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Originally published In Press as doi:10.1074/mcp.M600086-MCP200 on July 10, 2006.
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Molecular & Cellular Proteomics 5:1593-1609, 2006.
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

Research

Comprehensive Phosphoprotein Analysis of Linker Histone H1 from Tetrahymena thermophila*,S

Benjamin A. Garcia{ddagger},§, Swati Joshi, C. Eric Thomas||, Raghu K. Chitta{ddagger}, Robert L. Diaz, Scott A. Busby{ddagger}, Philip C. Andrews**, Rachel R. Ogorzalek Loo{ddagger}{ddagger}, Jeffrey Shabanowitz{ddagger}, Neil L. Kelleher||, Craig A. Mizzen§§, C. David Allis and Donald F. Hunt{ddagger},¶¶,||||

From the Departments of {ddagger} Chemistry and ¶¶ Pathology, University of Virginia, Charlottesville, Virginia 22904, Laboratory of Chromatin Biology, The Rockefeller University, New York, New York, 10021, Departments of || Chemistry and §§ Cell and Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, {ddagger}{ddagger} Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095, and ** Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48104


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Linker histone H1 is highly phosphorylated in normal growing Tetrahymena thermophila but becomes noticeably dephosphorylated in response to certain conditions such as prolonged starvation. Because phosphorylation of H1 has been associated with the regulation of gene expression, DNA repair, and other critical processes, we sought to use mass spectrometry-based approaches to obtain an in depth phosphorylation "signature" for this linker histone. Histone H1 from both growing and starved Tetrahymena was analyzed by nanoflow reversed-phase HPLC MS/MS following enzymatic digestions, propionic anhydride derivatization, and phosphopeptide enrichment via IMAC. We confirmed five phosphorylation sites identified previously and detected two novel sites of phosphorylation and two novel minor sites of acetylation. The sequential order of phosphorylation on H1 was deduced by using mass spectrometry to define the modified sites on phosphorylated H1 isoforms separated by cation-exchange chromatography. Relative levels of site-specific phosphorylation on H1 isolated from growing and starved Tetrahymena were obtained using a combination of stable isotopic labeling, IMAC, and tandem mass spectrometry.

Eukaryotic DNA is organized and condensed in chromatin by close association with histone proteins. Around 146 bp of DNA wrap around an octameric complex of the core histones H3, H4, H2A, and H2B to form the basic repeating unit of chromatin, the nucleosome (1, 2). A growing list of post-translational modifications to the core histone proteins has been identified using antibody-based methods, for example, acetylation (on Lys residues), methylation (mono-, di-, or trimethylation on Lys and mono- or dimethylation on Arg residues), phosphorylation (on Ser and Thr residues), and ubiquitination (on Lys residues). Within the past few years, MS has played an increasing role in the characterization of post-translational modifications on various histone proteins (3–8). These modifications can alter the structure of chromatin and have been demonstrated to regulate the activation or repression of target genes during various epigenetic processes by acting as binding platforms recruiting downstream "effector" proteins (Histone Code hypothesis) (9). For example, acetylation of Lys residues or methylation of Lys-4 or Lys-79 on histone H3 is generally linked to gene expression, whereas methylation of H3 at Lys-9 or Lys-27 by recruitment of heterochromatin-associated protein 1 (HP1)1 or Polycomb, respectively, has been associated with heterochromatin assembly and gene silencing (10–12).

Distinct from the core histones in both structure and function, the linker histone H1 binds the linker DNA connecting nucleosomes in chromatin and is involved in the folding of nucleosomal filaments into higher order fibers (13, 14). H1 is known to be phosphorylated at multiple sites in a wide variety of organisms, although many details regarding the sites and function of H1 phosphorylation remain unclear (15). Early studies of H1 phosphorylation in animal cells revealed that it was progressive through the cell cycle, reaching maximum levels as chromosomes condensed during mitosis, suggesting a role for H1 phosphorylation in regulating global chromatin condensation (16–18). More recently H1 has been implicated in regulating transcription in a gene-specific fashion and the regulation of apoptosis and DNA repair (19–21), and it is intriguing to speculate that phosphorylation contributes to H1 function in these targeted processes also. Although they have diverged much more than the core histones during evolution, linker histones in a wide variety of species typically contain numerous (S/T)PXZ motifs (where X is any amino acid and Z is a basic amino acid), and accordingly cyclin-dependent kinases have been investigated for their roles in H1 phosphorylation. Toward this end, the addition of Cdc2 kinase has been shown to cause premature chromatin condensation in some cell types, and decreased H1 phosphorylation following inhibition of cyclin-dependent kinases is associated with chromosome decondensation (22, 23). However, because H1 phosphorylation and mitosis do not always occur simultaneously in certain organisms such as in Tetrahymena thermophila, the function of H1 phosphorylation has remained elusive. T. thermophila is a model organism to study H1 phosphorylation because only one protein isoform exists, and histone H1 of macronuclei is highly phosphorylated outside of mitosis during interphase. Macronuclei divide amitotically without overt chromosome condensation, implying that the function of H1 phosphorylation in this organism may be restricted to the regulation of gene transcription. Mutagenesis of known phosphorylation sites on T. thermophila macronuclear H1 has revealed that these phosphorylations are not essential for viability in this organism (24). However, Dou et al. (25) recently demonstrated that H1 phosphorylation modulates the expression of Cdc2 kinase- and protease-encoding genes during prolonged starvation in T. thermophila. As H1 phosphorylation is dramatically decreased as cells are starved prior to conjugation and increased during heat shock, it seems likely that these changes in H1 phosphorylation contribute to the transcriptional responses of cells under these conditions (26).

Phosphorylation of H1 in mammalian cells has been studied by mass spectrometry previously. Tomer and co-workers (27) studied H1 phosphorylation in rat cell lines treated with the glucocorticoid hormone dexamethasone. Dexamethasone treatment was found to lead to the dephosphorylation of specific H1 protein isoforms even though phosphorylation sites were not specifically determined on any of the isoforms. We previously used IMAC and tandem mass spectrometry to discover 19 sites of phosphorylation on six H1 isoforms from asynchronously grown HeLa cells, and several of these phosphorylation sites were found on non-(S/T)PXZ-containing sequences (28). However, because H1 is involved in many important aspects of cellular growth, it is important to study the site-specific phosphorylation of H1 in both the normal growing state and following cellular perturbation.

Here we used mass spectrometry to investigate the phosphorylation of macronuclear H1 prepared from vegetative growing and starved T. thermophila. Utilizing chemical derivatization, enzymatic digestion, IMAC, and MS/MS experiments on an LTQ-FT mass spectrometer, we confirmed all five phosphorylation sites identified previously. Moreover we discovered two novel sites of phosphorylation and two novel sites of acetylation. Additionally separation of phosphorylated H1 species by cation-exchange chromatography and analysis by tandem mass spectrometry revealed information concerning the precise hierarchy of phosphorylation on histone H1 in this organism. Finally we used stable isotope labeling, IMAC, and mass spectrometry to relatively quantify differential phosphorylation of sites on H1 from growing and starved Tetrahymena cell cultures.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Histone Purification—
For vegetative growth, T. thermophila cells were grown to log phase to a cell density of 2.0–2.5 x 105 cells, and macronuclei were extracted under standard conditions as described previously (29). The same process was repeated under starved physiological conditions in which the cells were starved for 24 h in 10 mM Tris-HCl (pH 7.4) at 30 °C with gentle shaking (50 rpm). Highly purified macronuclei were prepared as described by Gorovsky et al. (29) with the exception that the isolation buffer did not contain spermidine. Macronuclei were washed in nuclear wash buffer, pH 7.5 (250 mM sucrose, 10 mM Tris, 3 mM calcium chloride, 1 mM magnesium chloride, 10 mM butyric acid, 1 mM PMSF). Total histones were extracted in 0.4 N H2SO4, TCA-precipitated, and put through consecutive washes of acidified acetone (0.1% HCl) and acetone. Purified macronuclear H1 was obtained by RP-HPLC using a C8 column (Aquapore RP-300, 4.6 x 220 mm, PerkinElmer Applied Biosystems) as described previously (30). Histone H1 was eluted at 1.0 ml/min with a gradient of 0–100% B in 100 min (solvent A, 5% acetonitrile in 0.1% TFA; solvent B, 70% acetonitrile in 0.1% TFA). H1 recovered from the RP-HPLC fractions were dried by lyophilization and redissolved in H2O. Alternatively crude H1 was prepared by making 0.4 N H2SO4 extracts 5% (w/v, final) in perchloric acid (PCA) to selectively precipitate core histones followed by recovery of H1 from the PCA-soluble fraction by TCA precipitation and consecutive washes with acetone/0.1% HCl and acetone.

Digestion of Tetrahymena Histone H1—
Dried histone H1 was redissolved in 50 µl of deionized water. Asp-N was purchased as sequence grade enzyme (Roche Applied Science) as was trypsin (Promega, Madison, WI). About 1 µg of H1 was diluted with 50 µl of a 100 mM ammonium bicarbonate buffer solution (pH = 8) and digested with Asp-N (substrate:enzyme ratio of 10:1) for 6 h at 37 °C or by trypsin (substrate:enzyme ratio of 20:1) for 30 min at 37 °C. All reactions were quenched by the addition of concentrated acetic acid and freezing.

Chemical Derivatization of Histone H1 Peptides—
H1 protein digests were incubated with propionylation reagent to convert endogenously unmodified amino groups on the side chain of lysine residues and peptide amino termini to propionyl amides (31). The propionylation reagent consists of mixing 75 µl of MeOH and 25 µl of propionic anhydride (Aldrich). An equal aliquot of propionyl reagent and protein digest solution were mixed and allowed to react for 15 min at 37 °C, and pH was adjusted to 8 with addition of NH4OH. Due to the high content of lysine residues on histone H1, this reaction was repeated twice to ensure full conversion of all amino groups.

Immobilized Metal Affinity Chromatography—
IMAC was also utilized to enrich for phosphopeptides as described previously (32). Samples were then lyophilized to dryness and redissolved in a 1:1:1 mixture of MeOH/MeCN/0.1% acetic acid following conversion to methyl esters as described below. The IMAC columns (360-µm outer diameter x 100-µm inner diameter with 8 cm of POROS 20 MC resin, Applied Biosystems, Framingham, MA) were first activated with several column volumes of a 100 mM FeCl3 solution (Aldrich) for 20 min. Samples were next loaded onto the columns, washed with several volumes of 0.01% acetic acid, and eluted with 250 mM Na2HPO4 (pH = 6, Aldrich) onto C18 packed capillary columns.

Mass Spectrometry—
Histone H1 digest mixtures were loaded onto capillary precolumns (360-µm outer diameter x 75-µm inner diameter, Polymicro Technologies, Phoenix, AZ) packed with irregular C18 resin (5–20 µm, YMC Inc., Wilmington, NC) and washed with 0.1% acetic acid for 5 min. Precolumns were connected to analytical columns (360-µm outer diameter x 50-µm inner diameter, Polymicro Technologies) packed with regular C18 resin (5 µm, YMC Inc.) constructed with integrated electrospray emitters as described previously (33). All samples were analyzed by nanoflow HPLC-microelectrospray ionization on a Finnigan linear quadrupole ion trap (LTQ)-FT-ICR mass spectrometer (Thermo Electron, San Jose, CA). The gradient used on an Agilent (Palo Alto, CA) 1100 series HPLC solvent delivery system consisted of 0–45% B in 60 min, 45–100% B in 20 min (A, 0.1% acetic acid; B, 70% acetonitrile in 0.1% acetic acid), or other similar gradients. The LTQ-FT mass spectrometer was operated in the data-dependent mode and acquired MS spectra using the FT-ICR with R = 100,000 at m/z 400. All MS/MS spectra were manually interpreted. Intact H1 protein mass spectra acquired on the LTQ-FTMS were deconvoluted using a demo version of the ProMass 2.3 software. Additionally electron capture dissociation (ECD) mass spectra of intact histone H1 were acquired on a custom 8.5-tesla quadrupole-FT-ICR mass spectrometer with a nano-ESI source operated in positive ion mode (34). Intact histone H1 protein was resuspended in an electrospray solution consisting of equal parts water and methanol plus 1% formic acid, and a NanoMate 100 (Advion BioSciences, Ithaca, NY) was used to automatically establish the nanospray. The ECD data set consists of around 300 scans with an ion accumulation time of 2 s. The entire 27+ charge state, containing all protein forms, was selectively enhanced using the instrument’s quadrupole and further filtered using stored waveform inverse Fourier transform (SWIFT). ECD was performed with a filament bias of 1.2 V using five loops of 500-ms electron pulses. Data were collected using the modular ICR data acquisition system (MIDAS) (35).

Stable Isotope Labeling for Relative Quantitative Analysis of Histone H1 Phosphorylation—
To compare H1 phosphorylation in growing and starved Tetrahymena cultures, a stable isotope labeling derivatization procedure that centered on conversion of peptide carboxylic groups to their corresponding methyl esters was used (36). First all samples were completely dried by lyophilization. Propionylated histone peptides from growing cultures were converted to d0-methyl esters by reconstituting the lyophilized sample in 100 µl of 2 M d0-methanol/HCl, whereas histone peptides from starved cultures were converted to d3-methyl esters by reconstituting the lyophilized sample in 100 µl of 2 M d3-methanol/HCl. Both reaction mixtures were allowed to stand for 1 h at room temperature. Solvent was removed by lyophilization, and the procedures were repeated using a second 100-µl aliquot of 2 M d0-methanol/HCl or d3-methanol/HCl, respectively. Solvent was removed by lyophilization, and the residues from each sample were then redissolved in a 1:1:1 mixture of MeOH/MeCN/0.1% acetic acid. Aliquots of each solution were then equally mixed for comparative analysis by mass spectrometry and loaded onto IMAC columns as stated above.

Cation-exchange Chromatography Separation of Phosphorylated H1 Proteins—
H1 molecules differing in the level of phosphorylation and in some cases phosphorylation isoforms were resolved by cation-exchange chromatography as described previously (37). Crude H1 recovered from the PCA-soluble fraction of acid extracts was chromatographed on a Poly CAT A column (4.6 x 100 mm, PolyLC, Columbia, MD). H1 forms were resolved with a shallow gradient of sodium perchlorate in 10 mM sodium phosphate, pH 6.5, at room temperature using a flow rate of 0.8 ml/min. Protein elution was detected at 214 nm. Fractions of interest were recovered by TCA precipitation followed by consecutive washes in acetone/0.1% HCl and acetone.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Global Mapping of Tetrahymena Histone H1 Post-translational Modification Sites
T. thermophila histone H1 contains high levels of phosphorylation during normal vegetative growth but is dephosphorylated during prolonged starvation periods (24, 26, 38). This drastic modification change in Tetrahymena suggests that phosphorylation of H1 may regulate the expression of certain genes during normal growth. This aspect of H1 phosphorylation is demonstrated in Fig. 1. Fig. 1A shows an SDS-PAGE separation of histone H1 from growing (G) and starved (S) cultures stained for total protein with Coomassie Blue. The electrophoretic mobility of H1 from growing cultures is retarded relative to that of H1 from starved cells. Total H1 phosphorylation was also assayed by immunoblotting using an antibody shown previously to be specific for phosphorylated Tetrahymena H1 (37). Greater phosphorylation was observed in the sample from growing cells (Fig. 1B). Lastly intact protein mass spectrometry of Tetrahymena from growing and starved cells also showed that more phosphate groups were present in the growing sample (Fig. 1, C and D). Broadband mass spectra of the +27 charged state revealed that up to five phosphate groups could be detected on the linker histone H1 from starved Tetrahymena albeit at low abundance compared with the unmodified species (Fig. 1C). However, as shown in Fig. 1D, a greater abundance of phosphorylation was readily observed on H1 from growing cells. Five phosphorylation sites in the amino terminus of H1 were identified previously by Edman sequencing of H1 prepared from growing Tetrahymena labeled in vivo with [32P]orthophosphate (24). Mutation of these sites in combination suggested that they represented the major if not the entire repertoire of phosphorylation sites found on this protein (24). Three sites (Thr-34, Thr-46, and Thr-53) were observed to be canonical proline-directed sites ((S/T)PXZ where X is any amino acid and Z is a basic amino acid residue) phosphorylated by Cdc2-related kinases. Two other non-Cdc2 motif phosphorylation sites at Ser-42 and Ser-44 were also identified and found to be phosphorylated only if one of the three proline-directed sites was also phosphorylated. This probable hierarchy of H1 phosphorylation suggested the possibility that additional, minor sites of phosphorylation that are dependent on the prior phosphorylation of one or more serine or threonine residues may have been overlooked in this previous analysis. Therefore, we used a mass spectrometry-based approach (Fig. 2) to thoroughly investigate the phosphorylation of histone H1 from growing and starved cells. To better facilitate differential modification mass spectrometry experiments to monitor phosphorylation dynamics in growing and starved cells, we first performed a comprehensive global analysis of all post-translational modifications of H1 to confirm all the known modifications and discover any unknown modifications.


Figure 1
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  		FIG. 1. A, SDS-PAGE of the RP-HPLC fraction containing histone H1 from growing (G) and starved (S) T. thermophila cell cultures on a 15% acrylamide gel with Coomassie Blue staining. B, Western blot analysis of H1 from growing and starved cells using antisera raised against hyperphosphorylated Tetrahymena H1. C, broadband mass spectrum of histone H1 from starved Tetrahymena cells (+27 charge). Up to five low level phosphate groups (P) can be observed. D, broadband mass spectrum of histone H1 from growing Tetrahymena cells (+27 charge state). Zoom inset shows the profile of the unmodified H1 protein. Note the increased abundance of phosphate groups compared with the sample from starved cells. Calc., calculated; Exp., experimental.

 

Figure 2
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  		FIG. 2. Strategy for the differential analysis of histone H1 from growing and starved Tetrahymena cultures using enzymatic digestions, chemical derivatization, stable isotope labeling, immobilized metal affinity chromatography, and mass spectrometry. H1 samples are first digested with either trypsin or Asp-N and then propionylated on Lys residues. Propionylated H1 peptides are then screened by MS and converted to their corresponding methyl esters, equally mixed, and loaded onto IMAC columns for phosphopeptide enrichment. H1 phosphopeptides are then quantitatively analyzed by high mass accuracy tandem mass spectrometry on an LTQ-FT mass spectrometer.

 
Histone H1 was analyzed on the LTQ-FT mass spectrometer according to the strategy depicted on Fig. 2. Histone H1 from both growing and starved cultures was digested with either Asp-N or trypsin, and the resulting peptides were derivatized with propionic anhydride. Derivatization with propionic anhydride increases the hydrophobicity and reduces the charge state of peptides, aiding MS/MS analysis (31). Additionally Asp-N digestion releases 10 major peptides from Tetrahymena H1 that are easily monitored by mass spectrometry for the identification of post-translational modifications. For example, Fig. 3A shows the MS/MS spectrum of the doubly charged ion at m/z 461.2213 from the Asp-N proteolytic digest of H1 from growing Tetrahymena cultures. This peptide ion was fragmented, and the corresponding sequence was found to be DVpTPVKA (where pT is phosphothreonine; propionyl amide Pr group (+56 Da) on amino terminus and on the Lys residue) possessing phosphorylation at Thr-53. Furthermore analysis of the peptides from a limited trypsin digest of H1 from starved cultures resulted in the MS/MS spectrum of the [M + 2H]2+ ion at m/z 654.8267 (Fig. 3B). This ion was sequenced and found to represent residues 40–50 of H1. This peptide (AASASTpTPVKK; Pr groups on amino terminus and Lys residues) was observed to contain a phosphorylation site at Thr-46. Another phosphorylation site at Thr-34 was also found in our analysis from both growing and starved cultures (Table I). These three sites all possess the consensus cyclin-dependent kinase recognition motif ((S/T)PXZ where X is any amino acid and Z is a basic amino acid residue), and all three sites were identified previously by Mizzen et al. (24). To detect minor sites of phosphorylation on H1, peptides generated in the proteolytic digest were converted to methyl esters and enriched for phosphopeptides using IMAC as described in Fig. 2. IMAC enrichment and mass spectrometry has been used previously to confirm known phosphorylation sites and identify novel phosphopeptides generated from enzymatic digestion of propionylated histone proteins (28, 39, 40). The use of IMAC resulted in the confirmation of Ser-42 and Ser-44 as lower level phosphorylation sites on histone H1 (Table I) as both have been characterized previously as phosphorylation sites on H1 (24). Shown in Fig. 4 is the MS/MS spectrum of the doubly charged ion at m/z 701.8182 from the tryptic digest of H1 from growing cultures following methyl ester derivatization and IMAC enrichment. This peptide ion has the sequence AApSASTpTPVKK (where pS is phosphoserine; Pr groups on the amino terminus and Lys residues and methyl ester on the carboxyl terminus). A doubly phosphorylated peptide containing phosphorylation at Ser-44 and Thr-46 and a triply phosphorylated peptide containing phosphorylation at Ser-42, Ser-44, and Thr-46 were also detected after IMAC enrichment (Table I) of the trypsin-digested samples. It is worth noting that the phosphorylation sites at Ser-42 and Ser-44 were only found to be phosphorylated on peptides containing Thr-46 phosphorylation as well. This supports the hierarchy of phosphorylation proposed previously based on Edman sequencing of tryptic peptides prepared from radiolabeled H1 (24).


Figure 3
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  		FIG. 3. ESI-MS/MS characterization of histone H1 phosphorylation sites containing (S/T)PXZ motif sequences (cyclin-dependent kinase sites). A, MS/MS spectrum of a doubly charged peptide ion at m/z 461.2213 derived from an Asp-N digest of H1 isolated from growing Tetrahymena cultures. B, MS/MS spectrum of a precursor [M + 2H]2+ ion at m/z 654.8267 from a tryptic digest of H1 from starved Tetrahymena cultures. Note our chemical derivatization modification (Pr) on amino groups adds 56 Da per each lysine side chain residue and amino terminus. All data were obtained on an LTQ-FT mass spectrometer. Calc., calculated; Exp., experimental.

 

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  		TABLE I Peptides identified by both ESI-MS/MS and accurate mass from Tetrahymena histone H1

Selected peptides from Asp-N and trypsin digests of histone H1 from growing Tetrahymena cells are shown. All endogenously unmodified and monomethylated lysine residues were modified with our propionyl reagent (+56 Da) after enzymatic digestion. All observed peptides were detected within 5 ppm of their calculated masses. Charge state of the peptide ion is in parentheses. Sequence coverage for histone H1 was 95%. pS, phosphoserine; pT, phosphothreonine; Ac, acetylation. All MS and MS/MS data were obtained on a Finnigan LTQ-FT mass spectrometer.

 

Figure 4
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  		FIG. 4. IMAC enrichment of a minor phosphorylation site on histone H1. Shown is the MS/MS spectrum of the precursor [M + 2H]2+ ion at m/z 701.8182 derived from a tryptic digest of H1 from starved Tetrahymena. This ion was identified as the methyl ester of the double phosphopeptide AApSASTpTPVKK. This phosphorylation site at Ser-42 was only observed after IMAC enrichment of the H1 sample. Calc., calculated; Exp., experimental.

 
Confirmation of the previously identified canonical Cdc2 kinase (Thr-34, Thr-46, and Thr-53) and non-canonical (Ser-42 and Ser-44) phosphorylation sites validates our MS approach for mapping phosphorylation sites on histone H1. However, consistent with the notion that a strength of MS over conventional biochemical methods is the ability to detect novel modifications sites in a sensitive, rapid, and accurate manner, we also discovered novel modifications sites on Tetrahymena histone H1. Displayed in Fig. 5A is the MS/MS spectrum of the [M + 2H]2+ phosphopeptide ion at m/z 969.4908 from the Asp-N digest of growing Tetrahymena H1. This phosphopeptide was determined to have the sequence APRSpSTSKSATREKK (Pr groups on Lys residues and amino terminus), corresponding to residues 1–15 of H1, and possessed a novel phosphorylation site at Ser-5. However, careful inspection of the fragment ions in the MS/MS spectrum indicated that Ser-4 is also a phosphorylation site on H1 as well. For example, the b4' ion at m/z 548, the b4'-H3PO4 ion at m/z 450, and the y11' ion at m/z 1390 confirm that a small population of fragment ions show Ser-4 phosphorylation. Novel modification sites on lysine residues were also revealed in our MS studies. Fig. 5B displays the MS/MS spectrum of the [M + 2H]2+ ion at m/z 812.4531 from a trypsin digest of H1 from starved cultures. This peptide ion was found to span residues 152–163 and contains a low level novel acetylation site at Lys-154. The high mass accuracy of the LTQ-FT mass spectrometer (<5 ppm) can easily distinguish between trimethylated and acetylated peptides ({Delta}M = 0.0364 Da). The mass recorded on the peptide ion at m/z 812.4531 is consistent with an acetylation mark (+1.97 ppm) and not trimethylation. Listed in Table I are selected peptides from the Asp-N and trypsin digests of histone H1 from growing Tetrahymena cells. In general, the Asp-N digests produced more sequence coverage of the H1 protein than the trypsin digests as trypsin cleavage at the abundant lysine residues of H1 produced numerous peptides that are too small for MS analysis. However, high sequence coverage (95%) was obtained from the combined use of both proteases with H1.


Figure 5
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  		FIG. 5. Identification of novel post-translational modifications on Tetrahymena histone H1. A, tandem mass spectrum of an [M + 2H]2+ ion at m/z 969.4908 from an Asp-N digest of propionylated H1 from growing Tetrahymena cells identifying Ser-5 as a phosphorylation site. Fragment peaks (b4', b4'-H3PO4, and y11') also show evidence of a phosphorylation mark on residue Ser-4 as well. B, MS/MS spectrum of an [M + 2H]2+ ion at m/z 812.4531 generated from an Asp-N digest of propionylated H1 from starved Tetrahymena cells. The high mass accuracy of LTQ-FT mass spectrometer distinguishes Lys-154 as an acetylation site and not trimethylation. Calc., calculated; Exp., experimental.

 
Cation-exchange Chromatography Separation of Multiply Phosphorylated H1 Proteins Reveal Hierarchy of Phosphorylation Sites—
As reported previously, the hierarchy of Tetrahymena H1 phosphorylation has been suggested (24), but the exact sequential nature of H1 phosphorylation has not been established. To determine the order of phosphorylation on histone H1, highly phosphorylated Tetrahymena H1 was first separated by cation-exchange chromatography. As shown in Fig. 6, numerous peaks were resolved when the PCA-soluble extract from growing cells was chromatographed. Peaks labeled from 6 to 15 contained histone H1 proteins that varied in either phosphate:protein stoichiometry or in the site of modification by one or more phosphates. The most heavily phosphorylated H1 protein eluted in fraction 6, whereas the least phosphorylated H1 protein (unphosphorylated) eluted in the last peak (fraction 15). Mass spectra of each fraction were acquired on LTQ-FT and home-built 8.5-tesla FT mass spectrometers to obtain the approximate molecular weight of the intact H1 species in all fractions. For example, the molecular mass of the H1 in fraction 14 was found to be 17,733 Da as shown in Fig. 7A. In comparison, the molecular weight of the unphosphorylated H1 protein in fraction 15 was determined to be 17,653 (Table II). We observed some mass discrepancies in most fractions that may be a result of the formation of oxidation products and/or metal ion adducts following cation-exchange chromatography as analysis of fractionated intact H1 protein species by ECD (Fig. 7B) confirmed the predicted amino acid sequence (Fig. 7C). Listed in Table II are the molecular weights recorded from the ESI-MS analysis of the cation-exchange fractions 6–15. The number of phosphate groups on each H1 protein in the fractions was estimated using 17,653 Da as the base molecular mass of unphosphorylated H1 and 80 Da as the mass increase due to the addition of a phosphate molecule. Histone H1 proteins containing zero to seven phosphate groups were observed in the cation-exchange fractions. The most highly phosphorylated H1 protein in fraction 6 contained seven phosphate groups, consistent with our detection of seven total sites of phosphorylation from the "bottom-up" global analysis of H1 peptides. Nevertheless the localization of these phosphate groups on the H1 protein from any cation-exchange fraction could not be determined from the measurement of the intact H1 protein molecular weight alone.


Figure 6
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  		FIG. 6. Cation-exchange chromatography of histone H1 from growing Tetrahymena cells. Peaks labeled 6–15 correspond to various H1 species containing zero to seven phosphate groups. Peak 15 was found to be the unphosphorylated H1 species, whereas peak 6 was found to contain the most highly phosphorylated form of H1 (seven phosphate groups). mAU, milliabsorbance units.

 

Figure 7
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  		FIG. 7. A, ESI-MS of fraction 14 from the cation-exchange chromatography separation of histone H1 from growing Tetrahymena cells. Inset MS shows SWIFT isolation of the +27 charge state species with a molecular mass consistent with the addition of one phosphate group and an oxidation modification (17,733.4 Da). B, ECD fragmentation of the SWIFT-isolated +27 charge state species. Results of approximately 200 summed scans are shown. C, ECD fragmentation map of histone H1 from fraction 14 showing a phosphorylation modification at Thr-53 and a possible oxidized residue on Met-69. Calc., calculated; Exp., experimental.

 

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  		TABLE II Molecular weights of intact Tetrahymena H1 proteins from growing cells following cation-exchange separation determined by ESI-LTQ-FT-MS

The number of phosphate groups on each H1 protein in the fractions was estimated using 17,653 Da as the base molecular mass of unphosphorylated H1 and 80 Da as the mass increase due to the addition of a phosphate molecule. The gene-derived protein sequence predicts a molecular mass of 17,637 Da. Localization of phosphorylation sites was determined by digesting H1 with Asp-N and analysis of peptides by MS/MS experiments (CAD) or by ECD fragmentation of intact H1 protein (ECD).

 
To determine the location of the phosphorylation sites on the cation-exchange chromatography-separated H1 molecules, each H1 fraction was subjected to Asp-N digestion, propionylated, and analyzed by tandem mass spectrometry on an LTQ-FT mass spectrometer. Additionally intact H1 from a few selected fractions was also subjected to ECD fragmentation for localization of modification sites as well. All MS/MS spectra used in these identifications are displayed as supplemental data. Using this approach, the sites of phosphorylation on every H1 protein in all fractions were determined as listed in Table II. Fraction 14, which contained an H1 protein with one phosphate group, displayed peptides that were consistent with phosphorylation at Thr-34, Thr-46, or Thr-53. Therefore, three monophosphorylated H1 species were present in that fraction. Fractions containing two phosphate groups on histone H1 showed that several species were present with two major phosphorylation sites in various combinations utilizing residues Thr-34, Thr-46, or Thr-53. However, it should be noted that some minor phosphorylation at Ser-5 was detected on fractions containing H1 with two phosphate groups albeit at much lower levels. Fractions containing H1 protein with three phosphorylation sites primarily displayed those sites at Thr-34, Thr-46, and Thr-53 with a small population again containing Ser-5 phosphorylation as well. Histone H1 from fractions containing four phosphate groups generated peptides with phosphorylation sites at Ser-5, Thr-34, Thr-46, and Thr-53, whereas fractions containing five phosphorylation modifications displayed these marks at Ser-4, Ser-5, Thr-34, Thr-46, and Thr-53. Fraction 6 contained H1 species with six and seven phosphates attached. The hexaphosphorylated form possessed phosphorylation at Ser-4, Ser-5, Thr-34, Ser-42 or Ser-44, Thr-46, and Thr-53, whereas the heptaphosphorylated form was phosphorylated at all of the sites observed in our global analysis (Ser-4, Ser-5, Thr-34, Ser-42, Ser-44, Thr-46, and Thr-53). These results are consistent with the existence of a hierarchy of phosphorylation on linker histone H1 (24). Phosphorylation of H1 occurs first at the canonical proline-directed Cdc2-related kinase sites (Thr-34, Thr-46, or Thr-53) with little apparent difference in the propensity for phosphorylation among these three sites. However, once one of those three sites is phosphorylated, phosphorylation at Ser-5 commences at a low level. After the three canonical proline-directed sites are fully phosphorylated, the levels of Ser-5 phosphorylation vastly increase. The phosphorylation at Ser-4 is observed after phosphorylation of Ser-5, Thr-34, Thr-46, and Thr-53. Finally phosphorylation of H1 at Ser-42 is detectable after the phosphorylation of the previously mentioned five residues, and Ser-44 is the last residue to become phosphorylated. This order of phosphorylation was also suggested by our global analysis as phosphorylation at the three Cdc2-related kinase sites was qualitatively found to be in higher abundance than Ser-4 or Ser-5 phosphorylation, although all five sites could be detected by normal C18 MS analyses. However, Ser-42 and Ser-44 were only observed in our global analysis of H1 phosphorylation after IMAC enrichment of phosphopeptides, suggesting that these sites were minor phosphorylation sites.

Stable Isotope Labeling and IMAC for Monitoring Differential Expression Site-specific Phosphorylation of Histone H1—
We decided to use stable isotope labeling and mass spectrometry (Fig. 2) for the relative quantitative analysis of Tetrahymena histone H1 site-specific phosphorylation to overcome the problems inherent with the use of site-specific antibodies. These problems include cross-reactivity with similar modification sites on the same or different protein and epitope occlusion through interference by neighboring post-translational modifications, which have been demonstrated to be a significant problem for histone studies (40–42). As stated earlier, digesting histone H1 with Asp-N creates 10 peptides that allow for straightforward differential modification analysis by mass spectrometry. To compare phosphorylation sites on histone H1 from growing and starved Tetrahymena cells, the propionylated peptides from each sample were labeled with different isotope labels as depicted in Fig. 2. Histone H1 peptides from growing Tetrahymena were converted to d0-methyl esters, and those from starved Tetrahymena were converted to d3-methyl esters. After MS screening of the samples, aliquots containing equal amounts of the two samples were mixed, phosphopeptides were enriched by IMAC, and both IMAC-isolated phosphopeptides and non-phosphorylated peptides collected in the flow-through of the IMAC column were analyzed separately by nano-flow LC coupled to an LTQ-FT mass spectrometer. Using this methodology of stable isotope labeling allows for H1 peptides from the growing Tetrahymena (d0-methyl ester) and starved Tetrahymena (d3-methyl esters) samples containing identical modifications to appear as signal peak doublets (chemically identical peptides whose isotopic distribution are separated by 3-Da shifts per incorporated isotopic label) in the mass spectrometer. Although the d0- and d3-methyl esters do not exactly co-elute (offset by several seconds), the quantification of peptide species is straightforward after identification of the peptide by accurate mass and MS/MS fragmentation. Relative quantification of peptides involves measuring the area under the curve of a particular peptide and expressing the ratio of that modified form to the total area under the curve of all the forms of that peptide.

Shown in Figs. 8 and 9 are examples of differential phosphoproteomic IMAC-LC-MS/MS analyses of histone H1 peptides from growing and starved Tetrahymena cells. Fig. 8A shows a full mass spectrum from a differential IMAC analysis between the two H1 samples following Asp-N digestion. As can be seen, a set of [M + 2H]2+ doublet ion peaks at m/z 475.2398 and 478.2596 were detected (zoom MS inset) and found to be separated by a {Delta}M of 3 Da. MS/MS fragmentation of these peptide ions confirmed their sequence as DVpTPVKA with the incorporation of two methyl ester isotopic labels (d0 for growing Tetrahymena at m/z 475.2398 and d3 for starved Tetrahymena at m/z 478.2596) on the carboxyl terminus and aspartic acid residue as shown in Fig. 8B. Additionally the growing Tetrahymena peptide at m/z 475.2398 was found in 2-fold higher abundance compared with the starved Tetrahymena sample. The results of three independent differential IMAC-LC-MS/MS analyses of histone H1 from growing and starved Tetrahymena cells following Asp-N digestions are summarized in Table III. In general as expected, higher levels of phosphorylation were found on growing strains of Tetrahymena when compared with the starved cultures. More importantly, we detected differences in phosphorylation at specific sites between the two samples. For instance, the peptide containing the Thr-46 phosphorylation site (DTKPTPTKGKAASASTpTPVKK) was found to be ~7x enriched in the growing sample, whereas a similar peptide containing the Thr-34 phosphorylation site (DTKPpTPTKGKAASASTTPVKK) was found to be increased in the growing sample 4-fold compared with the starved sample. Furthermore the doubly phosphorylated peptide containing both Thr-34 and Thr-46 phosphorylation and the peptide APRSpSTSKSATREKK were only detected in the growing Tetrahymena sample. Analysis of the peptides collected from the flow-through of the IMAC column (non-phosphorylated peptides) also added some information. Non-phosphorylated peptides where no modified counterparts were detected in the IMAC enriched sample (such as DHKKAPIKKAIAKK) were found roughly in 1:1 ratios between the growing and starved Tetrahymena samples as expected for equally mixed samples. An exception to this was the peptide APRSSTSKSATREKK that was found at a ratio of 1.02 ± 0.15, although a phosphorylated version (APRSpSTSKSATREKK) was observed in the IMAC enriched growing Tetrahymena sample. This result may reflect the extremely low abundance of the phosphorylated peptide. Additionally some non-phosphorylated peptides were found in lower amounts in the growing versus the starved H1 samples (Table III). This included the peptides DTKPTPTKGKAASASTTPVKK and DVTPVKA, whose abundance is expected to be diminished in the growing samples because both were also present as phosphorylated forms.


Figure 8
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  		FIG. 8. A, full MS of a differential phosphoproteomic analysis of histone H1 from growing Tetrahymena (isotopically labeled with d0-methyl esters, m/z 475.2398) and starved Tetrahymena (isotopically labeled with d3-methyl esters, m/z 478.2596) from an Asp-N digest. Zoom inset shows the labeled pair of [M + 2H]2+ ions from peptide residues 51–57. This particular phosphorylation modification at Thr-53 (DVpTPVKA) was found to be present in a higher abundance (~2x) in the growing sample. B, MS/MS spectrum of the doubly charged peptide ion at m/z 478.2596 derived from the starved Tetrahymena sample confirming the sequence as DVpTPVKA. Note the heavy Me (d3) label present on the Asp residue as well as on the carboxyl terminus, whereas the amino terminus and lysine residue have been modified by the addition of propionyl amide groups.

 

Figure 9
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  		FIG. 9. A, full MS of a differential phosphoproteomic analysis of histone H1 from growing Tetrahymena (isotopically labeled with d0-methyl esters, m/z 661.8354) and starved Tetrahymena (isotopically labeled with d3-methyl esters, m/z 663.3458) from a trypsin digest. Zoom inset shows the labeled pair of [M + 2H]2+ ions from peptide residues 40–50. This particular phosphorylation modification at Thr-46 (AASASTpTPVKK) was found to be present in a higher abundance (~6x) in the growing sample. B, tandem mass spectrum of the doubly charged peptide ion at m/z 663.3458 generated from the starved Tetrahymena sample. The sequence was determined as AASASTpTPVKK. Note the heavy Me (d3) label on the carboxyl terminus, whereas the amino terminus and lysine residues have been modified by the addition of propionyl amide groups.

 

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  		TABLE III Summary of phosphoproteomic differential expression IMAC-MS analysis from Asp-N digests of histone H1 from growing and starved T. thermophila

pS, phosphoserine; pT, phosphothreonine. Selected peptides from Asp-N digests of histone H1 protein from growing and starved Tetrahymena cells after IMAC enrichment and analysis using LTQ-FT-MS are shown. All data were relatively quantified by the use of stable isotope labeling (d0- and d3-methyl esters) and mass spectrometry during three individual experiments completed.

 
Phosphorylation at residues Ser-42 and Ser-44 was not readily detected in Asp-N digests but was observed on H1 peptides derived from trypsin digests. This might be due to a lower ionization efficiency of multiply phosphorylated forms of the peptide representing residues 30–50 (DTKPTPTKGKAASASTTPVKK) as only two phosphorylation sites at Thr-34 and Thr-46 were routinely detected simultaneously on this fragment. Therefore, to obtain data regarding the levels of phosphorylation at Ser-42 and Ser-44 from both growing and starved Tetrahymena cell cultures we performed trypsin digestion, amino group propionylation, IMAC enrichment, and LC-MS/MS on these samples as these sites had been observed previously with these methods. Fig. 9A shows a full mass spectrum from a differential IMAC analysis between the two H1 samples following trypsin digestion. Zoom expansion of the region covering m/z 660–665 shows a set of doubly charged doublet peaks at m/z 661.8354 and 663.3458, respectively. The mass difference between these two sets of ion peaks is 1.5 Da, indicating that one methyl ester label is present on the peptide. MS/MS fragmentation of these ions validated the sequence of the peptide as AASASTpTPVKK (Thr-46 phosphorylation) as shown in Fig. 9B, and a 6-fold increase in phosphorylation at this site in the growing sample relative to the starved sample was quantified in agreement with the results from the Asp-N-generated peptide containing this phosphorylation site (~7-fold increase, Table III). Exhaustive digestion of histone H1 with trypsin is not ideal for MS-based analyses as trypsin digestion generates many peptides that are too small for MS analysis. Nevertheless partial trypsin digestion of histone H1 generated peptides spanning nearly all of the phosphorylation sites on this protein. Table IV summarizes the results of three independent differential expression IMAC-LC-MS/MS analyses of trypsin-digested histone H1 from growing and starved Tetrahymena cells. Major phosphorylation sites at Thr-34, Thr-46, and Thr-53 and minor sites at Ser-42 and Ser-44 were observed and relatively quantified in the trypsin-digested IMAC enriched samples (Table IV). The peptide DTKPpTPTK was found at an ~4-fold increase in the growing versus starved sample; this is similar to the increase found on the DTKPpTPTKGKAASASTTPVKK peptide generated from the Asp-N digest (also about 4-fold increase). Additionally the peptide containing the Thr-53 phosphorylation site (DVpTPVK) was quantified as having a 2-fold enrichment on the growing Tetrahymena H1 again in agreement to the Asp-N-generated peptide (DVpTPVKA) containing this same modification site (Table III). Trypsin digestion of histone H1 also allowed for the detection and quantification of phosphorylation sites at Ser-42 and Ser-44 as shown in Table IV. A triply phosphorylated peptide with the sequence AApSApSTpTPVKK was also only observed in the growing Tetrahymena sample. Finally non-phosphorylated peptides analyzed from the flow-through of the IMAC column also displayed the same trends as seen in their Asp-N-generated counterparts. Overall the differential phosphoproteomic IMAC analyses from the Asp-N and trypsin digestions of H1 from growing and starved cultures were in agreement and provided complementary information about the site-specific phosphorylation of H1 during normal growth and prolonged starvation conditions.


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  		TABLE IV Summary of phosphoproteomic differential expression IMAC-MS analysis from trypsin digests of histone H1 from growing and starved T. thermophila

pS, phosphoserine; pT, phosphothreonine. Selected peptides from trypsin digests of histone H1 protein from growing and starved Tetrahymena cells after IMAC enrichment and analysis using LTQ-FT-MS are shown. All data were relatively quantified by the use of stable isotope labeling (d0- and d3-methyl esters) and mass spectrometry during three individual experiments completed.

 

   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Post-translational modifications of histone proteins have become a focal point for several areas of chromatin research as these modifications can affect many distinct nuclear events such as transcriptional regulation and DNA damage repair. As different types of modifications and specific modification sites on all histones have important roles in chromatin structure and function, it is essential to have both biological and analytical tools to interrogate histone modification states and probe their possible cellular function. Mass spectrometry has recently established itself as an impressive technique capable of discovering novel histone modifications, most recently demonstrated with the identification of acetylation at Lys-56 of H3 and Lys-91 of H4. Both marks were shown to be significantly involved in global gene regulation, nucleosome assembly, and DNA repair (43–46). Histone methylation and acetylation have been well studied and found to act as binding platforms for certain effector molecules (e.g. H3 Lys-9 trimethylation attracts HP1 to silence chromatin regions). Nevertheless the exact function of histone phosphorylation remains unclear. One proposed function of phosphorylation is to act as a mechanism for regulating protein-protein interactions. Indeed Fischle et al. (47) showed that phosphorylation of Ser-10 during mitosis ejects HP1 molecules from H3, and a similar phenomenon was observed when HP1 was bound to the histone H1.4 variant methylated at Lys-25 upon Ser-26 phosphorylation (48). However, because phosphorylation of human H1 and other histones occurs on sites that are not adjacent to modified lysine residues, other functions for histone phosphorylation must exist. To this end, phosphorylation of histone H2B Ser-14 (Ser-10 in yeast) and phosphorylation of H2A.X at Ser-139 have been found to be critical processes linked to apoptosis and double strand DNA damage repair, whereas the phosphorylation of Ser-31 on the histone H3.3 variant was shown to be located only at distinct chromosomal regions adjacent to centromeres during mitosis (49–51). The acetyltransferase Tip60 also acetylates phospho-H2Av in Drosophila (H2A.X human) and exchanges it with an unmodified H2Av at sites of DNA double strand breaks, demonstrating a role for phosphorylation in the mechanism for selective histone exchange (52). Whereas H1 is phosphorylated in a cell cycle-dependent manner in many species, H1 is highly phosphorylated outside of mitosis in Tetrahymena, suggesting the possibility that H1 phosphorylation is predominantly linked to interphase processes in this organism. This notion is supported by the finding that the transcription of different sets of genes are up-regulated in Tetrahymena strains expressing forms of H1 mutated to mimic constitutive phosphorylation (25, 53). Although it seems likely that phosphorylation of H1 is involved in transcriptional regulation in higher eukaryotes in a similar fashion, the role of phosphorylation at mitosis and other processes remains obscure.

Therefore, using a combination of analytical approaches, we generated information regarding the phosphorylation states of histone H1 from both growing and starved T. thermophila cells. We confirmed five previously characterized sites of H1 phosphorylation in this organism through MS/MS analyses following enzymatic digestions, chemical derivatization, and IMAC enrichment. Chemical derivatization of the H1 generated peptides reduced the charge states of the peptides from being mostly higher than +4 charge states to 2+ and 3+ charge peptides whose MS/MS spectra could be interpreted with little difficulty. Three of these known phosphorylation sites at Thr-34, Thr-46, and Thr-53 are located on regions that fit the consensus motif for cyclin-dependent kinases ((S/T)PXZ motif regions). IMAC proved to be essential as the abundance of the two other previously characterized phosphorylation sites at Ser-42 and Ser-44 was too low to be observed using normal C18-based analyses and could only be detected after IMAC enrichment. The digestion of the linker histone with both trypsin and Asp-N was necessary to achieve maximal sequence coverage, which allowed us to identify two novel additional sites of phosphorylation on the amino terminus of H1 (Ser-4 and Ser-5 phosphorylation). Because the regions surrounding the phosphorylation sites at Ser-4, Ser-5, Ser-42, and Ser-44 do not seem to fit the required motif for cyclin-dependent kinases, it will be interesting to determine which kinase(s) phosphorylates those sites. Additionally new non-phosphorylation modification sites (Lys-77 or Lys-78 and Lys-154 acetylation) were also detected on both growing and starved Tetrahymena. The significance of these new modification sites remains to be determined.

Dynamics of Tetrahymena histone H1 phosphorylation were also uncovered using mass spectrometry-based techniques. Separation of highly phosphorylated histone H1 species by cation-exchange chromatography and tandem MS experiments elucidated information concerning the hierarchy of phosphorylation of Tetrahymena H1. We conclude that the order of phosphorylation is Thr-34, Thr-46, and Thr-53 first; Ser-4 and Ser-5 next; and Ser-42 and Ser-44 last. The hierarchy observed suggests that the phosphorylation sites at Thr-34, Thr-46, and Thr-53 may act as primer phosphorylation sites that once phosphorylated by cyclin-dependent kinases recruit other kinases to phosphorylate the remaining sites on H1. This system could be analogous to the phosphorylation of ß-catenin in the Wnt pathway by the priming kinase GSK-3ß, which creates an acidic patch that is recognized by a second kinase (casein kinase 1{epsilon}) resulting in further phosphorylation of ß-catenin and destabilization of the ß-catenin destruction complex (54). The phosphorylation of any one of the three cyclin-dependent sites was found to be enough to initiate the phosphorylation of Ser-4 and/or Ser-5, and the levels of Ser-4 and Ser-5 phosphorylation vastly increased after all three cyclin-dependent sites became fully phosphorylated. This sequence of phosphorylation is consistent with the earlier finding that mutation of the five phosphorylation sites identified previously by Edman degradation sequencing (Thr-34, Ser-42, Ser-44, Thr-46, and Thr-53) abolished all detectable 32P signal from radiolabeled H1 (24). Because phosphorylation of at least one of the three cyclin-dependent sites (Thr-34, Thr-46, and Thr-53) is required before Ser-4 or Ser-5 become phosphorylated, mutation of all three is predicted to abolish phosphorylation at Ser-4 and Ser-5. A recent report has also suggested a possible hierarchy of human histone H1 phosphorylation as phosphorylation of certain variants occurred non-randomly during interphase and mitosis (55).

Lastly stable isotope labeling, IMAC, and MS/MS analyses were utilized to gain perspective on the differential site-specific phosphorylation on histone H1 from growing and starved Tetrahymena cell cultures. Digestion of the linker histone with Asp-N was crucial in generating uniform fragments (both unphosphorylated and phosphorylated peptides) across the entire protein that could be easily detected and relatively quantified using mass spectrometry. Alternatively trypsin digestion of histone H1 also resulted in producing smaller peptides, however, that did contain the phosphorylation site regions. Collecting the flow-through from the IMAC columns allowed us to measure the ratio of unphosphorylated peptides in addition to phosphorylated peptides retained on the IMAC columns. Using these procedures, we were able to relatively quantify differentially phosphorylated sites between growing and starved Tetrahymena cells. Results were consistent between stable isotope labeling IMAC experiments and between the two different digests as well. The methodology used in these experiments can be carried over to investigate any protein that shows changes in phosphorylation (or other modification) levels upon cellular perturbation as recently demonstrated for histone modifications other than phosphorylation (56, 57). In sum, we completed a comprehensive analysis of Tetrahymena histone H1 phosphorylation and compared how phosphorylation at each site is regulated in growing and starved cells. These findings should contribute to future investigations of the roles of H1 phosphorylation in gene activation or silencing.


  FOOTNOTES
 
Received, March 13, 2006, and in revised form, July 7, 2006.

Published, MCP Papers in Press, July 10, 2006, DOI 10.1074/mcp.M600086-MCP200

1 The abbreviations used are: HP1, heterochromatin-associated protein 1; RP, reversed-phase; ECD, electron capture dissociation; LTQ, linear ion trap; PCA, perchloric acid; SWIFT, stored waveform inverse Fourier transform; Pr, propionyl amide. Back

* This work was supported in part by National Institutes of Health Grants GM40922 (to C. D. A.), GM067193 (to N. L. K.), and GM37537 (to D. F. H.) and by grants from the Roy J. Carver Charitable Trust (04-76) and University of Illinois (to C. A. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

S The on-line version of this article (available at http://www.mcponline.org) contains supplemental material. Back

§ Supported by Ford Foundation, Sigma Xi, and the Institute for Genomic Biology at the University of Illinois. Present address: Inst. for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801. Back

|||| To whom correspondence should be addressed. Tel.: 434-924-3610; Fax: 434-982-2781; E-mail: dfh{at}virginia.edu


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