Hydrophilic interaction liquid chromatography (HILIC) in proteomics

In proteomics, nanoflow multidimensional chromatography is now the gold standard for the separation of complex mixtures of peptides as generated by in-solution digestion of whole-cell lysates. Ideally, the different stationary phases used in multidimensional chromatography should provide orthogonal separation characteristics. For this reason, the combination of strong cation exchange chromatography (SCX) and reversed-phase (RP) chromatography is the most widely used combination for the separation of peptides. Here, we review the potential of hydrophilic interaction liquid chromatography (HILIC) as a separation tool in the multidimensional separation of peptides in proteomics applications. Recent work has revealed that HILIC may provide an excellent alternative to SCX, possessing several advantages in the area of separation power and targeted analysis of protein post-translational modifications

Fine Mapping of Posttranslational Modifications of the Linker Histone H1 from Drosophila melanogaster

The linker histone H1 binds to the DNA in between adjacent nucleosomes and contributes to chromatin organization and transcriptional control. It is known that H1 carries diverse posttranslational modifications (PTMs), including phosphorylation, lysine methylation and ADP-ribosylation. Their biological functions, however, remain largely unclear. This is in part due to the fact that most of the studies have been performed in organisms that have several H1 variants, which complicates the analyses. We have chosen Drosophila melanogaster, a model organism, which has a single H1 variant, to approach the study of the role of H1 PTMs during embryonic development. Mass spectrometry mapping of the entire sequence of the protein showed phosphorylation only in the ten N-terminal amino acids, mostly at S10. For the first time, changes in the PTMs of a linker H1 during the development of a multicellular organism are reported. The abundance of H1 monophosphorylated at S10 decreases as the embryos age, which suggests that this PTM is related to cell cycle progression and/or cell differentiation. Additionally, we have found a polymorphism in the protein sequence that can be mistaken with lysine methylation if the analysis is not rigorous

Comparative Analyses of SUV420H1 Isoforms and SUV420H2 Reveal Differences in Their Cellular Localization and Effects on Myogenic Differentiation

Methylation of histone H4 on lysine 20 plays critical roles in chromatin structure and function via mono- (H4K20me1), di- (H4K20me2), and trimethyl (H4K20me3) derivatives. In previous analyses of histone methylation dynamics in mid-gestation mouse embryos, we documented marked changes in H4K20 methylation during cell differentiation. These changes were particularly robust during myogenesis, both in vivo and in cell culture, where we observed a transition from H4K20me1 to H4K20me3. To assess the significance of this change, we used a gain-of-function strategy involving the lysine methyltransferases SUV420H1 and SUV420H2, which catalyze H4K20me2 and H4K20me3. At the same time, we characterized a second isoform of SUV420H1 (designated SUV420H1_i2) and compared the activity of all three SUV420H proteins with regard to localization and H4K20 methylation.Immunofluorescence revealed that exogenous SUV420H1_i2 was distributed throughout the cell, while a substantial portion of SUV420H1_i1 and SUV420H2 displayed the expected association with constitutive heterochromatin. Moreover, SUV420H1_i2 distribution was unaffected by co-expression of heterochromatin protein-1α, which increased the targeting of SUV420H1_i1 and SUV420H2 to regions of pericentromeric heterochromatin. Consistent with their distributions, SUV420H1_i2 caused an increase in H4K20me3 levels throughout the nucleus, whereas SUV420H1_i1 and SUV420H2 facilitated an increase in pericentric H4K20me3. Striking differences continued when the SUV420H proteins were tested in the C2C12 myogenic model system. Specifically, although SUV420H1_i2 induced precocious appearance of the differentiation marker Myogenin in the presence of mitogens, only SUV420H2 maintained a Myogenin-enriched population over the course of differentiation. Paradoxically, SUV420H1_i1 could not be expressed in C2C12 cells, which suggests it is under post-transcriptional or post-translational control.These data indicate that SUV420H proteins differ substantially in their localization and activity. Importantly, SUV420H2 can induce a transition from H4K20me1 to H4K20me3 in regions of constitutive heterochromatin that is sufficient to enhance myogenic differentiation, suggesting it can act an as epigenetic ‘switch’ in this process

Mapping H4K20me3 onto the chromatin landscape of senescent cells indicates a function in control of cell senescence and tumor suppression through preservation of genetic and epigenetic stability

Background: Histone modification H4K20me3 and its methyltransferase SUV420H2 have been implicated in suppression of tumorigenesis. The underlying mechanism is unclear, although H4K20me3 abundance increases during cellular senescence, a stable proliferation arrest and tumor suppressor process, triggered by diverse molecular cues, including activated oncogenes. Here, we investigate the function of H4K20me3 in senescence and tumor suppression. Results: Using immunofluorescence and ChIP-seq we determine the distribution of H4K20me3 in proliferating and senescent human cells. Altered H4K20me3 in senescence is coupled to H4K16ac and DNA methylation changes in senescence. In senescent cells, H4K20me3 is especially enriched at DNA sequences contained within specialized domains of senescence-associated heterochromatin foci (SAHF), as well as specific families of non-genic and genic repeats. Altered H4K20me3 does not correlate strongly with changes in gene expression between proliferating and senescent cells; however, in senescent cells, but not proliferating cells, H4K20me3 enrichment at gene bodies correlates inversely with gene expression, reflecting de novo accumulation of H4K20me3 at repressed genes in senescent cells, including at genes also repressed in proliferating cells. Although elevated SUV420H2 upregulates H4K20me3, this does not accelerate senescence of primary human cells. However, elevated SUV420H2/H4K20me3 reinforces oncogene-induced senescence-associated proliferation arrest and slows tumorigenesis in vivo. Conclusions: These results corroborate a role for chromatin in underpinning the senescence phenotype but do not support a major role for H4K20me3 in initiation of senescence. Rather, we speculate that H4K20me3 plays a role in heterochromatinization and stabilization of the epigenome and genome of pre-malignant, oncogene-expressing senescent cells, thereby suppressing epigenetic and genetic instability and contributing to long-term senescence-mediated tumor suppression

Comparing and Combining Capillary Electrophoresis Electrospray Ionization Mass Spectrometry and Nano-Liquid Chromatography Electrospray Ionization Mass Spectrometry for the Characterization of Post-translationally Modified Histones

We present the first comprehensive capillary electrophoresis electrospray ionization mass spectrometry (CESI-MS) analysis of post-translational modifications derived from H1 and core histones. Using a capillary electrophoresis system equipped with a sheathless high-sensitivity porous sprayer and nano–liquid chromatography electrospray ionization mass spectrometry (nano-LC-ESI-MS) as two complementary techniques, we characterized H1 histones isolated from rat testis. Without any pre-separation of the perchloric acid extraction, a total of 70 different modified peptides, including 50 phosphopeptides, were identified in the rat linker histones H1.0, H1a-H1e, and H1t. Out of the 70 modified H1 histone peptides, 27 peptides could be identified with CESI-MS only, and 11 solely with LC-ESI-MS. Immobilized metal-affinity chromatography enrichment prior to MS analysis yielded a total of 55 phosphopeptides; 22 of these peptides could be identified only by CESI-MS, and 19 only by LC-ESI-MS, showing the complementarity of the two techniques. We mapped 42 H1 modification sites, including 31 phosphorylation sites, of which 8 were novel sites. For the analysis of core histones, we chose a different strategy. In a first step, the sulfuric-acid-extracted core histones were pre-separated using reverse-phase high-performance liquid chromatography. Individual rat testis core histone fractions obtained in this way were digested and analyzed via bottom-up CESI-MS. This approach yielded the identification of 42 different modification sites including acetylation (lysine and N(α)-terminal); mono-, di-, and trimethylation; and phosphorylation. When we applied CESI-MS for the analysis of intact core histone subtypes from butyrate-treated mouse tumor cells, we were able to rapidly detect their degree of modification, and we found this method very useful for the separation of isobaric trimethyl and acetyl modifications. Taken together, our results highlight the need for additional techniques for the comprehensive analysis of post-translational modifications. CESI-MS is a promising new proteomics tool as demonstrated by this, the first comprehensive analysis of histone modifications, using rat testis as an example