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Comparing and Combining CE-ESI-MS and nano-LC-ESI-MS for

the Characterization of Post-translationally Modified Histones

Bettina Sarg1*

, Klaus Faserl1*

,

Leopold Kremser1, Bernhard Halfinger

1, Roberto

Sebastiano2 and Herbert H. Lindner

1¶

1Division of Clinical Biochemistry, Biocenter, Innsbruck Medical University, Innsbruck,

Austria; 2Politecnico di Milano, Department of Chemistry, Via Mancinelli, Milano, Italy.

* These authors contributed equally to this work.

¶ Corresponding author:

Herbert Lindner Ph.D.,

Biocenter, Division of Clinical Biochemistry

Innsbruck Medical University

Innrain 80-82

A-6020 Innsbruck, Austria.

Phone: 0043-512-9003-70310

Fax: 0043-512-9003-73300

E-mail: Herbert.Lindner@i-med.ac.at

Running Title: Characterization of Modified Histones by CESI-MS and LC-MS.

MCP Papers in Press. Published on May 29, 2013 as Manuscript M112.024109

Copyright 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

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List of Abbreviations:

CE - Capillary electrophoresis

CESI-MS - Capillary-electrophoresis electrospray-ionization mass spectrometry

CZE - Capillary zone electrophoresis

HILIC – hydrophilic interaction liquid chromatography

HPMC - hydroxypropylmethyl cellulose

PEI - polyethyleneimine

BGE - background electrolyte

EOF - electroosmotic flow

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Summary

We present the first comprehensive capillary-electrophoresis electrospray-ionization mass

spectrometric (CESI-MS) analysis of PTMs derived from H1 and core histones. Using a capillary

electrophoresis system equipped with a sheathless high sensitivity porous sprayer and nano-LC-

ESI-MS as two complementary techniques, we characterized H1 histones isolated from rat testis.

Without any pre-separation of the perchloric acid extraction, a total of 70 different modified

peptides, including 50 phosphopeptides, were identified in the rat linker histones H1.0, H1a-H1e,

and H1t. Out of the 70 modified H1 histone peptides, 27 peptides could be identified with CESI-

MS only and 11 solely by LC-ESI-MS. IMAC enrichment prior to MS analysis yielded a total of

55 phosphopeptides, 22 of these peptides could only be identified by CESI-MS and 19 only by

LC-ESI-MS, showing the complementarity of the two techniques. We mapped 42 H1

modification sites, including 31 phosphorylation sites of which eight were novel sites. For the

analysis of core histones we chose a different strategy where in a first step the sulphuric acid-

extracted core histones were pre-separated using RP-HPLC. Individual rat testis core histone

fractions obtained in this way were digested and analysed by bottom-up CESI-MS. This approach

yielded the identification of 42 different modification sites including acetylation (lysine and Nα-

terminal), mono-, di- and trimethylation and phosphorylation. When applying CESI-MS for the

analysis of intact core histone subtypes from butyrate-treated mouse tumor cells we were able to

rapidly detect their degree of modification and found this method very useful for the separation of

isobaric trimethyl and acetyl modifications. Taking together, our results highlight the need for

additional techniques for a comprehensive analysis of PTMs. CESI-MS has proved to be a

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promising new proteomics tool as demonstrated by this, the first comprehensive analysis of

histone modifications, using rat testis as example.

Introduction

Histones are the most intensively studied group of basic nuclear proteins and of great importance

with regard to the organization of chromatin structure and control of gene activity. They are

highly conserved during evolution, binding to and condensing eukaryotic chromosomal DNA to

form chromatin. The fundamental chromatin subunit is the nucleosome, in which 166 bp of DNA

are wrapped around a core histone octamer and a further ~ 40 bp comprises the linker between

one nucleosome core and the next. The histone octamer contains two molecules of each of the

core histones H2A, H2B, H3 and H4. A fifth type of histone, referred to as linker histone (H1,

H5), binds to both the DNA on the outer surface of nucleosomes and to the linker DNA.

There are numerous microsequence variants of linker and core histones (except H4) differing

only slightly in primary sequence. In rat testis, for example, six somatic H1 subtypes, designated

as H1a, H1b, H1c, H1d, H1e, H1.0, as well as germ cell specific subtypes, i.e. H1t, H1T2 and

HILS1 have been identified (1-3). Under various biological conditions all histone proteins, linker

and core histones, are subjected to post-translational modifications, including phosphorylation,

acetylation, methylation, ubiquitination, deamidation, glycosylation, and ADP ribosylation,

which have a great influence on the epigenetic control of gene expression (4-6). The multitude of

histone proteins resulting from closely related sequence variants and post-translational

modifications as well as their highly basic nature combined with hydrophobic properties provides

a major analytical challenge in current proteomics research. Over the last several years,

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considerable efforts have been expended to develop methods to identify the specific sites of

histone modifications. Mass spectrometry (MS) coupled to liquid chromatography (LC) is the

dominant technique for their characterization (7-14). However, due to the fact that histone

proteins contain up to nearly 35% basic amino acids, the analysis of histone peptides is still

problematic as digestion with many commonly used enzymes (e.g., trypsin, Lys-C, etc.) causes

the formation of many short and polar peptides that poorly interact with the RP material and go

undetected by conventional LC-ESI-MS. To overcome this problem, chemical derivatization such

as propionylation is often applied (15, 16).

Capillary electrophoresis (CE) overcomes this disadvantage as this technique allows separations

based on mass to charge ratio of peptides and does not utilize their hydrophobic nature as a

separation principle. The methods of electrophoresis and liquid chromatography and their

applicability for histone analysis are reviewed in detail by Lindner (17). CE has proven to be a

remarkably powerful method in separating individual histones and their modified forms due to

their different electrophoretic mobilities. Using a bare fused silica capillary and

hydroxypropylmethyl cellulose (HPMC) as buffer additive to avoid undesired protein adsorption

different core and linker histones as well as their multiply phosphorylated and acetylated forms

were successfully separated by CZE (18-22). So far, no data are published about identification of

histone modifications by capillary electrophoresis-electrospray ionization-mass spectrometry

(CE-ESI-MS). The reason why LC is given preference to CE is the difficulty in on-line

interfacing of CE with MS that allows stable electrospray processes without compromising the

quality of separation or the detection sensitivity. However, CE-MS is a promising technique with

constantly increasing importance, which is documented by numerous articles (23-26).

Various interfaces have been constructed to improve the CE-ESI-MS coupling (27, 28).

Sheathflow interfaces are most widely used and, despite the drawback of diluting the analyte

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which is inherent to this kind of interface, they offer stable electrophoretic separations and allow

greater versatility in the choice of BGE and range of flow rates (29-32). Sheathless interfaces

have generated interest as no sheath liquid is added leading to enhanced sensitivity of detection

(33, 34). However, they have not been frequently used due to their limited robustness, lack of

well-established interfaces and routine analysis protocols. The most widely used method for

establishing the terminating electrical contact is coating the outer surface of the CE capillary tip

with a conductive material (35-37). Unfortunately, lifetimes of such coatings are generally very

limited as they suffer from deterioration under influence of the high voltages applied.

A recently published concept of a sheathless interface based on a separation capillary with a

porous tip acting as nanospray emitter overcomes these disadvantages (38). The capillary tip is

etched using hydrofluoric acid until the capillary wall becomes so thin and porous that an electric

contact can be established. The performance of this so-called CESI-MS methodology, which

combines low flow characteristics of CE with an integrated ESI source, is described in (39-41).

Applications, such as the analysis of intact proteins (42), protein-protein and protein-metal

complexes (43) and ribosomal protein digests from E.coli (44) are published. Method-inherent

advantages of CESI-MS are highly efficient separations, low flow rates leading to reduced ion-

suppression and higher sensitivity (40). Compared with nanoLC, no column equilibration is

needed, there are no gradient effects and the instrumentation is less maintenance-intensive.

Our group recently described important features of CESI-MS and reported the comparison of this

method with LC-ESI-MS for the analysis of a 5% perchloric acid extraction of rat testis

consisting mainly of different histone H1 subtypes (39). The performance of both techniques was

evaluated regarding analysis time, protein sequence coverage, number and molecular mass

distribution of the identified peptides. The CESI-MS method developed provided shorter analysis

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times, narrower peaks yielding high signals and the identification of a greater number of low

molecular mass range peptides compared to LC-ESI-MS (39).

In the current study we investigated the analysis of post-translationally modified peptides,

particularly phosphopeptides, obtained from endoproteinase Arg-C digested histones from rat

testis, since this organ contains the whole set of somatic and germ cell specific H1 histones as

well as numerous modified core histone proteins. CESI-MS and LC-ESI-MS were compared

regarding number and type of identified modified peptides. Without any pre-separation of the

perchloric acid extraction we found numerous known and novel modification sites in linker

histones. In addition, IMAC experiments were utilized to enrich phosphopeptides prior to MS

analysis. CESI-MS was also used for rapid identification of post-translational modifications of rat

testis core histones, which were pre-fractionated by RP-HPLC and digested with Arg-C. Using

core histones from butyrate-treated mouse erythroleukemia cells we further demonstrate that our

method achieves excellent separations of intact histone subtypes and their multiply modified

forms and enables the detection of the extent of PTMs in a fast and reproducible way. Our work

represents the first detailed characterization of modified linker and core histone peptides and

clearly demonstrates that CESI-MS is a promising alternative tool for epigenetic studies.

Experimental Procedures

Materials. Hydrochloric acid and sodium tetraborate were purchased from Merck (Darmstadt,

Germany). The polyethyleneimine (PEI) coating trimethoxysilylpropyl(polyethyleneimine) was

provided by Beckman Coulter (Brea, USA), the coating reagent M7C4I (1-(4-iodobutyl) 4-aza-1-

azoniabicyclo[2,2,2] octane iodide) was prepared according to Sebastiano et al. (45). All other

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chemicals were purchased from Sigma-Aldrich (Vienna, Austria). Water was purified with a

Milli-Q water purification system (Millipore, Vienna, Austria).

Histone Preparation. Nuclei from rat testes (Sprague-Dawley) were extracted with perchloric

acid (5%, v/v) for H1 histone preparation followed by 0.2 M sulfuric acid for core histone

preparation (46). Mouse erythroleukemia cells were grown and treated with 1.75 mM sodium

butyrate (10 h) as described previously (47).

Capillary Zone Electrophoresis (CZE). CZE of whole H1 histones was performed on a

Beckman system P/ACE 5000 using an uncoated capillary (50 cm in length; 75 µm ID) and a 0.5

M sodium phosphate buffer (pH 2.0), containing 0.02% HPMC (21). For the separation of

deamidated H1.0, a 0.1 M sodium phosphate buffer (pH 3.5), containing 0.02% HPMC was used.

Enzymatic Cleavage. Histones were digested using endoproteinase Arg-C (EC 3.4.21.35) (1:20,

w/w; Sigma-Aldrich) in 5 mM NH4HCO3 buffer (pH 8.0). H1 histones were incubated for 1h at

37°C, core histones were incubated for 30 min at 37°C.

Phosphopeptide Enrichment. Immobilized metal-affinity chromatography (IMAC) was

performed using PHOS-Select™ Iron Affinity Gel (Prod. No. P9740) and the SigmaPrep Spin

column kit (Prod. No. SC1000) both obtained from Sigma-Aldrich (Vienna, Austria). 5 µg of

Arg-C H1 histone digest were applied and 140 µl 400 mM ammonium hydroxide was used as

elution solution.

Capillary Electrophoresis-Electrospray Ionization-Mass Spectrometry (CESI-MS). A

Beckman Coulter prototype CESI capillary electrophoresis system equipped with a sheathless

high sensitivity porous sprayer, was used for peptide separation and ionization upstream from

mass spectrometry characterization. Fused silica capillaries (total length: 100 cm, i.d: 30 µm, o.d:

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150 µm, Beckman Coulter) with a terminal 3 cm long porous segment were inserted into the

prototype sprayer interface enabling the electric contact via a secondary capillary (length: 80 cm,

i.d: 50 µm, o.d: 360 µm) filled with background electrolyte (BGE).

The separation capillaries were modified with three different coatings; one generated a non-

charged inner capillary surface, and the other two modifiers generated positively charged surfaces

(PEI and M7C4I). A positively charged surface induces a reversed EOF, which is directed to the

MS orifice when applying a negative voltage at the CE inlet. The non-charged, neutral surface

suppresses EOF. The procedure for PEI coating was: (i) Preconditioning; flushing each for 5 min

at 50 psi with methanol, MilliQ-water, 0.1 M NaOH, 0.1M HCl, and MilliQ-water, (porous tip

placed in MilliQ-water during this procedure). (ii) Coating; rinsing with methanol (10 min), air

(20 min), and 20% (v/v) coating solution in pure methanol (20 min) at 50 psi (porous tip placed

in methanol). The coating solution was left in the capillary for 12h. (iii) Postconditioning (next

day); flushing each for 20 min at 50 psi with air, methanol and MilliQ-water. Coating with

M7C4I was performed according to Elhamili et al. (48) with slight modifications. The capillary

was treated 8 min with 0.1 M NaOH and 2 min with 25 mM sodium tetraborate buffer at pH 9

followed by treatment with 4 mM M7C4I modifier solution in 25 mM sodium tetraborate buffer

at pH 9 for 20 min. All steps were carried out at 50 psi. The capillary was kept dry until use. The

neutral capillary was provided by Beckman Coulter, Inc.

Capillary electrophoresis conditions were as follows: The separation capillary and the conductive

liquid capillary were rinsed with BGE to refresh the buffer. The sample was injected for 10 sec at

5 psi (7.5 nL), followed by an injection plug of BGE (5 psi for 5 sec). Using positively charged

capillaries the separation was performed at -12.5 kV or -25 kV applied in a 0.5 min ramp

(reversed polarity mode); 0.1% to 0.6% (v/v) formic acid was used as BGE. Using neutral coated

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capillaries the separation was performed at +30 kV applied in a 1 min ramp (normal polarity

mode) and a pressure gradient applied at the capillary inlet. The gradient profile was: 0-43 min,

0.5 psi; 43-51 min, 2 psi and 51-60 min, 5 psi. Acetic acid 10% (v/v) was used as BGE for

separations using neutral coated capillaries.

Nano-HPLC. Protein digests were analyzed using an UltiMate 3000 nano-HPLC system

(Dionex, Germering, Germany) coupled to an LTQ Orbitrap XL mass spectrometer equipped

with a nanospray ionization source. A homemade fritless fused silica microcapillary column (75

µm i.d. x 280 µm o.d.) packed with 10 cm of 3 µm reverse-phase C18 material (Reprosil) was

used. The gradient (solvent A: 0.1% formic acid; solvent B: 0.1% formic acid in 85%

acetonitrile) started at 4% B. The concentration of solvent B was increased linearly from 4% to

50% during 50 min and from 50% to 100% during 5 min. A flowrate of 250 nL/min was applied.

Mass Spectrometry of Arg-C histone digests. The LTQ Orbitrap XL mass spectrometer was

operating in data dependent mode to switch between MS and MS² and MS³ acquisition. Survey

full scan MS spectra (from m/z 250 – 1800) were acquired in the Orbitrap with a resolution of R

= 15,000 (FTMS). Up to three of the most intense ions detected in the full scan MS were isolated

and fragmented in the linear ion trap (LTQ) using collision induced dissociation (CID). An

activation time of 30 ms was applied in MS/MS acquisitions. Normalized collision energy was

set to 35%. The ion selection threshold was 1000 counts with an activation q = 0.25.

Single charged ions were excluded from MS/MS. A neutral loss of 49, 32.66 or 24.5 detected in

one of the three most intense ions in MS² was decisive for a MS³, which was performed to

reaffirm the peptide sequence. Dynamic exclusion was enabled with a repeat count of 2 over a

duration of 3 s and an exclusion window of 30 s.

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Data base search. MS/MS and MS³ spectra were searched against a rat database (rat IPI, version

3.81, 39602 entries) via Sequest, ProteomeDiscoverer (Version 1.3, ThermoScientific). Database

search criteria were set as follows: Processing MSn: allowed cleavage sites Lys and Arg;

maximum miss cleavage sites 6 (linker histone samples) or 8 (core histone samples); precursor

tolerance 10 ppm; fragment mass tolerance 0.8 Da. Variable modifications were acetylation

(+42.011) at Lys and N-terminus, phosphorylation (+79.966) at Ser, Thr and Tyr, deamidation

(+0.984) at Asn, ubiquitination (+114.043) at Lys, methylation (+14.016), dimethylation

(+28.031) at Lys and Arg, and trimethylation (+42.047) at Lys. Up to four modifications were

allowed per peptide.

For Sequest search the identified peptides were further evaluated using charge state versus cross-

correlation number (Xcorr). The criteria for positive identification of histone peptides were Xcorr

> 2.0 for doubly charged ions, Xcorr > 2.5 for triply charged ions, and Xcorr > 3 for fourfold and

higher charged ions. Only best matches were considered. Phosphosites were localized at a false

localization rate (FLR) less than 5% using phosphoRS site probability ≥ 0.95. The searched

peptides and proteins were further validated by Percolator Peptide FDR based on the q-value.

Relative peptide quantification was performed using precursor ion areas, which were calculated

at a mass precision of 2 ppm.

Mass Spectrometry of intact core histones. Approximately 10 ng of each protein fraction pre-

fractionated by RP-HPLC were analysed by CESI-MS. Survey full scan MS spectra (from m/z

300 – 2000) were acquired in the Orbitrap with a resolution of R = 100,000 (FTMS). Protein

masses were determined by deconvolution using the integrated Xcalibur Xtract software

(ThermoScientific).

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Results

Occurrence of H1 Subtypes in Rat Testis – CE has already proved useful for the analysis of core

and linker histone proteins in order to determine both the proportion of the various subtypes and

the degree of post-translational modification (for review, see (17)). To verify the quality and the

composition of the linker histone sample prepared by 5% perchloric acid extraction, Figure 1

shows a separation of whole H1 histones isolated from rat testis using CZE with an uncoated

capillary and a 0.5 M sodium phosphate buffer (pH 2.0), containing 0.02% HPMC. Under these

conditions a complete resolution of the six somatic subtypes, designated as H1a, H1b, H1c, H1d,

H1e, H1.0, as well as the testis specific subtype H1t could be achieved (21). They are expressed

at very different levels and are modified (e.g., phosphorylated) to different degrees. Using

hydrophilic-interaction liquid chromatography (HILIC) we found that rat histone H1.0 consists of

a mixture of intact (H1.0 Asn-3) and in vivo deamidated forms (H1.0 Asp-3, H1.0 isoAsp-3). All

three forms appear acetylated and non-acetylated on their N terminus and both the N-terminally

acetylated and the deamidated forms accumulate with aging (49, 50). By applying a 0.1M sodium

phosphate buffer and increasing the buffer pH from 2.0 to 3.5, we are able to demonstrate for the

first time that CE is also capable of resolving these deamidated forms (as shown in the insert of

Figure 1). Due to their lower positive charge, the deamidated forms migrate slower than the

corresponding non-deamidated protein.

CESI-MS/MS Analysis of Arg-C Digested Histone H1 Peptides – Rat testis H1 histones were

digested with endoproteinase Arg-C, which preferentially cleaves after arginine residues although

hydrolysis proceeds to a minor degree in most Lys-containing substrates, creating a complex

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mixture of highly multiply charged H1 peptides with appropriate molecular mass for ESI-

MS/MS.

For CESI-MS analysis, two different types of positively charged coatings were compared,

polyethyleneimine (PEI), and 1-(4-iodobutyl) 4-aza-1-azoniabicyclo[2,2,2] octane iodide

hereinafter referred to as M7C4I (48). Using diluted formic acid as BGE both surface

modifications generate a reversed EOF towards the MS inlet when working in the reversed mode.

Under these conditions the positively charged peptide ions migrate towards the cathode inlet and

actually out of the capillary. However, due to the positive charge of the modified capillary

surface, the magnitude of the EOF generated is much greater than the electrophoretic mobility of

the peptides and, therefore, moves the bulk solution towards the inlet of the MS instrument.

Figure 2A shows the base peak electropherogram of 300fmol histone H1 digest obtained with the

PEI coated capillary, 0.1% formic acid as BGE and a separation voltage of -25 kV. Due to the

strong positively charged coating a rather high electroosmotic flow rate of approximately 120-

135 nL/min is generated resulting in very short migration times (39). The sample was run in

triplicate and MS/MS spectra obtained were searched against the IPI-rat database. Within a

separation window of about 3.5 min and a total analysis time less than 10 min, 83 histone

peptides (71 unmodified/12 modified peptides) could be identified (Figure 5A). Using the same

separation conditions but an M7C4I coated capillary, which is less positively charged than PEI

and generates flow rates in the range from 96 to 110 nL/min (39), peptides could be identified

within an 8.5 min separation window (Figure 2B), yielding 110 histone peptides (92

unmodified/18 modified peptides; Figure 5A). The effect of varying the CE separation voltage

from -25 to -10 kV was further investigated. At -12.5 kV, which was found to be the optimum

value regarding spray stability and increased separation time, the total number of histone peptides

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identified could be further improved to 127 (98 unmodified/29 modified peptides; Figure 5A)

within a separation window of about 17.4 min (Figure 2C).

LC-ESI-MS/MS Analysis of Arg-C Digested Histone H1 Peptides – For comparison with HPLC,

nano-LC-ESI-MS was performed using a homemade fritless column packed 10 cm with 3 µm

reverse-phase C18 resin. In Figure 3A, the base peak chromatogram of the histone H1 digest

using the same amount of sample as in CESI-MS is presented. After database search,

significantly fewer peptides were identified by LC-ESI-MS due to the lower signal intensity (73

peptides; 62 unmodified/11 modified; Figure 5A) than by CESI-MS. As the primary advantage of

LC over CE is a much higher mass loading capability, 10 times more sample (3 pmol; Figure 3B)

was loaded, permitting the identification of 114 histone H1 peptides (87 unmodified and 27

modified; Figure 5A), similar to the CE result obtained with the 300 fmol sample. In order to

identify as many H1 peptides as possible we injected 30 pmol of the Arg-C digest (Figure 3C), a

100-fold increase compared to the CESI-MS analyses. This resulted in broad peaks and yielded a

total number of 154 H1 peptides (111 unmodified and 43 modified; Figure 5A). This outcome

was significantly better than the result obtained by CESI-MS when the M7C4I coated capillary

with 0.1% formic acid as BGE and a separation voltage of -12.5 kV was used and indicates the

presence of a number of peptides that could not be identified by CESI-MS.

Improving CESI-MS/MS Analysis of Histone H1 Peptides – In addition to the effect of surface

modification and separation voltage on peptide identification by CESI-MS (shown in Figure 2),

the influence of buffer concentration was examined. Therefore, the 300fmol histone H1 digest

was analysed by increasing the BGE concentration from 0.1% formic acid (Figure 2C) to 0.3%

(Figure 4A) and to 0.6% (Figure 4B). As can be seen, higher buffer concentrations produce an

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increase in separation time, which is related to a decrease in the electroosmotic flow. Using 0.3%

formic acid instead of 0.1%, peptides could be identified within a 21.5 min separation window

and the number of identified peptides was raised further to 140 different H1 peptides (108

unmodified/32 modified peptides; Figure 5A). A further increase to 0.6% formic acid yielded in a

separation window of 25.7 min and 151 identified histone peptides (112 unmodified/39 modified

peptides; Figure 5A). This result is comparable to the one when 100 times more sample was

analysed by LC-ESI-MS, which clearly shows the importance of an appropriate length of the

separation window in CESI-MS due to the limited scan rate of the MS instrument.

A further reduction of the EOF can be successfully achieved by using a neutrally coated

capillary. Busnel et al. recently demonstrated that neutral capillaries are well suited for the

separation of peptide mixtures using CESI-MS (40). The analysis of the 300fmol histone H1

digest was performed using 10% acetic acid as BGE, a separation voltage of 30 kV and a

pressure of 0.5 psi applied at the capillary inlet, which is needed to provide a stable spray over

the entire duration of the experiment. The resulting base peak electropherogram is shown in

Figure 4C. Using this method, 157 H1 peptides could be successfully identified within a 38.5 min

separation window (105 unmodified/52 modified peptides; Figure 5A). In order to increase the

mass loading of the system, transient isotachophoresis (t-ITP) was integrated as an in-capillary

pre-concentration procedure (40). However, applying 10 times more sample (3pmol) did not

result in further increase in the number of peptide identifications (data not shown).

Comparing the Results of CE- and LC-ESI-MS Analyses of Modified H1 Histone Peptides - The

various analyses of the rat testis histone H1 sample revealed the presence of different types of

post-translational modifications including phosphorylation, acetylation and deamidation as well

as Nα-terminal acetylation, which is a co-translational process on the nascent polypeptide. Figure

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5B summarizes the results regarding modified peptide identification (including Nα-terminal

acetylated peptides) from six different CE and three LC experiments.

The number of modified peptides identified and type of modification can be seen for each

method. Using CESI-MS the highest number of different modified peptides was achieved when

either a neutrally coated capillary with 10% acetic acid as BGE and a separation voltage of 30 kV

(52 modified peptides), or a M7C4I coated capillary with 0.6% formic acid and -12.5 kV was

applied (39 modified peptides). Using LC-ESI-MS a comparable number of peptides (154 vs. 157

in CESI-MS) could only be obtained when 100 times more sample was injected than in CESI-

MS. Even in this case the number of modified peptides was significantly lower (43 vs. 52 in

CESI-MS). All mapped sites of post- translational modifications of rat testis H1 histones obtained

by CE- and LC-ESI-MS are given in Table 1. Combining all three datasets, each one comprising

triplicate runs, a total of 70 different modified H1 histone peptides were identified.

All H1 subtypes were found to be acetylated as well as non-acetylated at their N-Terminus (49,

51). The function of this type of modification is still unknown. The majority of the peptides

identified (50 out of 70) were phosphorylated. Some of them were common to most of the H1

subtypes, whereas others were restricted to single variants. Furthermore, some peptides were

found to be post-translationally acetylated or deamidated.

Applying CE- and LC-ESI-MS/MS, the rat testis histone subtypes H1a, H1d, H1e and H1t were

found to be modified at multiple sites, whereas only a few sites were identified on H1b, H1c and

H1.0. Most of the sites could be identified with both CE and LC, but some were found with one

method only. We confirmed all five phosphorylation sites we have recently reported for rat H1t

(14) and identified an additional three novel phosphorylation sites in this subtype. Two of these

sites could be assigned unambiguously to Ser-40 and Ser-79. Two diphosphorylated peptides of

different length (ASRSPKSSKTK and ASRSPKSSKTKVVK) were observed. One already known

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phosphorylation site was detected on Ser-140. The second site, however, could not be

distinguished but was localized to one of three possible amino acids (Ser-143, Ser-144 and Thr-

146). Furthermore, a novel acetylation site on Lys-170 of H1t could be detected. The novel

phosphorylation site on Ser-79 (H1t residue numbering) could also be found for histone H1a, c, d

and H1e. This site is outside of the consensus motif of CDK1 ((S/T)PXK), since most of the

phosphorylation sites identified in these experiments were found outside CDK-consensus

sequences. This further strengthens the notion that histone phosphorylation is mediated by

additional kinases targeting still unknown motifs (16, 51). The same is true for a novel non-motif

phosphorylation site found on Ser-28 on H1.0.

On the basis of peptide identifications obtained (Table 1), we investigated the overlap of the total

peptides and of the modified peptides identified between different experiments. First, the overlap

of peptides identified with CESI-MS was investigated. As can be seen in Figure 6, the overlap is

about 66% for total (Figure 6A) and 54% for modified peptides (Figure 6B) using either M7C4I/-

12.5 kV/0.6% formic acid or neutral/30 kV/10% acetic acid. When the CESI-MS results are

compared with the LC-ESI-MS analysis of the 30 pmol sample, the overlap decreases to only

46% ( M7C4I) and 47% (neutral), respectively, for total peptides (Figure 6A) and to 37%

(M7C4I) and 51% (neutral), respectively, for modified peptides (Figure 6B). Out of the 70

modified H1 histone peptides, 22 peptides could be identified either way, additional 7 with CESI-

MS using the M7C4I coating, 10 with CESI-MS using the neutral capillary and 11 by LC-ESI-

MS. With this approach, we identified 33 modification sites including 8 novel histone marks on

rat testis H1 histones (Table 1, highlighted in grey). Out of these 33 sites, 6 sites were only

identified by CESI-MS using the neutral capillary, just one with the M7C4I coated capillary and

5 solely by LC-ESI-MS (Figure 6C). These results suggest that both techniques possess

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complementary properties and that no one of the investigated methods alone is sufficient, by

itself, for a comprehensive analysis of peptide modifications.

Post-translational modification sites on non-histone proteins - The histone H1 sample contained

other modified proteins which were co-extracted with linker histones, including Hmgn1, HMG-

I/HMG-Y, Nuclear ubiquitous casein and cyclin-dependent kinase substrate 1, Trip12 protein,

PC4 and SFRS1-interacting protein, Serine/arginine repetitive matrix protein 1 and 5'-

nucleotidase cytosolic IB. A list of phosphorylated peptides found in the non-histone fraction is

shown in the supplemental section (Table S-1). Despite the fact that 100 times more material was

injected, the highest number of phosphopeptides was again achieved by CESI-MS using the

neutral capillary (27 phosphopeptides), whereas the high sample load LC-ESI-MS analysis

yielded just 9 phosphopeptides. As a result of this investigation, several phosphorylation sites

that have been reported in other species but not in rat were identified. Moreover, two novel

phosphorylation sites on 5'-nucleotidase cytosolic IB could be detected (details shown in Table S-

1). In order to compare relative peptide signal intensities eight phosphopeptides identified with

both CE- and LC-MS were evaluated (supplemental Figure S-1). With one exception the peptide

intensities are lowest in CESI-MS using the M7C4I coated capillary and highest in LC-ESI-MS.

A detailed comparison about complementary peptide identification in CE and nano-LC is

described in the discussion section.

Comparison of CE- and LC-ESI-MS analysis of IMAC enriched phosphopeptides – We have also

studied the suitability of CESI-MS for the analysis of phosphopeptides isolated by IMAC. For

this purpose, 5 ug Arg-C digested rat testis H1 histones were further purified with IMAC- Fe(III).

19

Due to the low sample complexity the fast CESI-MS method involving an M7C4I coated

capillary, 0.1% formic acid as BGE and a separation voltage of -25 kV was used (data not

shown). With triplicate runs 36 different phosphopeptides could be identified (Table 1). The

results obtained by CE were compared with LC-ESI-MS (data not shown), applying the same

method as shown in Figure 3. Eleven times more sample was injected in LC than in CE and as a

result 33 phosphopeptides were obtained (Table 1). Merging the results of CESI-MS and LC-

ESI-MS a total number of 55 phosphopeptides could be identified, 14 by both methods, 19 by LC

only and 22 solely by CE. Two additional phosphorylation sites of H1d could be identified,

which were found only with IMAC enrichment. One of which, Ser-186 was found on two low

mass peptides by CESI-MS only, and the other, Ser-1 or Thr-3, which could not be

unambiguously identified, by LC-ESI-MS only.

Very recently, we were able to demonstrate that CESI-MS is especially effective in analysing low

molecular weight peptides. These poorly interact with the reversed-phase material and elute in

the void volume, so they cannot be detected by LC-ESI-MS (39). This effect should be even

more pronounced with small and hydrophilic phosphopeptides. In Figure 7, the number of

identified phosphopeptides obtained by CE- and LC-ESI-MS obtained with IMAC is plotted

against their molecular mass. As can clearly be seen, more selective isolation of low molecular

mass range peptides was observed with CESI-MS compared to LC-MS, e.g. in the mass range

from 600 – 1400 Da, 21 peptides vs. 4, whereas LC-MS preferentially enriched larger

phosphopeptides, e.g. in the mass range from 1400 – 2400 Da, 27 peptides vs. 11.

Comparing the number of phosphopeptides identified, the result is only slightly better than

without IMAC enrichment, when 50 phosphopeptides were identified. However, a different set of

phosphopeptides was apparently enriched. Out of the 75 phosphopeptides identified in rat testis

H1 histone sample, 25 were detected only by IMAC (Table 1). Taking all results together, 29

20

phosphopeptides were obtained by LC and CE, 20 by LC and 26 by CE only. Out of the 23 small

phosphopeptides in the low mass range from 600 – 1200 Da, 19 could be identified by CESI-MS

only. This result also clearly demonstrates that CESI-MS complements LC-ESI-MS in an ideal

manner.

CESI-MS/MS Analysis of Arg-C Digested Core Histone Peptides – For the analysis of core

histones we chose a different strategy where in a first step the sulphuric acid-extracted core

histones from rat testis were pre-separated using RP-HPLC (supplemental Figure S-2) (46). This

procedure allows the separation of the complex core histone family into individual subtypes but

permits no resolution of post-translationally modified histones, they elute together with the

unmodified parent proteins (52). The collected fractions were subjected to Arg-C digestion and

the resulting peptides were analysed by bottom-up mass spectrometry applying CESI-MS using

an M7C4I coated capillary, 0.1% formic acid as BGE and a separation voltage of -25 kV. For

each sample, approximately 10 ng were injected into the capillary and analysed in a single run of

about 20 min (data not shown). The identified histones were H3.1, H3.1t, H3.3, H4 as well as

several H2A and H2B subtypes. CESI-MS analysis yielded individual sequence coverages

between 61% (H3.1t) and 98.4% (H2A.Z). In total, 77 modified peptides were identified

consisting of 54 peptides modified only by acetylation, one peptide by phosphorylation, 14

peptides by methylation only and 8 peptides, which were both acetylated and methylated

(supplemental Table S-2). In summary, we identified and located 42 different modification sites

including acetylation (lysine and Nα-terminal), mono-, di- and trimethylation. The

phosphorylation site was identified at Ser-139 of H2A.X.

21

CESI-MS Analysis of Intact Core Histones – It is an important challenge to detect the degree of

modification using larger core histone peptides or even intact proteins. It has been demonstrated

by our group that CE offers excellent separation of e.g. multiply acetylated intact core histones

(17, 18). We were interested, therefore, in developing a fast and reproducible analytical approach

for identifying and measuring intact histone variants and the extent of their PTMs, which is

crucial when comparing modification states under different biological conditions. In contrast to

histones from organs like liver, kidney, testis etc., which exhibit only a minor degree of

acetylation, for this investigation core histones were used from a mouse tumor cell line treated

with the deacetylase inhibitor sodium butyrate that produces hyperacetylated species (47). The

proteins were isolated using RP-HPLC as shown in supplemental Figure S-2 and further resolved

by CESI-MS using the same conditions as described for the core histone peptide analysis.

Intact histone H4 was clearly resolved into 5 peaks within 18 min by CESI-MS (Figure 8A). The

average deconvoluted intact mass of each peak revealed that CE separated H4 solely by

acetylation state into its non-, mono-, di-, tri- and tetraacetylated forms (Figure 8B). As

acetylation diminishes the positive charge of the protein, the non-acetylated form has the highest

electrophoretic mobility, however, due to the reversed polarity of the separation voltage applied

and the countercurrent electroosmotic flow, it shows the slowest migration time. The non-, mono-

, di-, and trimethylated forms were observed to co-migrate with the different acetylated states as

increasing methyl addition does not significantly alter the net positive charge of the histone

molecule. The selectivity of this separation is not only leading to less complicated MS spectra but

enables the assignment of isobaric trimethyl and acetyl modifications without the requirement of

high-resolution mass spectrometry as they can be confidently distinguished because of their

different migration time. The application of CESI-MS enables a fast evaluation and quantification

of the methylation and acetylation status of H4. Peak 5, therefore, consists of non-acetylated H4

22

that is mainly dimethylated but also mono- and trimethylated to a minor extent. Peak 4, which

consists of monoacetylated H4 closely resembles to peak 5. With increasing acetylation the

methylation status changes significantly (Figure 9). The higher acetylated peaks 1-3 consist of

less trimethylated forms, however, a substantial increase of non- and monomethylated forms is

observed. These results are in accordance with previous studies of our group that demonstrated a

decrease in the abundance of trimethylation (47) as well as an increase of monomoethylation

upon hyperacetylated H4 (53). The RP-CESI-MS assay reproducibility was tested through

triplicate analysis of histone H4. The differently acetylated proteoforms can be quantified

according to their MS1 precursor intensities with a standard deviation fewer than 3.5%

(supplemental Table S-3A), corresponding to a relative standard deviation ranging from 4.7 to

16.0%. The differently methylated H4 forms present in a single peak can be quantified with a

standard deviation fewer than 2% (supplemental Table S-3B) based on their deconvolution result.

The relative standard deviation ranged from 0.3 to 16.6%, depending on signal height. As a

result, the developed CESI-MS methodology has proved to be very reproducible and efficient for

assessing the degree of modification of intact H4 (Figure 9).

Using the same approach the H2A.2 fraction was separated into four peaks (Figure 8C).

Determination of the molecular masses revealed the presence of the H2A.2 variants H2A.2A,

H2A.2B and H2A.2C. In detail, peak No.4 contains non-acetylated H2A.2A; No.3,

monoacetylated H2A.2A, which is not well separated from non-acetylated H2A.2C; No.2,

diacetylated H2A.2A, monoacetylated H2A.2C and non-acetylated H2A.2B; No.1, diacetylated

H2A.2C and monoacetylated H2A.2B (supplemental Figure S-3). The CE procedure allows the

separation of the variants and their acetylated forms as well as the determination of relative

proportions of the different forms.

23

A similar result was obtained when the H2A.1 fraction was subjected to CESI-MS (Figure 8D).

In addition to H2A.1 this fraction contains the variants H2A.1H and H2A.X, which elute as a

single peak in RP-HPLC. In CE, the three variants were baseline separated from each other and

from their different acetylated forms. H2A.1 was found to be mainly non- and monoacetylated, a

diacetylated form was detected in very little amounts (supplemental Figure S-4). H2A.X was also

non-, mono-, and diacetylated. H2A.1H, present in very low amounts, could still be identified as

non- and monoacetylated.

CESI-MS of intact H2B resulted in six well separated peaks according to their acetylation status

(Figure 8E). The four major molecular masses observed in each peak are very close in mass

(separated by 14 – 16 Da) suggesting the possibility that this fraction contains multiple variants

of H2B or forms bearing multiple methylations. Top Down MS of human H2B isoforms

performed by Siuti et al. (54) proved that the heterogeneity of H2B is due to amino acid sequence

and not to methylation. They detected five major molecular masses containing seven distinct

H2B variants. The four molecular masses observed in our study can be assigned to the H2B

forms H2B.1C, H2B.1F, H2B.1B and H2B.2B, which differ by only one or two amino acids. The

treatment with sodium butyrate resulted in increased acetylation of each of the four major peaks.

The pattern of increased acetylation of the H2B isoforms is very similar regarding mono-, to di-,

tri-, and tetra-acetylation (supplemental Figure S-5). The penta-acetylated pattern differs between

the H2B isoforms, but is present in minute amounts only.

Finally, the HPLC fraction containing H3.2 and H3.3 was subjected to CESI-MS. The basepeak

electropherogram is shown in Figure 8F. The CE analysis revealed six well separated peaks

corresponding to the non-, mono-, di-, tri-, tetra- and pentaacetylated forms of the protein.

However, when analysing each peak spectrum with Xtract, we found that the extreme diversity of

H3 modified forms precludes assignment of most of the molecular masses detected (supplemental

24

Figure S-6). However, some lower methylated forms, e.g. dimethylated H3.2 ac0-ac5 and

trimethylated H3.3 ac4-ac5 could be designated.

Discussion

In this study, the suitability of CE-ESI-MS for the identification of post-translational

modifications of histones was evaluated for the first time. Moreover, to date no proteomic

analysis of purified H1 and core histones from rat testis has been performed. A porous sheathless

CESI-MS interface in combination with a Thermo Scientific LTQ Orbitrap XL was used. For the

analysis of H1 histones, the CESI-MS method developed was compared with nano-LC-ESI-MS.

The results clearly demonstrate that more modified peptides and more modification sites were

found by CESI-MS. However, both methods complement each other. Modified peptides from

biological samples are often present in minute amounts only. Therefore, sensitivity of the

instrument configuration is an important issue for the analysis of these compounds. Generally,

sensitivity increases with decreasing flow rates. For example, a 20 fold increase in sensitivity by

decreasing the flowrate from 330 nL/min to 10 nL/min has been shown by Busnel et al. (40).

Another advantage of minimal flow rates below 20 nL/min is the operation in the mass sensitive

range of the ESI process resulting in significantly reduced analyte suppression and improved

sensitivity (55, 56). However, in LC void volumes and instrument specifications usually limit the

broad application possibilities of flow rates below 100nL/min. In addition, co-elution with the

unmodified form associated with ion suppression effects may also affect sensitive detection of the

modified species in an adverse manner. The CE-MS configuration used for this investigation

does not suffer from these limitations. There is no dead volume and flow rate generated by the

EOF can easily be manipulated in a wide range from almost zero to over 100 nL/min by

25

appropriate capillary surface modification. As many covalent modifications such as

phosphorylation, acetylation alter the charge of peptides and thus their mobility, co-migration

with their unmodified form and hence possible ion-suppression effects usually do not occur.

Interestingly, linker histone methylation and ubiquitination was not observed. In a previous study

on post-translational modifications of H1 histones across different mouse tissues, a range of

modifications including methylation, ubiquitination and formylation was found (51, 57). These

modifications were mainly detected in mouse spleen and other tissues such as brain or kidney but

none were detected in mouse testis. As the testis is an organ composed of many different cell

types, including various types of somatic cells (e.g., Sertoli cells, Leydig cells, and peritubular

cells) as well as germ cells, performing a variety of distinct functions, this could be a reason for

the lack or under-representation of certain modifications. Even with pre-fractionation of the

linker histone subtypes via RP-HPLC before enzymatic digestion, very few modifications were

identified in mouse testis by LC-MS. These few included three phosphorylation sites on H1.1

(Ser-1, Thr-3 and Ser-43), one on H1.3 (Thr-18), one on H1.4 (Thr-18) and on H1.5 (Ser-18) and

one acetylation site on H1.1 (Lys-87) (51).

In contrast to these results we were able to identify 33 modification sites in rat testis by CESI-MS

without pre-separation of individual H1 subtypes by RP-HPLC. In addition, for low

concentration phosphopeptide analysis of rat H1 histones IMAC enrichment was applied, which

increases the number of modification sites up to 35. As a result, this investigation showed that

CESI-MS significantly increases the detection of phosphopeptides in the low molecular mass

range. Even in the high sample load analysis LC-ESI-MS is not able to detect this group of

peptides. Some of the larger phosphopeptides, however, are preferentially detected in LC-MS.

Our group recently described this effect for unmodified H1 peptides. We found that low

molecular mass peptides (below 1400 Da) were preferentially identified by CESI-MS, since this

26

group of peptides poorly interacts with the reversed phase material in the nano-LC system and,

therefore, are washed out (39).

In order to further evaluate the different outcome of both techniques, we compared relative

peptide abundances across the best CE and LC analysis (shown in supplemental Figure S-7). A

heat map of the relative peptide abundances was generated and illustrates that there is not only a

substantial difference between CE- and LC methods but also a difference between the two CE

methods, albeit to a lesser extent. The divergent peptide identifications among both CESI-

methods are mainly due to the different separation conditions of positive and neutral capillaries.

In a positively coated capillary the positively charged peptide ions migrate towards the cathode

CE inlet. As the magnitude of the EOF generated is much greater than the oppositely directed

electrophoretic mobility of the peptides, they are carried by the EOF towards the inlet of the MS

instrument. Under these conditions peptides with low m/z values (implying high electrophoretic

mobility and, therefore, long migration times) are well separated from each other and from other

peptides, which improves their identification and quantification (supplemental Figure S-7,

peptide groups 1 and 5). In contrast, a neutral capillary results in an almost complete suppression

of the EOF and, when working in normal CE mode (+30kV) a low flow rate (10nL/min) towards

the MS can be obtained. In this case, peptides with low m/z values are the fastest migrating

peptides and, therefore, less well separated, which favors the identification and quantification of

peptides with high m/z values (supplemental Figure S-7, peptide group 2 and 6). This includes

also phosphopeptides (peptide group 2) and Nα-terminal acetylated peptides (peptide group 6)

due to their reduced charge state.

When comparing CESI-MS with LC-ESI-MS, there is a clear bias of CE towards low molecular

mass peptides and phophopeptides. Because of their poor interaction with the column support

many of these peptides are not even detected in LC-MS although 100 times more sample was

27

injected. This can be clearly seen in supplemental Figure S-7 due to the color coding of relative

peptide signal intensities. The most significant difference in signal intensities was found for

peptide group 4 by LC-MS compared to CESI-MS reflecting the 100 fold increase in sample

amount injected in LC. This group comprises low abundant longer peptides and peptides, which

do not profit from the gain in sensitivity usually seen in CESI-MS.

Similar complementary peptide identification was found in recent CE and nano-LC studies by

our group and other researchers (32, 39, 58). There is a clear bias of CE towards basic,

hydrophilic peptides of low molecular mass. Additionally, the Yates Lab attributed gains in

sensitivity to lower noise levels with CE, illustrated by better signal-to-noise ratios of peptide

precursor ions and associated higher XCorr values of identified peptides when compared to LC

(58). These trends may be also explained by the difference in electrospray conditions between

both methods. The increasing organic composition of the solvent throughout the LC separation is

known to affect ESI response of different peptide hydrophobicities, while CE-MS conditions are

essentially constant. However, the results also show that the complementary nature of both

techniques is of great value for a comprehensive analysis of peptide modifications.

According to sample complexity the CESI analysis can be adjusted easily using different types of

coated capillaries. Using positively coated capillaries very fast separations suitable for low

complex samples can be achieved, as shown for phosphopeptides enriched by IMAC, for intact

core histones and for mapping core histone modifications. Neutrally coated capillaries result in

increased separation windows suitable for medium complex samples. In general, CESI-MS

analyses are faster than LC-MS as no column equilibration time is necessary. Depending on the

surface modification of the capillary, a total cycle time between 16 min (PEI) and 70 min

(neutral) was found. In contrast, the nano-LC method used for the linker histone analysis required

a total cycle time of 82 min, including 25 min for washing and reequilibration. It is also

28

important to note in this context that the carryover between analysis can be a major problem in

LC-MS requiring additional time consuming wash steps and analyses, which may increase total

analysis time substantially. In CESI-MS, after applying a one minute wash step with methanol

and background electrolyte carryover is actually not observable.

The second part of the present study evaluates the potential of CESI-MS for the characterization

of core histone subtypes and their modifications. Rat testis core histone fractions pre-separated by

RP-HPLC were analysed by bottom-up CESI-MS using a positively coated capillary, which

enables fast separations, allowing the annotation of acetylated, methylated, and phosphorylated

histone peptides. A large number of modification sites known from human and mouse core

histones were experimentally proved on rat core histones for the first time. Many of the recently

reported most frequent PTMs found in mouse brain core histones were also found with this

approach (59). Moreover, several sites on testis-specific subtypes like H2B1A and H3.1t could be

detected as well as specific sites not found in mouse brain. CESI-MS resulted in identification of

77 modified peptides corresponding to 42 sites, supporting the efficiency, sensitivity, and

specificity of the CESI technique. In order to get knowledge of the presence of multiple

modifications that can co-occur on different histone residues, one must analyze larger peptides or

intact proteins that encompass more modified sites. Only recently have new technological

advances based on ETD and ECD fragmentation begun to permit these studies. Top-down

proteomics has been successfully applied to the characterization and quantitation of histone forms

and PTMs by Kelleher and coworkers. Histone H2A, H2B and H3 variants were identified by use

of high mass-accuracy FT-ICR-MS along with their changes in relative expression and

modifications during the cell cycle (54, 60, 61). In order to increase throughput and sensitivity of

comprehensive histone modification characterization two-dimensional liquid chromatography is

advantageous prior to mass spectrometric investigation. It turned out that a HILIC method

29

developed by our group for the separation of histone variants and their modified forms (62, 63) is

very well suited for combination with top-down MS (64-66). HILIC is a powerful tool to separate

post-translational modifications of proteins. Combining HILIC and RP-HPLC enabled us to

isolate various H2A variants and their acetylated forms (63) as well as methylated and acetylated

H4 forms with high purity, whose identities were determined by using offline MS detection (46,

47). The method was successfully applied for the characterization of combinatorial histone codes

by the Kelleher and Garcia groups (64-66). They also showed that more detailed PTM occupancy

information can be obtained from longer peptides derived by Asp-N or Glu-C digestion using

middle-down MS (66). This strategy was found to be especially important for the analysis of

highly modified histone H3, which has proven to be a significantly more difficult analytical

problem than H2A, H2B or H4. This problem also occurs with our CESI-MS analysis of intact

H3 shown in supplemental Figure S-6. The peaks obtained were found to differ in the number of

acetyl groups, but the complex pattern of modified forms present within each peak did not allow

the assignment of all individual H3 forms. Recently, Young et el. presented an MS-friendly

version of this HILIC chromatography utilizing “saltless” pH gradient chromatography which is

on-line coupled with MS and results in improved analysis time, sample consumption and

dynamic range (67).

Our study on intact histone analysis presents an alternative method to HILIC. In contrast to

HILIC the CE method separates the histone proteins solely by acetylation state, the non-, mono-,

di-, and trimethylated forms co-migrate with the different acetylated species. Nevertheless, CESI-

MS also allows confident assignment between isobaric modifications like acetylation and

trimethylation without the requirement of high-resolution mass spectrometry, as they can be

confidently distinguished because of their different migration time. The combined use of CE and

MS has the potential to permit the characterization of complex mixtures such as hypermodified

30

histones and should couple nicely with ETD to make this method amenable to bench-top MS

instruments. Although CESI-MS detection of histone modifications is limited by the mass

loading capacity of the system, it has the advantage of providing a rapid overview of the

modification status of intact proteins, allowing fast and reproducible comparisons between

treated and untreated cells or different cell phases. Advances in several areas will be needed to

further increase the range of potential CE applications in this field, including continued

improvements in sensitivity, resolution of high molecular masses and scan rate of MS

instruments. It is noteworthy in this context that combining CE with MS reveals another

advantage: CE shows a remarkably higher separation efficiency for high molecular mass

compounds like larger peptides and proteins compared to any LC method.

Based on the results obtained, we envision the “CESI-method” being used in a broad range of

applications, not only for epigenetic studies of histones, but also for modified peptide

identification in general.

Acknowledgements

The authors thank Beckman Coulter, Inc. for providing the sheathless high sensitive porous

sprayer interface. We kindly thank Astrid Devich for excellent technical assistance and Jim

Thorn for his help with the manuscript.

31

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Figure Legends

Table 1: List of modified H1 histone peptides identified from rat testis. The peptide confidence

level (percolator q value) and the probability for the respective site being truly phosphorylated

(pRS probability) are given. Novel modification sites are highlighted in grey; a, acetylated; p,

phosphorylated; d, deamidated.

Figure 1. CZE separation of H1 histones from rat testis carried out in 0.5 M sodium phosphate

buffer at pH 2.0 containing 0.02% HPMC and shown in the insert, the separation of histone H1.0

carried out in 0.1 M sodium phosphate buffer at pH 3.5, containing 0.02% HPMC. Running

conditions were as follows: injection time, 2 s; UV detection at 200 nm; voltage, 12 kV;

untreated capillary (50 cm in length; 75 µm ID). (ac0, ac1: non-acetylated and monoacetylated

forms; p0, p1, p2: non-, mono-, di- and triphosphorylated forms).

Figure 2. Base peak electropherograms of rat testis H1 histones digested with endoproteinase

Arg-C using positively charged capillaries. (A) PEI coated capillary, separation voltage: -25 kV,

(B) M7C4I coated capillary, separation voltage: -25 kV, (C) M7C4I coated capillary, separation

voltage: -12.5 kV. BGE: 0.1% (v/v) formic acid. Sample amount: 6.15 ng (300 fmol). Capillary

length: 100 cm with porous tip, i.d.: 30 µm, o.d.: 150 µm.

Figure 3. Base peak chromatograms of rat testis H1 histones digested with endoproteinase Arg-C

using LC-ESI-MS. (A) sample amount: 6.15 ng (300 fmol), (B) sample amount: 61.5 ng (3 pmol)

and (C) sample amount: 615 ng (30.0 pmol). LC-ESI-MS was performed using a homemade

fritless column; packed 10 cm with 3 µm reversed-phase C18 (Reprosil). The gradient (solvent

A: 0.1% formic acid; solvent B: 0.1% formic acid in 85% acetonitrile) started at 4%B. The

41

concentration of solvent B was increased linearly from 4% to 50% during 50 min and from 50%

to 100% during 5 min. A flowrate of 250 nL/min was applied.

Figure 4. Base peak electropherograms of rat testis H1 histones digested with endoproteinase

Arg-C using (A) an M7C4I coated capillary, separation voltage: -12.5 kV, BGE: 0.3% (v/v)

formic acid (B) an M7C4I coated capillary, separation voltage: -12.5 kV, BGE: 0.6% (v/v)

formic acid and (C) a neutrally coated capillary, separation voltage: +30 kV, BGE: 10% (v/v)

acetic acid. Sample amount: 6.15 ng (300 fmol). Capillary length: 100 cm with porous tip, i.d.: 30

µm, o.d.: 150 µm.

Figure 5. Number of unmodified and modified histone H1 peptides identified by CE- and LC-

ESI-MS/MS analysis. (A) Number of identified histone H1 peptides (modified and non-modified)

were merged from triplicate analyses. (B) Within each column the total number of modified

peptides as well as the distribution of specific types of modifications is shown. The numbers

presented in the diagram are the sum of unique modified peptides found in triplicate runs.

Overlap of peptides identified with CESI-MS ranges from 75.0% - 84.1%, with LC-ESI-MS from

65.8% - 71.4%.

Figure 6. Venn diagram showing the overlap of (A) total histone H1 peptides, (B) modified H1

peptides and (C) modification sites identified by CESI-MS and LC-ESI-MS from triplicate runs.

Figure 7. Mass distribution of histone H1 phosphopeptides obtained with IMAC enrichment

using CESI-MS and LC-ESI-MS. Data originate from Table 1. Each analysis was performed

three times.

Figure 8. CESI-MS analysis of intact hyperacetylated core histones using an M7C4I coated

capillary. Conditions as described in Figure 2B. (A) Base peak electropherogram of intact H4.

42

(B) Mass deconvoluted spectra of individual H4 peaks (1-5). Average mass of histone H4

calculated for ac0me2: 11330.27 Da (UniProt accession P62806). (C) Base peak

electropherogram of intact H2A.2. (D) Base peak electropherogram of intact H2A.1. (E) Base

peak electropherogram of intact H2B. (F) Base peak electropherogram of intact H3.2+H3.3.

ac0,ac1,ac2,ac3,ac4: non-, mono-, di-, tri- and tetraacetylated forms; me0,me1,me2,me3: non-,

mono-, di- and trimethylated forms.

Figure 9. Acetylation (A) and methylation (B) levels of hyperacetylated histone H4 in mouse

erythroleukemia cells. Acetylation levels were determined by integrating the peak areas of MS1

precursors shown in Figure 8A and were calculated as percent area relative to the entire area of

H4. Methylation status of each acetylated proteoform was determined individually by computing

the monoisotopic mass intensities for the non-, mono-, di-, and trimethylated H4 present in each

peak. Analyses are based on triplicate runs.

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Table 1, page 1

44

Table 1, page 2

45

Figure 1

Figure 2

46

Figure 3

47

Figure 4

48

Figure 5

Figure 6

A B C

B

A

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Figure 7

Figure 8

50

Figure 9