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High resolution is not a strict requirement for characterization and quantification of histone post-translational modifications.

ABSTRACT: Mass spectrometry (MS) is a powerful tool to accurately identify and quantify histone post-translational modifications (PTMs). High-resolution mass analyzers have been regarded as essential for these PTM analyses because the mass accuracy afforded is sufficient to differentiate trimethylation versus acetylation (42.0470 and 42.0106 Da, respectively), whereas lower-resolution mass analyzers cannot. Noting this limitation, we sought to determine whether lower-resolution detectors are nonetheless adequate for histone PTM analysis by comparing the low-resolution LTQ Velos Pro with the high-resolution LTQ-Orbitrap Velos Pro. We first determined that the optimal scan mode on the LTQ Velos Pro is the Enhanced scan mode with respect to apparent resolution, number of MS and MS/MS scans per run, and reproducibility of label-free quantifications. We next compared the performance of the LTQ Velos Pro to the LTQ-Orbitrap Velos Pro using the same criteria for comparison, and we found that the main difference is that the LTQ-Orbitrap Velos Pro is able to resolve the difference between acetylation and trimethylation while the LTQ Velos Pro cannot. However, using heavy isotope labeled synthetic peptide standards and retention time information enables confident assignment of these modifications and comparable quantification between the instruments. Therefore, lower-resolution instruments can confidently be utilized for histone PTM analysis.

SUBMITTER: Karch KR

PROVIDER: S-EPMC4261946 | biostudies-other | 2014 Dec

REPOSITORIES: biostudies-other

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Project description:Histone post-translational modifications (PTMs) have a fundamental function in chromatin biology, as they model chromatin structure and recruit enzymes involved in gene regulation, DNA repair, and chromosome condensation. High throughput characterization of histone PTMs is mostly performed by using nano-liquid chromatography coupled to mass spectrometry. However, limitations in speed and stochastic sampling of data dependent acquisition methods in MS lead to incomplete discrimination of isobaric peptides and loss of low abundant species. In this work, we analyzed histone PTMs with a data-independent acquisition method, namely SWATH™ analysis. This approach allows for MS/MS-based quantification of all analytes without upfront assay development and no issues of biased and incomplete sampling. We purified histone proteins from human embryonic stem cells and mouse trophoblast stem cells before and after differentiation, and prepared them for MS analysis using the propionic anhydride protocol. Results on histone H3 peptides verified that sequential window acquisition of all theoretical mass spectra could accurately quantify peptides (<9% average coefficient of variation, CV) over four orders of magnitude, and we could discriminate isobaric and co-eluting peptides (e.g. H3K18ac and H3K23ac) using MS/MS-based quantification. This method provided high sensitivity and precision, supported by the fact that we could find significant differences for remarkably low abundance PTMs such as H3K9me2S10ph (relative abundance <0.02%). We performed relative quantification for few sample peptides using different fragment ions and observed high consistency (CV <15%) between the fragments. This indicated that different fragment ions can be used independently to achieve the same peptide relative quantification. Taken together, sequential window acquisition of all theoretical mass spectra proved to be an easy-to-use MS acquisition method to perform high quality MS/MS-based quantification of histone-modified peptides.

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High resolution is not a strict requirement for characterization and quantification of histone post-translational modifications.

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