Characterization of Phosphorylation Sites on Histone H1 Isoforms by Tandem Mass Spectrometry

Abstract

Histone H1 isoforms isolated from asynchronously grown HeLa cells were subjected to enzymatic digestion and analyzed by nano-flow reversed-phase high performance liquid chromatography (RP-HPLC) tandem mass spectrometry (MS/MS) on both quadrupole ion trap and linear quadrupole ion trap-Fourier transform ion cyclotron resonance mass spectrometers. We have observed all five major isoforms of histone H1 (H1.1, H1.2, H1.3, H1.4, and H1.5) as well as a lesser studied H1, isoform H1.X. MS/MS experiments confirmed N-terminal acetylation on all isoforms plus a single internal acetylation site. Immobilized metal affinity chromatography in combination with tandem mass spectrometry was utilized to identify 19 phosphorylation sites on the five major H1 isoforms plus H1.X. Fourteen of these phosphorylation sites were located on peptides containing the cyclin dependent kinase (CDK) consensus motif (S/T)-P-X-Z (where X is any amino acid and Z is a basic amino acid). Five phosphorylation sites were identified in regions that did not fit the consensus CDK motif. One of these phosphorylation sites was found on the serine residue on the H1.4 peptide KARKSAGAAKR. The adjacent lysine residue to the phosphoserine was also shown to be methylated. This finding raises the question of whether the hypothesized "methyl/phos" switch could be extended to linker histones, and not exclusive to core histones.

Liquid chromatography mass spectrometry profiling of histones

“…In addition, mono-phosphorylation was observed on H1.X and H1.1 but not on H1.0 and H1.3. These results are consistent with those reported previously by Hunt and coworkers who used immobilized metal ion affinity chromatography (IMAC) combined with tandem mass spectrometry to characterize histone phosphorylation [61]. The LC-MS profiles were able to detect the presence of phosphorylation without enrichment by IMAC.…”

“…The masses for all the H1 variants were 42 Da higher than their theoretical masses. H1 variants have been reported to be N-terminally acetylated, which would precisely account for this mass difference [61].…”

N- and C-terminal Domains Determine Differential Nucleosomal Binding Geometry and Affinity of Linker Histone Isotypes H10 and H1c

“…This structural flexibility could allow interaction with distinct protein binding partners as suggested in the case of Msx1 and HP1 (47,48). Recent studies have also mapped distinct post-translational modifications within the N-terminal domains of H1 (49,50). The N terminus of H1 0 differs from that of H1c in length (20 versus 32 residues) with shorter basic and distal subregions.…”

Mass spectrometry-based strategies for characterization of histones and their post-translational modifications

“…Phosphorylation presents a challenge for MS detection due to its much lower abundance than acetylation and methylation. Immobilized metal affinity chromatography (IMAC) has been demonstrated by Hunt and coworkers as an effective way to enrich these low-abundance peptides [149]. They combined IMAC and nano-LC/MS/MS to detect 19 phosphorylation sites on six H1 isoforms derived from asynchronously grown HeLa cells.…”