EpiProfile 2.0: A Computational Platform for Processing Epi-Proteomics Mass Spectrometry Data

Abstract

Epigenetics has become a fundamental scientific discipline with various implications for biology and medicine. Epigenetic marks, mostly DNA methylation and histone post-translational modifications (PTMs), play important roles in chromatin structure and function. Accurate quantification of these marks is an ongoing challenge due to the variety of modifications and their wide dynamic range of abundance. Here we present EpiProfile 2.0, an extended version of our 2015 software (v1.0), for accurate quantification of histone peptides based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. EpiProfile 2.0 is now optimized for data-independent acquisition through the use of precursor and fragment extracted ion chromatography to accurately determine the chromatographic profile and to discriminate isobaric forms of peptides. The software uses an intelligent retention time prediction trained on the analyzed samples to enable accurate peak detection. EpiProfile 2.0 supports label-free and isotopic labeling, different organisms, known sequence mutations in diseases, different derivatization strategies, and unusual PTMs (such as acyl-derived modifications). In summary, EpiProfile 2.0 is a universal and accurate platform for the quantification of histone marks via LC-MS/MS. Being the first software of its kind, we anticipate that EpiProfile 2.0 will play a fundamental role in epigenetic studies relevant to biology and translational medicine. EpiProfile is freely available at https://github.com/zfyuan/EpiProfile2.0_Family .

Keywords: acylation; epigenetics; histone; mutations; post-translational modification; quantification.

Figure 1.. Layouts on H3 9−17 KSTGGKAPR in the endogenous samples.

Two layouts are: K9me3 < K9me2 < K9ac < unmodified < K9me1, and K9me3K14ac < K9me2K14ac < K9acK14ac < K14ac < K9me1K14ac, where the later layout is earlier than the former layout. Subplots show the extracted ion chromatography (XIC) for each peptide based on the precursor m/z (i.e. x-axis is the retention time, y-axis is the intensity). PTM type and retention time of each peptide are above the corresponding chromatographic peak. Fragment ions have the same retention time as the precursor ion. Because fragment ions are usually much lower than the precursor ion, they are aside the precursor ion rather than under the precursor ion profile.

Figure 2.. Discrimination of H3 K9ac and K14ac by unique fragment ions.

(A) The same MS2 from DIA is matched to both H3 K9ac and K14ac. Unique fragment ions between K9 and K14 are extracted to calculate the ratio of K9ac to K14ac on this MS2. (B) During the retention time of the chromatographic peak there are 16 MS2. On each MS2 the ratio of K9ac to K14ac is calculated. (C) The whole chromatographic peak is split to two components by the ratios at each time point. The overall ratio of K9ac to K14ac is calculated by the area under curve.

Figure 3.. High fidelity quantification using EpiProfile 2.0 for high and low resolution data and for DDA and DIA acquisition methods.

(A) Correlation of areas of ion chromatograms for synthetic peptides extracted using EpiProfile 2.0 vs manually. (B) Same plot using signals of histone peptides from HeLa cells. (C) Coefficient of variation of three technical replicates for histone peptide relative abundance from HeLa cells acquired using DIA or DDA in high resolution MS (OT) or low resolution MS (IT). Below, number of peptides quantified. On the right, coefficient of variation estimated across all results from all acquisitions (using the average of technical replicates). (D) Relative abundance of single histone H3 and H4 PTMs using high resolution (left) and low resolution (right) acquisition from HeLa cells acquired by DIA. (E) Correlation (in green) and correlation significance p-value (in blue) for all replicates and acquisition methods, calculated using HeLa cells peptide relative abundance.