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High-Resolution, Accurate-Mass (HR/AM) and Intelligent Acquisition-Enabled Global Discovery and Quanti cation of Histones, Histone PTMS, and Histone
Modi cation Enzymes in Mesenchymal Stem Cells
Results
Intelligent Data Acquisition
Initial discovery experiments w
experiments (Figures 1, 2). Th
data-dependent acquisition ac
and peptides as well as the init
histones and histone modificat
experiments were performed t
identification based on theoreti
discovery experiments were u
precursor and product ion
m/z
relative retention time values.
based on known and novel PT
peptides was used to create a
perform qual/quan determinati
optimization of instrument par
capabilities resulting in signific
experiment. The final assay pe
histone and histone-related pe
approach enabled quantificatio
novel targets across different s
aging (replicative or genotoxic
Overview
Purpose:
Development of a real-time, intelligent acquisition strategy for HR/AM global
targeted quantification of histones, histone PTMs, and histone modification enzymes
Methods:
Mesenchymal stem cells were derived from human adipose tissue and
treated with bleomycin. Samples were analyzed on a Thermo Scientific™
Q Exactive™ mass spectrometer using an intelligent acquisition method.
Results:
The final assay performed qual/quan studies on 36 proteins and 154 histone
and histone-related peptides and modified analogs. The combined approach enabled
quantification of previously identified modified peptides as well as novel targets across
different samples and were correlated to somatic or stem cell aging (replicative or
genotoxic stress-induced senescence).
Introduction
Chromatin is viewed as an operational interface for almost all known nuclear
processes. Nucleosomal packaging and histone modifications dictate the different
degrees of primary chromatin compaction achieved by additional chromatin structural
proteins. For example, euchromatic chromatin fibers contain six nucleosomes per
11 nm; however, heterochromatin consists of 12–15 nucleosomes per 11 nm. A
dynamic balance between these two radically different chromatin compaction states is
at the very core of the high-level nuclear chromatin organization (nuclear architecture),
and is vital for maintaining cell-type identity over time. Functional differences between
the cells in an organism are defined by epigenetic factors and epigenetic programs,
which are critical for the preservation of functional integrity of the cellular phenotypes
(1). However, as a mediator of the external signals, chromatin is anything but static.
Nucleosome unwrapping and disassembly events, which must occur during DNA
replication, transcription, and DNA repair, can directly influence the state of chromatin
compaction. Several lines of evidence obtained in Drosophila, yeast, and plants
indicate that chromatin undergoes disassembly during the onset of DNA double-strand
breaks (DSB) and repair at the DSB sites. It is still an open question how to quantify
the chromatin changes. One of the accepted ways to do such is through cellular
fractionation. Based on the salt and detergent concentrations in buffer solutions, three
cellular fractions can be obtained: cytoplasmic, nuclear soluble (proteins that are
loosely bound to chromatin), and a chromatin fraction (containing tightly bound
proteins, or enzymes engaged in chromatin modifications). Tracing histones or
histone-modifying activities (HMT, HATs, and HDACs) dynamics in these fractions
during biological events permits the assessment of overall chromatin dynamics and
full characterization of the cellular stage (2).
In this study, we set out to quantify the changes in the chromatin composition in
primary human stem cells upon acute DNA damage and during drug-evoked
senescence. This assessment allowed insights into the “access, repair, restore” model
of chromatin dynamics upon DNA damage repair and a better understanding of
whether or not misregulation of this axis is one of the critical factors of cellular
senescence. In our model system, we compared two conditions: 1) Acute DNA
damage (2 hrs treatment with bleomycin) and 2) DNA damage-induced cellular
senescence (cellular recovery after 5 days of post-bleomycin treatment). Both
conditions were compared to the normally proliferating (self-renewing) cells.
Using a novel application of intelligent acquisition and HR/AM MS, we have developed
workflows for quantitative global profiling and targeted analysis of histones, histone
post-translational modifications (PTMs), and histone modification enzymes.
Methods
Samples
Mesenchymal stem cells were derived from human adipose tissue and treated with
bleomycin as follows: nuclear, cytoplasmic, and chromatin fraction samples were
isolated from proliferating (bleomycin –) cells as well as cells under conditions of acute
DNA damage (bleomycin 2 hrs) and drug-induced senescence (bleomycin 2 hrs
followed by a 5-day recovery).
MS Data Acquisition and Analysis
Samples were analyzed on a Q Exactive mass spectrometer. Initial discovery
experiments were performed to generate a list of peptides/protein IDs and
corresponding spectral libraries. A subset of peptides was selected for targeted
quantification across the different samples. Heavy labeled analogs were used for
qual/quan data processing. Qualitative and quantitative data analysis was performed
using Thermo Scientific™ Pinpoint™ software.
FIGURE 1. Intelligent data ac
intelligent data acquisition s
a targeted scanning window
product ion spectral acquisi
matching are performed to i
Theoretical
Isotope
Experimental
HR/AM MS
Spectrum
*
*
Measured Ion Intensity
Start time for “watc
Trig
Thre
1.
FIGURE 2. Extracted ion chr
A. Precursor ion charge stat