucleosomes.
Fig. 11. H/D exchange profile of nucleosomes assembled with H3CATD. The CATD (labeled with a red bar above the schematic of the H3CATD chimeric protein) is comprised of loop 1 (L1) and the a2 helix from CENP-A. Multiple charge states were detected for a subset of peptides, and in these cases each charge state is represented by its own horizontal color-coded block.
Fig. 12. Comparison of H/D exchange from an identical peptide corresponding to residues 101-110 of CENP-A (peptide retention time was in the range of 18-20 min in all cases) from nucleosomes assembled with CENP-A (Left) or H3CATD (Right). Exchange for this peptide is negligible over the entire timecourse, with only a small amount of exchange detectable at 1 ´ 106 s, in either nucleosome.
Fig. 13. Comparison of H/D exchange from an identical peptide from histone H4 (amino acids 65-70; peptide retention time was in the range of 7-10 min in all cases) from CENP-A-containing nucleosomes (Left) or H3CATD-containing nucleosomes (Right).
SI Methods
DNAs Used for Nucleosome Reconstitution.
For the human a-satellite DNA the sequence is 5'-ACCCCTTTGAGGCCTTCGTTGGAAACGGGATTTCTTCATATTATGCTAGACAGAATAATTCTCAGTAACTTCCCTGTGTTGTGTGTATTCAATTCACAGAGTTGAACGATCCTTTACAGAGAGCAGACTTGAAACACTCTTTTTGTGGAATTTGCAAGTGGAGATTTCAGCCGCTTTGAGTTCAATGGTAGAATAGGAAATATCTTCC-3' (the CENP-B box is shown in bold and underlined) and for rDNA the sequence is 5'-GGTATTCCCAGGCGGTCTCCCATCCAAGTACTAACCGAGCCCTATGCTGCTTGACTTCGGTGATCGGACGAGAACCGGTATATTCAGCATGGTATGGTCGTAGGCTCTTGCTTGATGAAAGTTAAGCTATTTAAAGGGTCAGGGATTTATGACGTCATCGGCTTATAAATCCCTGGAAGTTATTCGTTG-3'. The a-satellite DNA monomer sequence was amplified by PCR from a plasmid provided by A. Prunell (Paris) using the following primers: forward, 5'-ACCCCTTTGAGGCCTTC-3'; reverse, 5'-GGAAGATATTTCCTATTCTACC-3'. The rDNA monomer sequence was amplified by PCR from a plasmid that contains a single monomer and was itself derived from p12 ´ 208 (1) (provided by K. Luger, Fort Collins). The following primers were used: forward, 5'-GGTATTCCCAGGCGGTCTCC-3'; reverse, 5'-CAACGAATAACT TCCAGGG-3'. For a typical DNA preparation, PCRs from multiple 96-well plates were pooled, DNA precipitated, resuspended in TE, and FPLC-purified on an anion-exchange column. These preparations yield ~1 mg of DNA templates purified to near-homogeneity.Peptide Identification and Analysis for H/D Exchange MS.
Tentative identifications were tested with specialized DXMS data reduction software (2-4) developed in collaboration with Sierra Analytics (Modesto, CA). This software searches MS1 data for scans containing each of the individual peptides identified in previous experiments with control samples prepared without any deuteration, selects scans with optimal signal-to-noise, averages the selected scans, calculates centroids of isotopic envelopes, screens for peptide misidentification by comparing calculated and known centroids, then facilitates visual review of each averaged isotopic envelope allowing an assessment of "quality" (yield, signal-to-noise, and resolution), and confirmation or correction of peptide identity and calculated centroid (3). Peptides that score highly in the DXMS program were then checked for matching of calculated versus known mass, charge state, and the retention time of the peptide on the C18 column, and peptides that satisfied these criteria were chosen for further analysis (see below). Preliminary studies established that denaturation with 2 M guanidine hydrochloride (final concentration) resulted in proteolytic fragmentation optimal for this study (data not shown). Some differences, in terms of the peptides included in the final analysis, from one experiment to another (or between two different sets of timepoint data for different types of nucleosomes) were caused by variations in the pool of peptides that met our requirements in each and every one of the timepoints within a given experiment, variations in overlapping peptides that obscured the peptide of interest, small changes in protease digestion patterns, or a combination of these. In no case, although, is this a reflection of a change in the H/D exchange that we are measuring.H/D exchange was assessed by either direct comparison of mass spectra or calculation of the percentage of exchange at each time-point. In the latter case, corrections for loss of deuterium-label by individual fragments during DXMS analysis (after "quench") were made through measurement of loss of deuterium from reference samples that had been equilibrium-exchange-deuterated under denaturing conditions as described previously (3). These samples were then subjected to the same proteolysis, chromatography, and detection steps used for the on-exchange samples. The percentage of exchange is shown for each peptide [colored blocks spanning the region of each histone diagramed and labeled for known (canonical nucleosomes; ref. 5) or predicted (CENP-A-containing nucleosomes) secondary structural features] at each time point as an individual color-coded bar that is then grouped with those from each of the other time points and assembled in descending order of time. Each charge state available for analysis was displayed separately, and these typically acted as replicate data sets, although there were a small number of exceptional cases where we observed small differences in our calculated percentage of exchange. In all cases, although, we used data from peptides that partially overlap in sequence coverage to confirm behavior seen in a particular peptide. For our peptide profiles, each color gradation (see keys in Fig. 1) represents a range of 10% points (0-10%, 10-20%, and so on). Smaller peptides can show different exchange profiles than that seen in a larger overlapping peptide. For example, the shorter peptide might exhibit less exchange than the larger peptide, and this would be due to the smaller peptide containing protected residues while the larger peptide contains quickly exchanging residues in addition to the slow exchanging residues.
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4. Hamuro Y, Zawadzki KM, Kim JS, Stranz DD, Taylor SS, Woods VL, Jr (2003) J Mol Biol 327:1065-1076.
5. Luger K, Mader AW, Richmond RK, Sargent DF, Richmond TJ (1997) Nature 389:251-260.