Neutrophil elastase is the 'histone H2A-specific protease'

Fundamental changes in the epigenetic status of histones from hematopoietic stem cells might be one of the driving forces behind many malignant transformations and subsequent leukemia development. The amino-terminal tail of histones and the carboxy-tail of histone H2A protrude from the nucleosome and can be modified by many different posttranslational modifications (PTM) on at least 60 different residues, thereby mediating chromatin dynamics. During an iTRAQ proteome analysis on Chronic Lymphocytic Leukemia (CLL) B-cells we came across a specific kind of histone modification that has received only little attention in epigenetics until now: histone clipping. The clipping of the histone H2A C-tail at V114 was more abundant in the CLL B-cell clones compared to healthy B-cells. This specific proteolytic product was already described in the context of leukemia in the late 70’s and is still being referenced today. To specifically quantify this clipping product, we developed and optimized a sensitive and high throughput AQUA approach, based on two isotopically labeled synthetic peptides. We screened 36 patients to investigate any discriminative power of clipped H2A as a potential prognostic marker. In doing so, we found that clipping mainly occurs in the myeloid lineage and has no clear link to the CLL B-cell clone. Here we show that the responsible enzyme, until now known as the “H2A specific protease”, but previously not identified, actually is Neutrophil Elastase. With the growing interest in the epigenetic potential of histone clipping we emphasize its potential role in hematopoietic differentiation

PiTRAQ, a strategy to simultaneously correlate protein expression and phosphorylation stoichiometry between different samples: evaluation on different mass spectrometers

C

iTRAQ as a method for optimization : enhancing peptide recovery after gel fractionation

At the dawn of a new era in label-free quantitation on high-resolution MS instruments, classical methods such as iTRAQ continue to provide very useful insights in comparative proteomics. The potential to multiplex samples makes this reporter-based labeling technique highly suited for method optimization as demonstrated here by a set of standard series. Instead of studying ratios of annotated proteins, we propose an alternative method, based on the analysis of the average reporter ratios of all the spectra from a sample or a large distinct subset herein. This strategy circumvents the bias, associated with the annotation and iTRAQ quantitation, leading to increased adequacy in measuring yield differences between workflows. As gel electrophoresis prior to MS analysis is highly beneficial, for example, as a fractionation step, the approach was applied to evaluate the influence of several parameters of the established in-gel digestion protocol. We quantified the negative effect of SYPRO Ruby staining and the positive effect of gel fixation prior to digestion on peptide yield. Finally, we emphasize the benefits of adding CaCl2 and ACN to a tryptic in-gel digest, resulting in an up to tenfold enhanced peptide recovery and fewer trypsin missed cleavages

Neutrophil elastase in the capacity of the 'H2A-specific protease'

The amino-terminal tail of histones and the carboxy-tail of histone H2A protrude from the nucleosome and can become modified by many different posttranslational modifications (PTM). During a mass spectrometric proteome analysis on haematopoietic cells we encountered a histone PTM that has received only little attention since its discovery over 35 years ago: truncation of the histone H2A C-tail at V-114 which is mediated by the "H2A specific protease" (H2Asp). This enzyme is still referenced today but it was never identified. We first developed a sensitive AQUA approach for specific quantitation of the H2AV(114) clipping. This clipping was found only in myeloid cells and further cellular fractionation lead to the annotation of the H2Asp as Neutrophil Elastase (NE). Ultimate proof was provided by NE incubation experiments and by studying histone extracts from NE Null mice. The annotation of the H2Asp not only is an indispensable first step in elucidating the potential biological role of this enzymatic interaction but equally provides the necessary background to critically revise earlier reports of H2A clipping

Immunization with the immunodominant Helicobacter suis urease subunit B induces partial protection against H. suis infection in a mouse model

Helicobacter (H.) suis is a porcine and human gastric pathogen. Previous studies in mice showed that an H. suis infection does not result in protective immunity, whereas immunization with H. suis whole-cell lysate (lysate) protects against a subsequent experimental infection. Therefore, two-dimensional gel electrophoresis of H. suis proteins was performed followed by immunoblotting with pooled sera from H. suis- infected mice or mice immunized with lysate. Weak reactivity against H. suis proteins was observed in post-infection sera. Sera from lysate-immunized mice, however, showed immunoreactivity against a total of 19 protein spots which were identified using LC-MS/MS. The H. suis urease subunit B (UreB) showed most pronounced reactivity against sera from lysate-immunized mice and was not detected with sera from infected mice. None of the pooled sera detected H. suis neutrophil-activating protein A (NapA). The protective efficacy of intranasal vaccination of BALB/c mice with H. suis UreB and NapA, both recombinantly expressed in Escherichia coli (rUreB and rNapA, respectively), was compared with that of H. suis lysate. All vaccines contained choleratoxin as adjuvant. Immunization of mice with rUreB and lysate induced a significant reduction of H. suis colonization compared to non-vaccinated H. suis-infected controls, whereas rNapA had no significant protective effect. Probably, a combination of local Th1 and Th17 responses, complemented by antibody responses play a role in the protective immunity against H. suis infections

Quantitative proteomics to characterize specific histone H2A proteolysis in chronic lymphocytic leukemia and the myeloid THP-1 cell line

Proteome studies on hematological malignancies contribute to the understanding of the disease mechanism and to the identification of new biomarker candidates. With the isobaric tag for relative and absolute quantitation (iTRAQ) method we analyzed the protein expression between B-cells of healthy people and chronic lymphocytic leukemia (CLL) B-cells. CLL is the most common lymphoid cancer of the blood and is characterized by a variable clinical course. By comparing samples of patients with an aggressive vs. indolent disease, we identified a limited list of differentially regulated proteins. The enhanced sensitivity attributed to the iTRAQ labels led to the discovery of a previously reported but still not clarified proteolytic product of histone H2A (cH2A) which we further investigated in light of the suggested functional properties of this modification. In the exploratory proteome study the Histone H2A peptide was up-regulated in CLL samples but a more specific and sensitive screening of a larger patient cohort indicated that cH2A is of myeloid origin. Our subsequent quantitative analysis led to a more profound characterization of the clipping in acute monocytic leukemia THP-1 cells subjected to induced differentiation

Quantitative Proteomics to Characterize Specific Histone H2A Proteolysis in Chronic Lymphocytic Leukemia and the Myeloid THP-1 Cell Line

Proteome studies on hematological malignancies contribute to the understanding of the disease mechanism and to the identification of new biomarker candidates. With the isobaric tag for relative and absolute quantitation (iTRAQ) method we analyzed the protein expression between B-cells of healthy people and chronic lymphocytic leukemia (CLL) B-cells. CLL is the most common lymphoid cancer of the blood and is characterized by a variable clinical course. By comparing samples of patients with an aggressive vs. indolent disease, we identified a limited list of differentially regulated proteins. The enhanced sensitivity attributed to the iTRAQ labels led to the discovery of a previously reported but still not clarified proteolytic product of histone H2A (cH2A) which we further investigated in light of the suggested functional properties of this modification. In the exploratory proteome study the Histone H2A peptide was up-regulated in CLL samples but a more specific and sensitive screening of a larger patient cohort indicated that cH2A is of myeloid origin. Our subsequent quantitative analysis led to a more profound characterization of the clipping in acute monocytic leukemia THP-1 cells subjected to induced differentiation