Global Quantification of Histones, Histone PTMs, and Histone Modification Enzymes in Mesenchymal Stem Cells
Using a Multiplexed SRM Assay
Victoria Lunyak2, Bryan Krastins1, Alejandra Garces1, Benny Blackwell2, David Sarracino1, Amol Prakash1, Maryann S. Vogelsang1, Gouri Vadali1, Shadab Ahmad1,
Scott Peterman1, Mary F Lopez1
1Thermo Fisher Scientific, Cambridge, MA USA; 2Buck Institute for Age Research, Novato, CA, USA
Overview
Purpose: Develop a multiplexed, quantitative SRM assay for histones and histone
modification proteins for profiling protein abundance in self-renewing and
senescent mesenchymal stem cells.
Methods: A multiplexed SRM assay for gamma Histone H2Ax and nine histone
modification proteins was developed in one day using heavy-labeled synthetic
peptides (gamma Histone H2Ax ) and recombinant proteins (nine histone
modification proteins). Mesenchymal stem cell lysates from self-renewing and
senescent cultures were digested with trypsin and analyzed on a triple quadrupole
mass spectrometer. Protein abundance patterns were analyzed with software that
facilitates quantitative verification of putative biomarkers and general quantitative
proteomics, as well as with pathway analysis software.
Results: Dramatic changes in protein abundance were observed between the
groups.
Introduction
Histone post-translational modifications (PTMs) are a central theme in the
regulation of gene expression. A rapidly growing list of modifications confirms that
they play fundamental roles in chromatin remodeling processes. Chromatin
structure plays a key role in DNA damage repair. The response to double-strand
breaks (DSB) induced by radiation or DNA-damaging agents typically results in
DNA damage response (DDR) and accumulation of the proteins involved in DNA
repair in subnuclear foci. The rapid phosphorylation of Ser139 at the C terminus of
the H2Ax sequence functions in the recruitment of DDR proteins after DNA
damage. These processes are also thought to play a role in stem cell development
and senescence (1). To date, most studies in this area have been carried out by
genomic analysis, immunostaining, or top-down LC-MS/MS analysis, and as such
are not fully quantitative.
Over the years, mass spectrometry (MS) has emerged as a powerful analytical
technique for the identification of unknown compounds, determination of molecular
weights, elucidation of molecular structures, and quantification of a wide variety of
analytes. Although MS has been applied to protein biomarker discovery for at
least a decade, one of the most difficult problems has been the precise
quantification and characterization of PTMs and isoforms detected in “shotgun”
LC-MS/MS experiments (2,3). Recently, a different type of MS experiment,
selected-reaction monitoring (SRM), is increasingly being applied to the
measurement of protein biomarkers (4,5). Advantages of SRM-based MS
experiments include very high throughput, sensitivity, specificity, and absolute
quantification using internal isotopically labeled standards. SRM experiments are
also a powerful technique for the measurement and quantification of PTMs and
disease-specific alterations. MS is the only technique that can deliver the
specificity required to detect isoforms associated with protein sequence
microheterogeneity and many clinically relevant variants (6). Such assays could
potentially be used for disease diagnosis and prognosis, and they are typically
robust and selective, even in complex matrices (7).
In this study, we developed a multiplexed, quantitative SRM assay for nine histone
modification proteins and histone gamma H2Ax sequences. The assay was used
to interrogate mesenchymal stem cells derived from human adipose tissue.
Lysates were prepared from cell cultures at different ages (self-renewing (SR) and
senescent (S)). This approach was extremely fruitful, allowing us to confirm and
quantify previously identified PTMs occurring on histone gamma H2Ax, and to
demonstrate the differential abundance of histone modification proteins not
previously reported in conjunction with somatic stem cell aging.
Methods
Sample Preparation
Human adipose derived stem cells (hADSCs) were isolated from human
subcutaneous white adipose tissue collected during liposuction procedures and
prepared as previously published (8). Nuclear and cytoplasmic proteins were
isolated from self-renewing hADSCs using the Thermo Scientific ProteoJET
Cytoplasmic and Nuclear Protein Extraction Kit, according to the manufacturer’s
protocol. Samples were normalized to total protein. Samples were digested with
trypsin and prepared for MS as described in (4,5).
SRM Assay Development and Data Analysis
SRM assays were developed using Thermo Scientific Pinpoint software and a
Thermo Scientific TSQ Vantage triple quadrupole mass spectrometer as previously
described (4,5). Normalized protein abundance data were analyzed using
TABLE 1. Proteins and peptide sequences quantified in the multiplexed SRM
assay.
ID and Description
Peptide sequence
gi|21361095 histone-lysine N-methyltransferase EZH2 isoform a
YDC[Carboxymethyl]FLHR
TEILNQEWK
gi|4504253 Histone H2AX human
KATQASQEY[Phosphoryl]
KATQASQEY
KATQAS[Phosphoryl]QEY
gi|153791535 histone acetyltransferase KAT2A
NLLAQIK
LGVFSAC[Carboxymethyl]K
VIGGIC[Carboxymethyl]FR
KLFVADLQR
YETTHVFGR
SQAEDVATYK
DPDQLYTTLK
gi|5901962 histone acetyltransferase MYST2
VGSPERPLSDLGLISYR
gi|13699813 histone-lysine N-methyltransferase NSD3 isoform short
LIISTPNQR
A.
B.
gi|19913358probable histone-lysine N-methyltransferase NSD2 isoform 1 YNVGDLVWSK
GIGTPPNTTPIK
gi|50345997 histone acetyltransferase p300
SPMDLSTIK
gi|4507321 histone-lysine N-methyltransferase SUV39H1
FAYNDQGQVR
gi|50659082 histone-lysine N-methyltransferase SUV420H1 isoform 1
NM[Oxid]VVNGR
MNTSAFPSR
SSVGVKKNSK
EHVFIYLR
Conclusion
TABLE 2. Functional description of the proteins quantified in the SRM assay
The experiments presented in this poster present evidence suggesting differential
distribution and abundance of histone H2Ax and histone modification proteins in
senescent and self-renewing mesenchymal stem cells.
KAT2A K(lysine) acetyltransferase 2A; Functions as a histone acetyltransferase (HAT) to promote
The data demonstrate:
transcriptional activation. Acetylation of histones gives a specific tag for epigenetic transcription activation.
Has significant histone acetyltransferase activity with core histones, but not with nucleosome core particles.
Component of the ATAC complex, a complex with histone acetyltransferase activity on histones H3 and H4.
MYST2 MYST histone acetyltransferase 2; Component of the HBO1 complex which has a histone H4specific acetyltransferase activity, a reduced activity toward histone H3 and is responsible for the bulk of
histone H4 acetylation in vivo. Through chromatin acetylation, it may regulate DNA replication and act as a
coactivator of TP53-dependent transcription. Specifically represses AR-mediated transcription.
EP300
Functions as histone acetyltransferase and regulates transcription via chromatin remodeling.
Acetylates all four core histones in nucleosomes. Histone acetylation gives an epigenetic tag for
transcriptional activation. Mediates cAMP-gene regulation by binding specifically to phosphorylated CREB
protein.
SUV42H1 Suppressor of variegation 4-20 homolog 1 (Drosophila); Histone methyltransferase that
specifically trimethylates 'Lys-20' of histone H4. H4 'Lys-20' trimethylation represents a specific tag for
epigenetic transcriptional repression. Mainly functions in pericentric heterochromatin regions, thereby playing
a central role in the establishment of constitutive heterochromatin in these regions. SUV420H1 is targeted to
histone H3 via its interaction with RB1 family proteins (RB1, RBL1 and RBL2) by similarity.
WHSC1L1 Wolf-Hirschhorn syndrome candidate 1-like 1; Histone methyltransferase. Preferentially
methylates 'Lys-4' and 'Lys-27' of histone H3. H3 'Lys-4' methylation represents a specific tag for epigenetic
transcriptional activation, while 'Lys-27' is a mark for transcriptional repression.
EZH2 Enhancer of zeste homolog 2 (Drosophila); Polycomb group (PcG) protein. Catalytic subunit of the
PRC2/EED-EZH2 complex, which methylates 'Lys-9' and 'Lys-27' of histone H3, leading to transcriptional
repression of the affected target gene. Able to mono-, di- and trimethylate 'Lys-27' of histone H3 to form
H3K27me1, H3K27me2 and H3K27me3, respectively. Compared to EZH2-containing complexes, it is more
abundant in embryonic stem cells and plays a major role in forming H3K27me3, which is required for
embryonic stem cell identity and proper differentiation.
WSC1 Wolf-Hirschhorn syndrome candidate 1; Probable histone methyltransferase (by similarity). May act
as a transcription regulator that binds DNA and suppresses IL5 transcription.
SUV39H1 Suppressor of variegation 3-9 homolog 1 (Drosophila); Histone methyltransferase that
specifically trimethylates 'Lys-9' of histone H3 using monomethylated H3 'Lys- 9' as substrate. Also weakly
methylates histone H1 (in vitro). H3 'Lys-9' trimethylation represents a specific tag for epigenetic
transcriptional repression by recruiting HP1 (CBX1, CBX3 and/or CBX5) proteins to methylated histones.
Mainly functions in heterochromatin regions, thereby playing a central role in the establishment of constitutive
heterochromatin at pericentric and telomere regions.
Histone H2Ax H2A histone family, member X; Variant histone H2A which replaces conventional H2A in a
subset of nucleosomes. Nucleosomes wrap and compact DNA into chromatin, limiting DNA accessibility to the
cellular machineries which require DNA as a template. Histones thereby play a central role in transcription
regulation, DNA repair, DNA replication and chromosomal stability. DNA accessibility is regulated via a
complex set of post-translational modifications of histones, also called histone code, and nucleosome
remodeling.
FIGURE 1. Abundance ratios of histones and histone modification proteins
in self-renewing (SR) and senescent (S) mesenchymal stem cells.
A. cytoplasmic fraction, B. nuclear fraction
A.
B.
Ingenuity Pathways Analysis (IPA®) software.
  Development of a multiplexed, quantitative SRM assay for nine histone
modification proteins and histone gamma H2Ax peptides.
  Successful application of the assay to cytoplasmic and nuclear fractions derived
from senescent and self-renewing adipose-derived mesenchymal stem cells.
It has been shown that unresolved persistent DNA damage occurs upon
senescence [1]. This is consistent with the apparent increase in ser 139
phosphorylated H2Ax in our data. In order to understand the importance of
chromatin dynamics in aging, we next plan to investigate the changes in the
chromatin-associated histone chaperones and chromatin-remodeling complexes
upon replicative senescence of human adult mesenchymal stem cells. It is
known that both core histone chaperones and ATP-utilizing motor proteins
incorporated into histone remodeling complexes can interact with repair
factors and transmit signaling to cell cycle control machinery [1]. Several
distinct histone chaperones, such as CAF-1 (chromatin assembly factor-1)
Asf1 (anti-silencing function-1), NAP1 (nucleosome assembly protein-1) and
HirA (histone regulatory protein A) 50 participate in the deposition of the histones
onto the DNA and/or mediate the translocation of core histones from the
cytoplasm to the nucleus [1].
References
1.  Tollervey J, Lunyak VV. Epigenetics: Judge, jury and executioner of stem cell
fate. Epigenetics. 2012 Aug 1;7(8). [Epub ahead of print]
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3.  Kamath KS, Vasavada MS, Srivastava S. Proteomic databases and tools to
decipher post-translational modifications. J Proteomics. 2011 Sep 29. [Epub
ahead of print]
4.  Prakash A, Rezai T, Krastins B, Sarracino D, Athanas M, Russo P, Zhang H,
Tian Y, Li Y, Kulasingam V, Drabovich A, Smith CR, Batruch I, Oran PE, Fredolini
C, Luchini A, Liotta L, Petricoin E, Diamandis EP, Chan DW, Nelson R, Lopez
MF. Interlaboratory reproducibility of selective reaction monitoring assays using
multiple upfront analyte enrichment strategies. J Proteome Res. 2012 Aug 3;11
(8):3986-95. Epub 2012 Jul 3.
5.  Lopez MF, Sarracino DA, Prakash A, Athanas M, Krastins B, Rezai T, Sutton JN,
Peterman S, Gvozdyak O, Chou S, Lo E, Buonanno F, Ning M. Discrimination of
ischemic and hemorrhagic strokes using a multiplexed, mass spectrometrybased assay for serum apolipoproteins coupled to multi-marker ROC algorithm.
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6.  Borges CR, Rehder DS, Jarvis JW, Schaab MR, Oran PE, Nelson RW. Fulllength characterization of proteins in human populations. Clin Chem. 2010 56
(2):202-11.
Results
Differential Abundance of Histones and Histone Modification Proteins in
Cytoplasmic and Nuclear Fractions from Self-Renewing and Senescent
Mesenchymal Stem Cells
Table 1 shows the proteins and peptide sequences used in the multiplexed assay.
Surrogate peptides were chosen based upon optimization of parameters in
iterative experiments using recombinant proteins or based upon previously
obtained discovery data. Table 2 provides the functional description for the
quantified proteins. Figure 1 shows the protein abundance ratios of senescent(S)/
self-renewing(SR) in cytoplasmic and nuclear fractions. As is evident in the figure,
the abundance ratio profile was dramatically different between these groups.
WHSC1L1, WHSC1 and EP300 had abundance ratios
>1 in cytoplasmic senescent fractions, whereas SUV39H1, EP300, H2Ax
phosphorylated on ser 139 (KATQAS(phosphoryl)QEY and EZH2 had increased
abundance in nuclear senescent fractions. SUV420H1, MYST2 and H2Ax
phosphorylated on the C-terminal tyrosine (KATQASQE(phosphoryl)Y were not
significantly differentially expressed. The H2Ax abundance profile was consistent
with the previously published evidence that rapid phosphorylation of Ser139 at the
C terminus of the H2AX sequence functions in the recruitment of DDR proteins
after DNA damage. It is hypothesized that increased DNA damage occurs upon
senescence.
FIGURE 2. IPA analysis of histone H2Ax and histone modification proteins
in self-renewing (SR) and senescent (S) mesenchymal stem cell fractions.
A. Cytoplasmic fraction. B. Nuclear fraction. The IPA analysis identified the
top network functions as DNA replication, recombination and repair, cellular
assembly and organization, and gene expression. Proteins with increased
abundance are in red and proteins with decreased abundance are in green.
7.  Lopez MF, Rezai T, Sarracino DA, Prakash A, Krastins B, Athanas M, Singh RJ,
Barnidge DR, Oran P, Borges C, Nelson RW. Selected reaction monitoring-mass
spectrometric immunoassay responsive to parathyroid hormone and related
variants. Clin Chem. 2010 56(2):281-90. Epub 2009 Dec 18.
8.  Blackwell BJ, Lopez MF, Wang J, Krastins B, Sarracino D, Tollervey JR, Dobke
M, Jordan IK, Lunyak VV. Protein interactions with piALU RNA indicates putative
participation of retroRNA in the cell cycle, DNA repair and chromatin assembly.
Mob Genet Elements. 2012 Jan 1;2(1):26-35.
IPA Analysis of SR and S Abundance Ratios in Cytoplasmic and Nuclear
Fractions
Figures 2 A and B show the IPA analysis of the cytoplasmic and nuclear protein
abundance profiles. Figure 2A shows the cytoplasmic fraction. The IPA analysis
identified the top network functions as amino acid metabolism, post-translational
modification and small molecule biochemistry. Figure 2B shows the nuclear
fraction. Here, the IPA analysis identified the top network functions as DNA
replication, recombination and repair, cellular assembly and organization and gene
expression.
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