7 research outputs found
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Using mass spectrometry to quantify changes in histone post-translational modifications as a result of DNA double-strand breaks
The structure of DNA is under constant threat from both extracellular and intracellular sources, causing mutations that could result in the reduced viability of the cell. In the nucleus of eukaryotes, DNA wraps around histone proteins 1.67 times. Two copies of Histone H2A, H2B, H3 and H4 congregate to form an octomer: along with the DNA strand together make the nucleosome. It has been shown that via histone post-translational modifications (HPTM), the nucleosome will signal to recruit biological machinery to process DNA such as during transcription or repair. One of the most characterised HPTM is phosphorylation of serine 139 of H2AX (γH2AX), which is indicative of a DNA strand break — single or double. Subsequent to the phosphorylation, the cell’s DNA damage response (DDR) will proceed to recruit proteins that repair the damage.
Recent studies have implicated a myriad of HPTMs in the DDR. However, many of the putative DDR HPTM were described with various immunoassays such as western blot and immunofluorescence. Immunoassays are notorious for reproducibility issues and experimental design biases. Comparatively, mass spectrometry is renowned for its precision and its ability to accommodate high throughput analysis of many datasets in an unbiased manner. We utilise MS to quantify γH2AX changes after DNA damage and show that H2AX tyrosine 142 phosphorylation is not as abundant as previously shown. We also developed a method for simultaneously quantifying many HPTMs changes that occur as a result of the DDR at both proximal to the site of damage and a genomic scale. The developed method of quantifying DDR HPTM accommodates future experiments such as determining whether DDR HPTMs are old or new (with SILAC) or be adapted to analyse HPTMs as a result of transcription, translation or development. A translational aspect would be to quantify the epigenetic marks of cancer patients before and post-treatment
Histone H2AX Y142 phosphorylation is a low abundance modification
We employ targeted mass spectrometry to compare the levels of H2AX S139 phosphorylation (γH2AX) and Y142 phosphorylation. We use synthetic peptides to facilitate MS optimisation and estimate relative detection efficiencies for the different modifications. Despite phosphopeptide enrichment from large amounts of starting material, we are unable to detect endogenous H2AX Y142 phosphorylation, indicating that it is present in low abundance (<1%). We also calculate the relative levels of H2AX compared to other H2A isoforms and quantify the proportion of H2AX that is phosphorylated on S139 (γH2AX) after ionising radiation
Evaluation of Acquisition Modes for Semi-Quantitative Analysis by Targeted and Untargeted Mass Spectrometry
RATIONALE: Analyte quantitation by mass spectrometry underpins a diverse range of scientific endeavors. The fast growing field of mass spectrometer development has resulted in several targeted and untargeted acquisition modes suitable for these applications. By characterizing the acquisition methods available on an ion mobility (IM) enabled orthogonal acceleration time-of-flight (oa-ToF) instrument, the optimum modes for analyte semi-quantitation can be deduced. METHODS: Serial dilutions of commercial metabolite, peptide, or crosslinked peptide analytes were prepared in matrices of human urine or E. coli digest. Each analyte dilution was introduced into an IM separation enabled oa-ToF mass spectrometer by reversed phase liquid chromatography and electrospray ionization. Data were acquired for each sample in duplicate using nine different acquisition modes, including four IM enabled acquisitions modes, available on the mass spectrometer. RESULTS: Five (metabolite) or seven (peptide/crosslinked peptide) point calibration curves were prepared for analytes across each of the acquisition modes. A non-linear response was observed at high concentrations for some modes, attributed to saturation effects. Two correction methods, one MS1 isotope-correction and one MS2 ion intensity-correction, were applied to address this observation, resulting in an up to two-fold increase in dynamic range. By averaging the semi-quantitative results across analyte classes, two parameters, linear dynamic range (LDR) and lower limit of quantitation (LLOQ), were determined to evaluate each mode. CONCLUSION: Comparison of the acquisition modes revealed that data independent acquisition and parallel reaction monitoring methods are most robust for semi-quantitation when considering achievable LDR and LLOQ. IM enabled modes exhibited sensitivity increases, but a simultaneous reduction in dynamic range which required correction methods to recover. These findings will assist users in identifying the optimum acquisition mode for their analyte quantitation needs, supporting a diverse range of applications and providing guidance for future acquisition mode developments
Multi-omic profiling reveals the ataxia protein sacsin is required for integrin trafficking and synaptic organization
Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a childhood-onset cerebellar ataxia caused by mutations in SACS, which encodes the protein sacsin. Cellular ARSACS phenotypes include mitochondrial dysfunction, intermediate filament disorganization, and progressive death of cerebellar Purkinje neurons. It is unclear why the loss of sacsin causes these deficits or why they manifest as cerebellar ataxia. Here, we perform multi-omic profiling in sacsin knockout (KO) cells and identify alterations in microtubule dynamics and mislocalization of focal adhesion (FA) proteins, including multiple integrins. Deficits in FA structure, signaling, and function can be rescued by targeting PTEN, a negative regulator of FA signaling. ARSACS mice possess mislocalization of ITGA1 in Purkinje neurons and synaptic disorganization in the deep cerebellar nucleus (DCN). The sacsin interactome reveals that sacsin regulates interactions between cytoskeletal and synaptic adhesion proteins. Our findings suggest that disrupted trafficking of synaptic adhesion proteins is a causal molecular deficit in ARSACS
Evaluation of Acquisition Modes for the Quantitative Analysis of Cross-Linked Peptides by Targeted and Untargeted Mass Spectrometry
Cross-linking
mass spectrometry (XL-MS) is a structural biology technique that can provide
insights into the structure and interactions of proteins and their complexes,
especially those that cannot be easily assessed by other methods. Quantitative
XL-MS has the potential to probe the structural and temporal dynamics of
protein complexes; however, it requires further development. Until recently,
quantitative XL-MS has largely relied upon isotopic labeling and data dependent
acquisition (DDA) methods, limiting the number of biological samples that can
be studied in a single experiment. Here, the acquisition modes available on an
ion mobility (IM) enabled QToF mass spectrometer are evaluated for the
quantitation of cross-linked peptides, eliminating the need for isotopic labels
and thus expanding the number of comparable studies that can be conducted in
parallel. Workflows were optimized using metabolite and peptide standards
analyzed in biological matrices, facilitating modelling of the data and addressing
linearity issues, which allow for significant increases in dynamic range. Evaluation
of the DDA acquisition method commonly used in XL-MS studies indicated
consistency issues between technical replicates and reduced performance in quantitative
metrics. On the contrary, data independent acquisition (DIA) and parallel
reaction monitoring (PRM) modes proved more robust for analyte quantitation.
Mobility enabled modes exhibited an improvement in sensitivity due to the added
dimension of separation, and a simultaneous reduction in dynamic range, which
was largely recovered by correction methods. Hi[3] and probabilistic
quantitation methods were successfully applied to the DIA data, determining the
molar amounts of cross-linked peptides relative to their linear counterparts.</div