Abstract
Histone post-translational modifications (PTMs) are fundamental players of chromatin regulation, as they contribute to editing histone chemical properties and recruiting proteins for gene transcription and DNA repair. Mass spectrometry (MS)-based proteomics is currently the most widely adopted strategy for high-throughput quantification of hundreds of histone PTMs. Samples such as primary tissues, complex model systems, and biofluids are hard to retrieve in large quantities. Because of this, it is critical to know whether the amount of sample available would lead to an exhaustive analysis if subjected to MS. In this work, we assessed the reproducibility in quantification of histone PTMs using a wide range of starting material, that is, from 5 000 000 to 50 000 cells. We performed the experiment using four different cell lines, that is, HeLa, 293T, human embryonic stem cells (hESCs), and myoblasts, and we quantified a list of 205 histone peptides using ion trap MS and our in-house software. Results highlighted that the relative abundance of some histone PTMs deviated as little as just 4% when comparing high starting material with histone samples extracted from 50 000 cells, for example, H3K9me2 (40% average abundance). Low abundance PTMs such as H3K4me2 (<3% average abundance) showed higher variability, but still ∼34%. This indicates that most PTMs, and especially abundant ones, are quantified with high precision starting from low cell counts. This study will help scientists to decide whether specific experiments are feasible and to plan how much sample should be reserved for histone analysis using MS.