Expanding phosphorylation and post translational site mappingin proteomics using CESI-MS
Sensitive workflow for the detection of PTMs in biological samples from small sample volumes
Bettina Sarg,1 Klaus Faserl,1 Bernhard Halfinger,1 Leopold Kremser,1 Herbert H. Lindner,1 and Stephen Lock2
1Division of Clinical Biochemistry, Medical University of Innsbruck, Austria and 2Sciex UKOverview
Who Should Read This: Senior Scientists, Lab Directors andProteomics researchers.
Focus: Expanding Post Translational Modification (PTMs)
identification using capillary electrospray ionization (CESI) incombination with accurate MS detection in Histone analysis.
Goals: Show how CESI-MS can detect additional PTMs
compared to nano LC-MS and how it is a complementary andan orthogonal technique which can expand your PTM coveragewhen used together with nano LC-MS analysis.
Problem: Traditional nano LC-MS methods involve the injectionof µLs of sample to obtain peptide mapping of biological digests.
Repeating injections for additional analysis is not always possible

Key Features:

if sample volume is limited and additional PTM coverage from• Sample consumption by CESI-MS was 1% of that by LC-MS
allowing the analysis of limited samples firstly by CESI-MS
and then by LC-MS increasing peptide coverage.

repeat analysis is therefore not available. Nano LC-MS/MS
separates peptides based on hydrophobic interactions with theLC columns stationary phase and sometimes polar PTMs andvery hydrophobic peptides can be missed depending on thegradient used.

• CESI-MS was able to detect unique PTM sites which weremissed when only LC-MS was used.

Results: The Sciex CESI 8000 system in combination withExperimental Designaccurate MS detection provides a complementary solution

Sample Preparation: Nuclei from rat testes (Sprague-Dawley)

to nano LC-MS for the characterization of post translational
modifications sites in H1 histone samples. A total of 70

were extracted with perchloric acid (5%, v/v) for H1 histone
preparation.1 Once extracted the Histones were digested using

different modified peptides, including 50 phospho-peptides,

endoproteinase Arg-C (Sigma-Aldrich) in 5 mM ammonium

were identified in rat histones and out of the 70 modified

bicarbonate buffer (pH 8.0, 1 h at 37° C).

H1 histone peptides, 27 peptides could be identified solely
by CESI-MS with11 solely by LC-MS. CESI-MS was able
to detect 157 histone H1 peptides compared to 154 fromLC-MS but only consumed 300 fmol of sample comparedwith 30 pmol by LC-MS.

Key Challenges:

CESI-MS methods. For CESI-MS analysis a CESI 8000
equipped with positively coated or neutrally coated fused-silica
capillaries was coupled to a Thermo Scientific LTQ Orbitrap
XL mass spectrometer.2 Before each analysis the separation
capillary and the conductive liquid capillary were rinsed with BGEto refresh the buffer. Samples were injected for 10 s at 5 psi(7.5 nl) followed by an injection plug of BGE (5 psi for 5 s).

• Separation and detection of modifications sites for
H1 Histones.

For the separation the conditions depended on the capillary• Expanding PTM coverage with limited sample volume.

performed at 30 kV (normal polarity mode), with a pressure

coating but for the neutrally coated capillaries separations werep1

gradient of 0 - 43 min, 0.5 psi; 43 - 51 min, 2 psi; andMS methods. Both separations used the same Thermo Scientific51 - 60 min, 5 psi. Acetic acid 10% (v/v) was used as theLTQ Orbitrap XL mass spectrometer to detect the peptides. The

BGE for neutral coated capillaries.

MS was operated in data-dependent mode, switching between

2Nano LC methods. Nano-HPLC—Protein digests were analyzed
using an UltiMate 3000 nano-HPLC system coupled to an LTQ
Orbitrap XL mass spectrometer. A homemade frit less fused silica
micro-capillary column (75 µm inner diameter x 280 µm outerdiameter) packed with 10 cm of 3 µm RP C18 material was usedwith a gradient elution at a flowrate of 250 nl/min. The gradient(solvent A: 0.1% formic acid; solvent B: 0.1% formic acid in 85%

MS, MS2, and MS3 acquisition. Survey full-scan MS spectra(m/z 250–1800) were acquired in the Orbitrap with a resolution of
15,000 and the 3 most intense ions detected in the survey scan(above a threshold of 1000 counts) were fragmented in the linear
ion trap (LTQ) using collision-induced dissociation. All the dataacquired was searched against a rat database using Sequest,
Proteome Discoverer search engine.2acetonitrile) started at 4% B with B increased linearly from 4% to50% over 50 min and then from 50% to 100% over 5 min.

Analysis of Histone H1 digests by CESI-MS
When positively coated capillaries are used for the separation

analyzed by CESI-MS 157 H1 peptides were identified withinof protein digests the electroosmotic flow (EOF), which isa 38.5 minute separation window (Figure 1). Of these peptides

dependent on the level of charge on the capillary surface,

105 were unmodified and 52 were modified this was an increase

speeds up separations reducing the separation length which canwhen compared to when a positively charged (polyethyleneimine)adversely affect peptide identification. Neutral coated capillaries

coated capillary was used where only 83 peptides were detected

can reduce EOF lengthening separation windows and improving

due to its shorter separation window.2

peptide detection. When 300-fmol of histone H1 digest was

Figure 1. Base peak electropherogram of rat testis H1 histones digested with endoproteinase Arg-C analysed by CESI-MS using a neutrally coatedcapillary, a separation voltage of 30 kV, and a BGE of 10% (v/v) acetic acid. Sample amount consumed was 6.15 ng (300 fmol).

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Analysis of Histone H1 digests by nano LC-MS
Nano LC-MS was performed using a homemade fritless 10 cm

Nano LC has a much larger loading capacity than CE 30 pmol ofcolumn packed with 3 µm reverse-phase C18 resin. In Figure

the Arg-C digest was injected (a 100-fold increase relative to the2A, the base peak chromatogram of the histone H1 digest forCESI-MS analyses) this resulted in broader peaks but yielded athe same amount of sample as in the CESI-MS experiments issimilar amount of identified H1 peptides, 154 (111 unmodified anddisplayed. Due to the low signal in the nano LC- MS analysis

43 modified).

only 73 peptides were identified (62 unmodified and 11 modified).

Figure 2. Base peak chromatograms of rat testis H1 histones digested with endoproteinase Arg-C using nano LC –MS. A) represents an injection of 6.15 ng(300 fmol) and C 615 ng (30.0 pmol). Analyses were performed using a homemade fritless 10 cm column packed with 3 µm reversed-phase C18.

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Comparison of nano LC-MS with CESI-MS forHistone 1 digest analysis
When the CESI-MS analysis was compared with LC-MS analysisof the total peptides detected between the CESI-MS analysis

more modified peptides were detected when only 1% of the(neutrally coated capillary) and the LC-MS analysis (30 pmolsample was injected (300fmol compared to 30 pmol). Figureinjected). When the modified peptides were compared 10 were

3 shows a comparison of both techniques. Each analysis wasonly detected by CESI-MS and 11 only by LC-MS highlighting

repeated in triplicate and combining all the data sets a total

the complementary nature of these techniques.2 Of this group ofof 70 different modified peptides were identified the majority

modified peptides CESI-MS detected 3 times the amount of theof which were phosphorylated. When the individual peptide

phosphorylated peptides (27 compared to 11) due to polar nature

identifications were compared there was an overlap of 47%of this modification.

2AB

Figure 3. Comparison of CESI-MS using a neutrally coated capillary and LC-MS analysis of Histone H1 digests. A) The total number of histone
peptides detected; B) Breakdown of the modified peptides detected by both techniques. For each technique the data from triplicate runs wasmerged to give the overall number of identifications.
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Further Information
For further information on this topic we would like to refer readers
to the full scientific publication on which this application noteis based.2

References
1. Sarg, B., Koutzamani, E., Helliger, W., Rundquist, I., andLindner, H. H. (2002) Post synthetic trimethylation of histone
H4 at lysine 20 in mammalian tissues is associated with aging.
J. Biol. Chem. 277, 39195–39201.
2. Sarg, B., Faserl, K., Kremser, L., Halfinger, B., Sebastiano, R.,
and. H. H. Lindner (2013). Comparing and Combining
Capillary Electrophoresis Electrospray Ionization Mass
Spectrometry and Nano–Liquid Chromatography Electrospray
Ionization Mass Spectrometry for the Characterization ofPost-translationally Modified Histones. Mol. Cell Proteomics;
12(9): 2640–2656.

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