In this report, we detail a one-pot method to quantitatively analyze the majority of histone proteins and PTMs in a single MS experiment (). Traditional methods involve extensive purification through HPLC or SDS-PAGE prior to MS analysis of each individual histone variant (, left panel). While very effective, these methods are lengthy and prone to several drawbacks such as inherent sample loss in HPLC methods, while SDS-PAGE suffers from being more laborious work for extracting peptides as well as potentially inducing exogenous chemical modification artifacts on proteins that may be mistaken for endogenous modification.34
These off-line fractionation methods prior to MS analysis also vastly reduce the high-throughput abilities of the entire platform and increase the starting material amount needed, especially for HPLC purification. Our streamlined protocol entails direct derivatization and stable isotope labeling of bulk histones in unfractionated total acid extracts by two rounds of propionylation using either d0
-propionyl anhydride followed by comparative analysis through LC-MS/MS using CAD alone or together with ETD fragmentation (“decision tree” driven fragmentation used to choose in real time whether to perform either ETD or CAD based on precursor m/z
and charge state) 32
to select the optimum fragmentation method for all peptides (, right panel). Through our method, we are able to reduce sample loss, preparation time and MS acquisition time.
Figure 1 Flowchart showing the characterization of histone PTMs through chemical derivatization and stable isotope peptide labeling. Histones are acid extracted from nuclei and can be processed through two different methods. One of these methods (standard approach) (more ...)
To test this one-pot approach, we compared MS experiments from HPLC purified individual histones against unfractionated bulk histones isolated from total acid extracts. Identical acid extracts derived from the same number of HeLa cells were subjected to either HPLC purification or directly processed by our one-pot propionylation method. For one-pot propionylation, ~5% of the total amount of acid extract (~ 5 µg) was used to obtain sufficient material for MS analysis. In the traditional approach, 100% of the acid extract (~ 100 µg) was used for analytical scale HPLC purification and approximately 1 µg of individual histone was used for further analysis. Our initial objective was to determine if we could detect the same post-translational modifications on histones H3 and H4, as well as a similar number of histone H2A and H2B variants using both approaches. We chose these goals, as the vast majority of histone PTMs are higher in number and abundance on histones H3 and H4, while the complexity on histones H2A and H2B is derived from the multiple variant family members with modest to low level PTMs. Overall, our one-pot approach on total acid extracts is able to detect a similar number of histone H2A and H2B variants identified through analysis of HPLC purified histones (Supplemental Table 1
). Our results show that our method can identify many histone H2A/H2B variants in a single two hour run demonstrating that the one-pot approach does not significantly qualitatively suffer from a predicted decrease in dynamic range or sensitivity. Additionally, and of equal importance, we demonstrate that HPLC or SDS-PAGE purification of individual histone family members prior to Bottom Up MS analysis is no longer absolutely required for histone analysis. With regards to histone H3 PTMs, our new protocol is able to detect virtually all of the methylation and acetylation sites that are detected on individual histone H3.2 purified through the traditional HPLC-purification method (Supplemental Table 2
). We are also able to detect all acetylation sites on histone H4, plus all methylation states on K20 (data not shown). In agreement with prior reports,35
we find ETD MS/MS are more effective than CAD for characterizing longer or higher charged peptides (data not shown).
Another more analytical objective of our experiments was to make sure that we could obtain the same quantitative content from the one-pot approach as could be obtained from analysis of purified histones. This was an initial concern for us as in the one-pot approach we have a more complex mixture of peptides generated from many histone proteins potentially resulting in ion suppression effects, especially for less abundant modified peptides. To examine this possibility, we decided to quantitate the various modified forms of a histone H3 peptide. shows data from the 9–17 residue fragment (KSTGGKAPR) from histone H3 obtained through both the one-pot and standard MS approaches. This particular peptide is somewhat challenging because it spans two modification sites (K9 and K14) that can be modified with all possible degrees of methylation on K9 and acetylation on K9 or K14 in all several combinations. Furthermore, these modified peptides are usually in lower abundance (~5X) than most other H3 peptides due to signal dilution across the many modified forms. Shown in are the base peak and extracted ion chromatograms (XICs) for several modified forms of the 9–17 peptide obtained through the standard (HPLC purified H3.2 variant, ) and one-pot MS analyses (Acid Extract, ). The H3.2 variant was chosen, as it is arguably the most abundant H3 variant in human cells. As can be seen, the base peak chromatogram of purified histone H3.2 compared to a raw acid extract is markedly different, and as expected the acid extract chromatogram has many abundant peaks resulting from other non-histone H3 peptides (, first panel). Nevertheless, the peaks for the particular histone modified forms of the H3 9–17 peptides have similar retention time patterns (, lower panels). As mentioned before, all modified forms of this peptide observed in the purified sample are also detected in the total acid extracted sample. Despite some variation in the elution times, the overall retention time pattern for the various modified forms persists between samples.
Figure 2 Total ion chromatogram of the base peak and extracted ion chromatograms for various modified peptides ([M+2H]2+ ions) spanning the 9–17 residues KSTGGKAPR for (a) HPLC purified histone H3.2 and (b) whole acid total histone extracts after chemical (more ...)
Relative quantification of the histone peptides can be accomplished by measuring the area under the XIC peak corresponding to a specific peptide and expressing that as a fraction of the total sum of the peak areas corresponding to all observed modified forms. The relative abundance values for K9 and K14 modifications are shown in . Abundance values for all the modified forms of the 9–17 peptide of histone H3 overlap well between samples within a standard deviation. The average standard deviation of peptide abundances across all modified 9–17 peptides is ±1.76 for the HPLC purified H3 sample, and ± 1.87 for the total acid extracted one-pot sample. Similar results were obtained for many other histone H3 peptides (data not shown). Therefore, we feel that we can obtain similar quantitative information for histone peptides from acid extracted total histones analyzed by our one-pot shotgun approach as one would through the analysis of purified histones. However, it is important to note that our method is unable to link PTMs to specific variants for some histones, including some H2A and all H3 members. Consequently, our one-pot protocol is not appropriate for applications in which this information is sought, and thus the traditional method must be used to separate out specific H2A, and H3 variants followed by MS interrogation to gather this PTM information. Additionally, if endogenous histone propionylation36
is the main research emphasis, then a different approach should be employed.
Table 1 Relative quantification of individual post-translational modifications on the histone H3 peptide (K9STGGK14APR) from HPLC purified and one-pot methods shown in . Relative quantification of histone modifications was achieved by measuring the area (more ...)
We then desired to improve the relative quantification of histone peptides through Bottom Up analysis across multiple samples by the integration of a stable isotope labeling step into the second propionylation derivatization. Normally, a second round of propionylation derivatization using d0
-propionic anhydride is performed after trypsin digestion to cap the newly generated N-termini of the peptides with a propionyl amide bond that improves retention on C18 columns and helps limit the charge on the peptides (a very good procedure for producing mostly 2+ peptides for CAD fragmentation). For a comparative one-pot shotgun approach, we use d10
-propionic anhydride on one of the samples in the second round of derivatization to incorporate a stable isotope d5
-propionyl amide label on the newly formed free peptide N-termini. The resulting peptides from two samples (d0
-propionyl amides) can be mixed and analyzed together through LC-MS/MS. Histone PTM levels between samples can be directly compared as peptides from the d0
-propionyl and d5
-propionyl will appear as peak doublet pairs separated by a +5 Da mass difference. For doubly and triply charged peptides, this mass difference translates into a 2.5 or 1.67 m/z
shifts, respectively. This labeling has been previously used to measure phosphorylation stoichiometry through propionylation of the N-termini of all peptides in a mixture.26
Our labeling improves on traditional methods by combining the second propionylation step with the isotopic-labeling step, as previous methods involve a second d0
-propionylation step followed then by an isotopic labeling esterification step of carboxylic acid groups.10
We demonstrate the utility of this labeling to investigate the differences in histone PTMs profiles from wild-type and set2
deletion Saccharomyces cerevisiae
strains. Set2 is the only methyltransferase responsible for the mono-, di- and trimethylation of histone H3 K36 in yeast.37, 38
We used our one-pot approach to explore the effects of set2
deletion on K36 methylation levels for wild type (d0
-labeled) and mutant (-set2
-labeled) yeast strains. Unmodified K36 levels are found to be higher in the Set2 knockout strain (). Unsurprisingly, we observe that all degrees of methylation on K36 are abolished in the set2
deletion strain compared to the wild type yeast sample (). Also as expected, we did not detect any other significant changes in the modification level of other well known methylation sites on yeast histone H3 at either K4 or K79 (). In yeast, these sites are methylated by methyltransferases Set1 and Dot1 respectively, and thus deletion of Set2 does not affect modification of these sites.39
In contrast, we observe changes in the extent of acetylation on particular sites on both histone H3 and H4. Through our quantitative procedures, we determined that Set2 knockout also results in a 2-fold and 3-fold increase for the mono-and diacetylated forms of the histone H4 4–17 peptide respectively (). A smaller but reproducible effect occurs on histone H3 K23 and K18 acetylation, which increase in the Set2 deletion strain as well (). Set2 has been recently implicated in the regulation of histone deacetylation, as the HDAC Rpd3S recognizes the Set2 methylated histones and deacetylates histones within transcribed sequences.38, 40
Rpd3S is one of two forms of Rpd3, and S. cerevisiae
Rpd3 is involved in global, untargeted histone deacetylation.38, 41, 42
deletion strains deficient in K36 methylation have higher histone acetylation amounts resulting from the lack of recruitment of Rpd3 to nucleosomes.43
The increased histone H4 and H3 acetylation levels we observe in set2
deletion strains are consistent with this previous observation. Through this example, we show that our streamlined method is capable of detecting direct and even minor secondary histone PTM changes in biologically complex samples.
Figure 3 Side-by-side comparison of K36 methylation levels for wild type (d0-labeled) and Set2 knockout (d5-labeled) yeast strains as determined through a one-pot shotgun approach. Full Mass spectrum for the [M+3H]3+ peptide ions (27–40 residues, KSAPSTGGVKKPHR) (more ...)
Figure 4 Relative abundance of post-translational modified histone peptides for Set2 knockout and wild type yeast strains on histone (a) H3 and (b) H4. As before, quantification of histone modifications was achieved by measuring the area under the chromatogram (more ...)