pr7b00544_si_003.xlsx (83.08 kB)
Assessment of Quantification Precision of Histone Post-Translational Modifications by Using an Ion Trap and down To 50 000 Cells as Starting Material
dataset
posted on 2017-11-09, 00:00 authored by Qi Guo, Simone Sidoli, Benjamin A. Garcia, Xiaolu ZhaoHistone
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.