Previously, the relative levels of phosphorylation on specific H1 isoforms in asynchronously dividing mouse tumor cells following prolonged treatment with dexamethasone and after exposure of cells to staurosporine, a potent kinase inhibitor, were investigated using mass spectrometry.19,20
From these studies, it was determined that specific mouse histone H1 isoforms showed differential changes in phosphorylation based on treatment. These data pointed specifically to the mouse H1.3, H1.4, and H1.5 isoforms as targets of CDK-specific phosphorylation. This information suggests that the H1 isoforms may have differing functions. Thus, identification of the sites of phosphorylation of the specific isoforms may be important in defining their roles in such activities as transcription.
Identification of H1.4 Phosphorylation Sites in Mouse 1471.1 Cells
Initially, we have investigated the sites of phosphorylation on the mouse histone H1 isoforms that showed changes in phosphorylation after prolonged treatment with dexamethasone (e.g., H1.3 and H1.4). Because these isoforms co-purify by HPLC and because of the high sequence homology between these two isoforms (nearly 87% identical), endoproteinase Arg-C is the only enzyme readily available that will yield unique peptides with CDK2 consensus phosphorylation sites upon proteolysis. shows the amino acid sequences for mouse H1.3 and H1.4 with the CDK2 consensus sequences outlined in boxes, and the arrows indicate Arg-C cleavage sites. The Arg-C peptides which would yield unique sequences containing CDK2 consensus phosphorylation sites are Arg-C fragments one and six from H1.4 and Arg-C fragments one and four from H1.3. Unfortunately, the mass of some of these peptides are too large (greater than 5000 Da) for good MS/MS sequencing using traditional mass spectrometric instrumentation. Therefore, to determine the sites of phosphorylation unique to these peptides, the H1.3/H1.4 HPLC fractions were pooled and subjected to Arg-C digestion and the resulting peptides were separated by HPLC (data not shown). All of the HPLC fractions were lyophilized, reconstituted in water, and analyzed by MALDI/MS to determine which fractions contain the unique H1.3 or H1.4 peptides with the CDK2 consensus phosphorylation sites. shows the linear-mode MALDI mass spectrum of one of the HPLC fractions. The major ion observed has an average m/z of 9702 and corresponds in mass to Arg-C fragment 6 of mouse histone H1.4 (average theoretical m/z = 9703). Also observed are ions corresponding in mass to the addition of one and two phosphate groups (80 Da) to the Arg-C fragment 6 peptide (ions of m/z 9783 and 9862, respectively). Under the chromatographic conditions used, the phosphorylated H1.4 peptides were separated from the H1.3 peptides. All of the phosphorylated H1.3 peptide fractions, however, also contained some phosphorylated H1.4 peptides.
Figure 1 (A) Sequence alignment for mouse histone H1.3 and H1.4 isoforms. CDK consensus sites ((S/T)PX(R/K)) are outlined with a box. These histones are nearly 87% identical (shaded in gray). The arrows indicate endoproteinase Arg-C cleavage sites. (B) MALDI mass (more ...)
The HPLC fractions containing the unique H1.4 Arg-C fragments were further subjected to tryptic digestion followed by LC/MS and LC/MS/MS analyses. The MS/MS spectra of three tryptic phosphopeptides containing CDK2 consensus sites are shown in . The MS/MS spectrum of the ion of m/z 411.21 which corresponds in mass to monophosphorylated tryptic peptide SPK of Arg-C fragment 6 of histone H1.4 is shown in . A nearly complete series of both y and b ions and the loss of HPO3 (80 Da) from the a2 and b2 ions and the loss of H3PO4 (98 Da) from the a2 ion are observed. Abundant ions corresponding to the loss of H3PO4, HPO3, and H3PO4 + H2O from the molecular ion are observed. From these data, phosphorylation is verified and the site of phosphorylation in this peptide can be assigned to Ser-172 of histone H1.4.
Figure 2 Tandem mass spectra of phosphorylated tryptic peptides derived from Arg-C fragment six of mouse histone H1.4. (A) MS/MS data of the ion of m/z 411.21 corresponding in mass to monophosphorylated SPK; (B) MS/MS data of the ion of m/z 482.24 corresponding (more ...)
The MS/MS spectrum of the ion of m/z 482.24 () which corresponds in mass to monophosphorylated tryptic peptide SPAK of Arg-C fragment 6 of histone H1.4 was acquired. Again, both y and b ions as well as the loss of HPO3 and H3PO4 from y and b ions are observed. In addition, the loss of H3PO4, HPO3, and H3PO4 + H2O from the molecular ion, verifying phosphorylation is observed. These structurally informative fragment ions allow the assignment of the site of phosphorylation in this peptide to Ser-187 of histone H1.4.
shows the MS/MS spectrum of the (M + H)+ ion of m/z 524.27 which corresponds in mass to monophophorylated tryptic peptide TPVK from Arg-C fragment 1 of histone H1.4. The most abundant fragment ion observed in this spectrum corresponds to the loss of H3PO4 from the molecular ion. Other structurally informative ions observed include y1, y2, and y3 as well as the loss of H3PO4 from the b2 and b3 ions. These data allow the assignment of the phosphorylation site in this peptide to Thr-18 of histone H1.4.
Characterization of Histone H1 Isoform Phosphorylation in Human UL3 Cells
Concurrent with LC/MS/MS analyses of the mouse H1 isoforms, we also investigated the post-translational modification status of human H1 isoforms in osteosarcoma UL3 cells. H1 proteins were acid-extracted, purified by reverse phase HPLC, and screened by MALDI/MS analyses. From the mass of the ions observed from these data, it was determined that two chromatographic peaks contained H1 isoforms; the first peak contained the H1.0 isoform, whereas the H1.2 and H1.4 isoforms co-eluted in the second HPLC peak (data not shown). Interestingly, we did not find ions corresponding in mass to the H1.1, H1.3, or H1.5 isoforms in the UL3 cells in any of the fractions analyzed. This is not unexpected, as it had been previously reported that in cells in culture and in mammalian tissue, the H1.2 and H1.4 isoforms are more broadly expressed in all cell types, whereas expression of other H1 isoforms are often down-regulated in differentiated cells.27,28
To determine the phosphorylation status of these human H1 isoforms, the two HPLC fractions were analyzed by electrospray mass spectrometry and the data are summarized in . The ESI/MS analyses of the first HPLC peak show ions which correspond in mass to the unmodified H1.0 isoform. The deconvoluted ESI mass spectrum of the second HPLC peak () shows ions which correspond in mass to acetylated H1.2 as well as the mono- and diphosphorylated forms of acetylated H1.2. In addition, ions corresponding in mass to the acetylated H1.4 isoform were observed as well as the mono-, di-, and triphosphorylated forms of this protein.
Observed Average Masses from the Deconvoluted ESI Mass Spectra for the Human Histone H1 Isoforms Isolated from UL3 Cells
Figure 3 Deconvoluted electrospray mass spectra of intact H1 proteins detected in UL3 cells following treatments with dexamethasone and two CDK inhibitors. Mass spectra from (A) control cells and cells following treatment with (B) dexamethasone for 48 h, (C) 10 (more ...)
CDK Inhibitor Studies of Human H1 Isoform Phosphorylation
Using Western blot analyses, we have previously reported16
that the CDK2 inhibitor, CVT313, blocked H1 phosphorylation in mouse 1471.1 cells and are interested in determining whether the same phenomenon is observed for human H1 isoforms. Additionally, the recent development of a CDK1 inhibitor, CGP74514, allows one to determine the role of CDK1 in H1 phosphorylation. To determine the effects of CDK inhibitors on global phosphorylation of the human H1 isoforms, we used electrospray ionization mass spectrometry to analyze the intact H1 proteins following treatment of the cells with these CDK inhibitors. For these treatments, cells were treated for 24 h with either 0.01% DMSO (as a control), 10 μ
M CVT313, 25 μ
M CVT313, or 2.5 μ
M CGP74514. Additionally, cells were treated for 48 h with 10 nM dexamethasone. Following treatment, the cells were harvested and the H1 proteins were isolated, purified by HPLC, and analyzed by ESI/MS. From the resulting ESI mass spectra, the relative phosphorylation of the human H1 isoforms following 24 h treatments with each inhibitor was determined. Comparison of these data show that, when cells are pretreated with dexamethasone, the CDK2 inhibitor CVT313, or the CDK1 inhibitor CGP74514, the global levels of phosphorylation of both the H1.2 and H1.4 isoform were significantly decreased by all treatments ().
To determine the relative quantitative levels of the individual phosphorylated isoforms present, the ESI/MS analyses were repeated in triplicate from two independent biological experiments. Although the MS analysis of proteins, in general, is not very quantitative, relative changes in phosphorylation on a specific protein can be determined. Generally speaking, the addition of negatively charged modifications on a protein (i.e., phosphorylation) would reduce its ionization potential in the mass spectrometer. In the case of the histone H1 proteins, however, these proteins have large overall positive charges (e.g., over 50 positively charged amino acid residues per isoform). The addition, therefore, of one or two negatively charged phosphate groups per molecule is not expected to significantly affect the ionization potential of the protein, and this is not expected to significantly affect the relative sensitivity of the differentially phosphorylated protein ions.
The relative quantitation of each isoform of the individual human H1.2 and H1.4 proteins was computed by first determining the relative height abundance of the ions corresponding in mass to each specific isoform. These heights were then normalized to the total amount of the H1 isoform observed, thereby, reflecting the relative percentage of each species (; Supplementary Tables 1 and 2
). For example, the relative amounts of each of the H1.4 isoforms present in control cells was determined to be 39%, 34%, 18%, and 8% for acetylated H1.4, acetylated monophosphorylated H1.4, acetylated diphosphorylated H1.4, and acetylated triphosphorylated H1.4, respectively. For the H1.2 isoform in control cells, the relative amounts of each species observed is 58% acetylated, 34% acetylated monophosphorylated, and 8% acetylated diphosphorylated.
Figure 4 Relative quantitation of phosphorylation in each human H1 isoform following treatment with dexamethasone or CDK inhibitor. (A) Relative amounts of each isoform detected for human H1.4 protein; (B) relative amounts of each individual isoform detected for (more ...)
Similarly, the relative quantitation of each species was determined after treatment of the cells with the various drugs ( and ; Supplementary Tables
). For the H1.4 isoform, only the nonphosphorylated isoform and a relative small amount (15% or less) of the monophosphorylated isoform were observed from cells treated with dexamethasone and CVT313, whereas both the monophosphorylated (19%) and the diphosphorylated (4%) forms were observed after the CGP745145 treatment. For the H1.2 isoform, for all drug treatments studied, only relative small amounts (20% or less) of the monophosphorylated species were observed in addition to the acetylated form. Overall, these data suggest that CDK kinase activity is responsible for nearly all of the phosphorylation of the human histone H1 isoforms in these cells. Collectively, the overall level of phosphorylation can be determined for the H1.2 and H1.4 proteins based on treatment (; Supplementary Table 3
). For the H1.4 protein in the control cells, 60% of the H1.4 total protein detected was observed in a phosphorylated form. The total phosphorylation level in H1.4 decreased to 11%, 15%, 4%, and 23% after treatment with dexamethasone, 10 μ
M CVT, 25 μ
M CVT, and 2.5 μ
M CGP74514, respectively. Similar results were observed for the H1.2 isoform; 42% of the total protein was observed in a phosphorylated form in control cells. Following the same treatments as with H1.4, these total phosphorylation levels dropped to 15%, 16%, 8%, and 20%.
These data clearly demonstrate that the in vivo levels of phosphorylation of both the H1.2 and H1.4 isoforms are greatly reduced upon treatment for 48 h with dexamethasone. To determine whether these changes could be correlated with CDK activity, the levels of phosphorylation of H1.2 and H1.4 from control and dexamethasone-treated cells were compared to cells treated with either a CDK1 or a CDK2 inhibitor. Following treatment with 10 μM CVT313 or 2.5 μM CGP74514, the level of phosphorylation of H1.2 and H1.4 was reduced to a comparable level observed following dexamethasone treatment. Treatment with 25 μM CVT313 decreased the level of phosphorylation to an even greater extent, although a small amount of phosphorylation was still observed following all treatment conditions. These results suggest that the phosphorylation events of histone H1 proteins are complex and are a dynamic process which occurs throughout the cell cycle, with possibly a variety of factors influencing these modifications.
Identification of H1.4 Phosphorylation Sites in Human UL3 Cells
For the mouse H1.4 isoforms in 1471.1 cells, three CDK consensus sites of phosphorylation were identified; therefore, we were interested in determining the CDK-dependent sites of phosphorylation in the human H1.4 isoform. In the UL3 cells, both H1.2 and H1.4 were observed as phosphorylated. shows the amino acid sequences for human H1.2 and H1.4 with the CDK consensus sequences outlined in boxes. The consensus target residues correspond to threonine-18, -146, -154, and serine-174 and -187. To identify sites of phosphorylation in the human H1.4 protein, the HPLC purification of the intact histone H1 proteins from control cells was repeated, collecting 10 s fractions. By doing so, we could collect a fraction that contained mostly the H1.4 isoform with little or no H1.2 isoform. From this fraction, we performed LC/MS analyses of the tryptic digest of the H1.4 fraction. From a theoretical digest of the H1.4 isoform, those masses which contained the consensus CDK sites were searched in the LC/MS analyses. The potential consensus CDK-containing tryptic peptides and the theoretical nonphosphorylated protonated masses and theoretical phosphorylated protonated masses are as follows: TPVK (threonine-18; 444.2822, 524.2485), ATGAATPK (threonine-146; 716.3943, 796.3606), TPK (threonine-154; 345.2138, 425.1801), SPK (serine-174; 331.1981, 411.1645), and SPAK (serine-187; 402.2353, 482.2016). Of these, ions were observed which correspond in mass to all of the nonphosphorylated form of the peptides, whereas only masses corresponding to phosphorylated TPVK (m/z obs = 524.28), SPK (m/z obs= 411.19), and SPAK (m/z obs = 482.19) were observed. No ions were observed which would correspond in mass to phosphorylated ATGAATPK or TPK. Additional LC/MS analyses were performed where the instrument was set to acquire MS/MS spectra of specific masses throughout the entire LC/MS run. shows the MS/MS spectra of three tryptic peptides that were found to be phosphorylated in H1.4. In , fragment ions are observed which correspond to the loss of HPO3, H3PO4, and H3PO4 + H2O from the protonated molecule. The observation of these fragment ions indicates the presence of a phosphate group within these peptides. In addition, a series of b and y ions and the loss of phosphate (either as the loss of 80 Da or the loss of 98 Da) from the b ions are observed. These structurally informative fragment ions allow the assignment of the site of phosphorylation to Ser-172 and Ser-187 in these two peptides, respectively. shows the MS/MS spectrum of the (M + H)+ ion of m/z 524.28 which corresponds in mass to monophosphorylated tryptic peptide 2 (T2), TPVK (aa 18–21). Although the S/N ratio in this spectrum is low, structurally diagnostic ions, especially y3 and b3-98, are observed. In addition, several of these same structural ions are observed in the MS/MS spectrum of mouse monophosphorylated T2:18–21 (). These data allow confirmation of a phosphate group on the threonine residue within this tryptic peptide. From these data, we can confirm that T18, S172, and S187 are phosphorylated in the histone H1.4 isoform of UL3 cells.
Sequence alignment for human H1.2 and H1.4 histones. CDK consensus sites ((S/T)PX(R/K)) are outlined with a box. These histones are more than 86% identical (shaded in gray).
Figure 6 Tandem mass spectra of phosphorylated tryptic peptides derived from human histone H1.4. (A) MS/MS data of the ion of m/z 411.19 corresponding in mass to monophosphorylated SPK; (B) MS/MS data of the ion of m/z 482.19 corresponding in mass to monophosphorylated (more ...)
Tryptic digests of both the mouse and human isolated H1.4 isoforms resulted in the identification of phosphorylated peptides corresponding to three CDK consensus sites in both the N- and C-terminal tails of H1.4. These sites of phosphorylation were determined to be located at Ser-174, Ser-187, and Thr-18. Sites of post-translational modifications of H1 have been previously addressed using similar MS/MS techniques on the H1 isoforms.29–31
With this approach, Hunt et al.29
identified seven serine or threonine residues (including Ser-174, Ser-187, and Thr-18) that were phosphorylated in human H1.4 in asynchronous HeLa cells. Four of the identified H1.4 phosphorylation sites contain a putative cyclin-dependent kinase motif; however, three of the sites were identified from non-CDK consensus motifs. Conversely, Mann et al.31
identified two (possibly three) sites of phosphorylation in human H1.4 in both human MCF7 and HeLa cells. In addition to the two cell lines, Mann et al.31
also analyzed and compared the post-translational modifications of histone H1 in nine mouse tissues. From this work, these authors showed that differences in the post-translational modifications of histone H1 proteins could be determined between different tissues and different cell types. Several of the serine and threonine phosphorylation sites identified by these groups29,31
were found within peptides which did not contain CDK-consensus sites. This, therefore, implies that other kinases can influence H1 phosphorylation. Indeed, previous research has implicated that H1 phosphorylation is influenced by MAP kinases as well.32,33
Although this study has focused on the phosphorylation of CDK consensus sites because of the correlation between CDK2 inhibition and loss of MMTV activation found in our previous work,15,16
the results from the H1.4 analyses from the cell lines reported by Mann et al.31
are similar to the number of sites of phosphorylation found in this work, that is, three sites. Therefore, we hypothesize that different phosphorylation sites are utilized differentially between cells and are unique to particular isoforms with functional consequences.
Quantitative Changes in Specific Sites of Phosphorylation in H1.4
LC/MS data from one each of the tryptic digests of mouse H1.4 isoforms from 1471.1 control cells versus cells treated for 48 h with dexamethasone were compared to see if changes in the relative abundance of the phosphorylated peptides could be determined. Because the absolute amount of protein in each experiment is not known, only relative changes in the abundances can be stated. First, the ratio of ion counts of the extracted ion chromatograms of five different peptides, which do not contain a CDK2 consensus site, from the tryptic digest of Arg-C fragment 6 of H1.4 were compared. The change in the counts between the control cells versus cells treated for 48 h with dexamethasone for each of these peptides was relatively the same (average ratio of counts of dexamethasone-treated cells/control cells = 0.9). shows the extracted ion chromatograms of the protonated ion of m
784.5 (amino acids KPAAAAGAK) from both the control cells and the 48 h dexamethasone-treated cells. The ion counts for each of these masses is 329 and 308 for the control cells and the dexamethose-treated cells, respectively. Conversely, for the tryptic peptides containing phosphorylated Ser-172 (pSPK) and phosphorylated Ser-187 (pSPAK) of Arg-C fragment 6 of H1.4, an overall decrease in the counts of the extracted ion chromatograms of these ions was observed in the 48 h dexamethasone-treated cells versus the control cells (average ratio of counts of dexamethasone-treated cells/control cells = 0.5). The extracted ion chromatograms of the protonated ion of m
411.2 (pSPK) from the control cells (ion count of 124) versus dexamethasone-treated cells (ion count of 71) are shown in . Additionally, the ion counts from the extracted ion chromatograms of ions corresponding in mass to the nonphosphorylated tryptic peptides SPK and SPAK containing the CDK consensus sites showed an overall increase in the 48 h dexamethasone-treated cells treatment versus control cells (average ratio of counts of dexamethasone-treated cells/control cells/dexamethasone-treated cells = 1.1), albeit a slight increase (data not shown). Because of the small size of some of these tryptic peptides (3 or 4 amino acids in length), the peptides were not well-retained under the chromatographic conditions used. Therefore, quantitation of these ions is difficult. Nonetheless, these observations in the relative quantitation of these data are consistent with the observed changes in the relative phosphorylation levels of the full-length proteins reported previously.19
Figure 7 Extracted ion chromatograms of ions from the LC/MS/MS analyses of control cells and cells treated for 48 h with dexamethasone. (A) EIC of the protonated ion of m/z 784.5 (amino acids KPAAAAGAK); and (B) EIC of the protonated ion of m/z 411.2 (phosphorylated (more ...)
Phosphorylation Site-Specific Antibodies
To explore the dynamics of each H1 phosphorylation event in more detail, we generated phosphorylation site-specific antibodies to the serine and threonine residues found by MS to be phosphorylated in vivo. Up to now, the most widely used technique for assessing changes in the levels of histone modifications has been the use of Western blots with antibodies that specifically recognize the modification of interest. A significant advantage of this approach is that these antibodies can be highly sensitive. Potential disadvantages of this type of analysis are the lack of specificity of the antibody, the ability to only monitor a single modification at a time, and the modification of a residue near the site of interest may affect the specificity of the antibodies. Nonetheless, we generated polyclonal antisera to specific phosphorylation sites identified by MS/MS to accurately quantitate the changes in the levels of phosphorylation at specific residues.
Peptides corresponding to phosphorylated CDK consensus sites within both mouse H1.3 or H1.4 isoforms were used to immunize rabbits. One resulting antiserum (designated H1.4pT18 and/or 1754 antibody) recognized phosphorylated peptides corresponding to mouse H1.3 and H1.4 isoforms with phosphorylation on Thr-18, but did not recognize nonphosphorylated peptides of the same primary sequences (data not shown). The antibody was also tested by Western blot for recognition of four mutations of the mouse H1.3 isoforms (T18E, T18A, T18Q, and T18D) that were transiently expressed in UL3 cells. Each of these mutations abrogated recognition by the H1.4pT18 antibody (). This antiserum was then used to probe immunoblots of acid extracted H1 proteins from UL3 cells following various time hormone treatments. As shown in , the anti-H1.4pT18 antibody strongly recognizes a band corresponding to the H1.4 isoform in asynchronous UL3 cells not treated with hormone. Following 24 and 48 h of hormone treatment, band intensity diminished significantly to nearly undetectable levels by 48 h. After the 48 h treatment, hormone was washed away and cells were allowed to recover for 24 h, at which time band intensity returned to basal levels.
Figure 8 Anti-H1.4pT18 antibody used to detect hormone-associated changes in H1 phosphorylation in UL3 cells. Two micrograms of acid-extracted proteins was separated by SDS-PAGE and analyzed by Western blot using the anti-H1.4pT18 (1754) antibody. Coomassie stained (more ...)
To determine if threonine-18 in human H1.4 is a target for either CDK1 or CDK2 in vivo
, UL3 cells were treated with either CVT313 or CGP74514 at two concentrations each for 24 h, after which, Western blot analyses with the H1.4pT18 antibody were performed. Treatment of UL3 cells with dexamethasone or CVT313 showed a dose-dependent decrease in H1.4pT18 phosphorylation after 24 h relative to the phosphorylation observed in untreated cells. Treatment with the inhibitor CGP74514 also showed a decrease in H1.4pT18 phosphorylation levels relative to that observed in untreated cells, but what appears to be a lesser extent than with CVT313 treatment (). To quantitate the amount of total H1.4 that is phosphorylated, densitometry was used to compare each band detected by the polyclonal H1.4pT18 antibody to the corresponding H1.4 band on the coomassie-stained gel. The average relative quantitation of two independent experiments of phosphorylated H1.4 to total H1.4 (pH1.4/H1.4) was determined and is shown in . On the basis of densitometry, treatment of the cells with dexamethasone reduced levels of H1.4pT18 by 51% compared to untreated cells. The CDK2 inhibitor CVT313 reduced H1.4pT18 by 40% and 96% at 10 and 25 μ
M doses, respectively. The CDK1 inhibitor CGP74514 did not significantly reduce H1.4pT18 at 1 μ
M (11%), but reduced levels by 58% at the 2.5 μ
M concentration. For comparison, the ratio of phosphorylated isoforms relative to the total H1 isoform (normalized to untreated cells) was calculated from the MS analyses (Supplementary Table 4
). Although these numbers arise from different biological samples on different days, similar results were obtained from the MS analyses and the densitometry analyses, that is, a decrease in the ratio of phosphorylated H1.4 to total H1.4 was observed following treatment with the cdk inhibitors.
The H1.4pT18 polyclonal antibody was shown to be specific for phosphorylated Thr-18 of H1.4, and because UL3 cells only express the H1.4 isoform, these antisera allowed us to track phosphorylation of H1.4 in vivo in control cells and cells treated with hormone and/or CDK inhibitors. This antibody strongly recognizes a band corresponding to the H1.4 isoform in control cells, and this band is greatly reduced following treatment of UL3 cells with dexamethasone for 24 or 48 h. The intensity of the band returns to control levels if, after the 48 h treatment, the cells are washed of the hormone and allowed to recover for 24 h.
Similarly, the antibody was useful for determining if this specific site of phosphorylation (threonine-18) in human H1.4 is a target for either CDK1 or CDK2 in vivo
. Western blot analyses with the H1.4pT18 antibody on cells treated with either CVT313 or CGP74514 at two concentrations each for 24 h showed a dose-dependent decrease in H1pT18 phosphorylation after 24 h. Treatment with the inhibitor CGP74514 also showed a decrease in H1pT18 levels, but to a lesser extent than was seen with CVT313 treatment. Densitometry comparing each band detected in the Western analyses was used to quantitate the amount of total H1.4 that is phosphorylated. From this, an average relative quantitation was determined and compared to the relative quantitation levels obtained using mass spectrometry. A decrease in the relative quantitation levels of phosphorylation following CDK inhibitor treatments were detected by both the MS analyses and the Westerns with the H1.4pT18 antibody, thereby suggesting that the remaining phosphorylation seen by MS is perhaps, at least in part, due to residual T18 phosphorylation. The ability to accurately quantitate changes in the levels of specific phosphorylation sites in response to different hormone or CDK treatments is essential to fully understand the functional role of histones and/or their modifications. Although mass spectrometry is a useful tool for the quantitation of post-translational modifications of peptides, the presence of the modification can affect the ionization potential of the peptide. Recently, the use of stable isotope labeling in conjunction with mass spectrometric analyses was reported for the quantitation of changes in specific histone modifications.34
Perhaps this approach may prove to be the most useful technique for the quantitation of localized changes in histone post-translational modifications.