Detecting Challenging Post Translational Modifications (PTMs)
using CESI-MS
Sensitive workﬂow for the detection of PTMs in biological samples from small sample volumes
Klaus Faserl,1 Bettina Sarg,1 Verena Maurer,2 Herbert H. Lindner1 and Stephen Lock3
1 Division of Clinical Biochemistry, Medical University of Innsbruck, Austria
2 Department of Pharmacology and Toxicology, Leopold-Franzens University of Innsbruck, Austria
3 SCIEX, UKOverview
Who should read this: Senior scientists, lab directors andproteomics researchers
Focus: Detect post translational modifications (PTMs) usingcapillary electrospray ionization (CESI) in combination withaccurate MS detection.
Goals: Detect challenging PTMs (such as asparagine
deamidation, aspartate isomerization, arginine citrullination andphosphorylated peptide isomers) in biological samples wheresample volume is limited and the complete depletion of asample needs to be avoided to allow multiple analyses fromthe same sample.
Problem: Traditional LC-MS methods struggle to identify andquantify aspartate and iso-aspartate isomers separately, asThe CESI 8000 Plus Systemthey have the same mass and similar fragmentation patterns

Key Challenges:

using CID or HCD. Phosphorylated peptides are often polar• Separation and detection of phosphopeptide isomers.

and elute early in traditional LC analyses. Positional isomersof phosphorylated peptides are identical in mass and may
have very similar fragmentation patterns, which makes theidentification of individual modification sites by traditional
LC-MS approaches challenging.
Results: The CESI 8000 Plus High Performance Separation-ESI
Module from SCIEX in combination with accurate MS detection
was able to detect and quantify aspartate and iso-aspartateisomers and citrullinated proteoforms in biological extracts. In
addition, positional isomers of mono-phosphorylated peptides
were successfully separated, allowing unambiguous identificationand quantification of individual protein modification sites.

• Separation and detection of aspartate and iso-aspartate
isomerization in proteins• Detection of arginine citrullination.

Key Features:• Direct combination of capillary electrophoresis with mass
spectrometry, enabling the charge-based separation of
peptides from minute (<20 nL) injection volumes• The separation and detection of mono-phosphorylated
peptide isomers, sites of citrullination and other challenging
PTMs in a single analysis of biological samples

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Experimental design
Sample preparation: Histone H1 proteins were extracted froma human placenta according to Lindner et al.,1 solubilized in
5 mM ammonium bicarbonate buffer (pH 8.0) and digested for
1 h at 37 °C using endoproteinase Arg-C at a ratio of 1:20. Corehistones were extracted from blood cells and fractionated byreversed-phase HPLC according to Helliger et al.2 The histone
H4 fraction was dissolved in 10 mM sodium phosphate buffer (pH7.8) and digested for 8 h at 37 °C using endoproteinase Glu-C at
a ratio of 1:10. Resulting peptides were desalted using PerfectPure C-18 pipet tips (Eppendorf, Austria). The growth of SILAC
labeled yeast strains, protein extraction thereof and subsequent
sample preparation was performed as described elsewhere.3
All samples were lyophilized, re-solubilized in 50 mM ammonium

of 0.1 M acetic acid or 0.1% (v/v) formic acid for 3 min at
50 psi. The sample was injected by applying pressure (5 psi for
20 s equivalent to 17 nL) followed by an injection plug of BGE(5 psi for 5 s). The separations were performed at +30 kV witha 2-stage pressure profile (0–10 min at 1 psi and 10–52 min at
1.5 psi). The Q Exactive Plus mass spectrometer was operating
in data-dependent mode and set to switch between MS and MS/
MS acquisition. The full scan survey spectra were acquired witha resolution of R = 70,000. To generate MS/MS spectra, the10 highest precursors were selected for higher energy collision
dissociation (HCD). Product ion spectra were acquired with aresolution of R = 35,000 (unassigned singly charged peptides
and peptides with a charge state higher than +7 were excluded).

acetate buffer (pH 4.0) and stored at -20 °C until analysis.

ImportantCESI-MS methods. For CESI-MS analysis, a CESI 8000 Plus

• A separation current above 5 μA might cause permanent
damage to the separation capillary.

System equipped with a bare or neutrally coated fused-silica
capillary was coupled to a Thermo Scientific Q Exactive Plus orLTQ Orbitrap XL ETD mass spectrometer.4 Prior to each analysis,• In general, please do not apply >2,000 V to generate
electrospray as it may result in capillary damage.

the CESI system was rinsed with a background electrolyte (BGE)

Figure 1. (A) CESI-MS separation of 3 mono-phosphopeptide isomers of the SPTLASTDDINSASASVNSHATSVK peptide detected in a fraction of aLys-C cleaved yeast sample. (B) Quantification spectra and H/L ratios of the 3 SILAC labeled phosphopeptide pairs.
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Separation of phosphorylated peptides

Identification of deamination products in vivo

It is known that capillary zone electrophoresis (CZE) is able toCESI-MS was used to investigate the in vivo deamidation rate ofseparate peptides with identical amino acid composition but withhistone H1, which is known to be deamidated at position 3.4 H1

different sequences as long as the sequence variation causes

histones were extracted from human placenta and digested witha variation in the pKa values of the corresponding peptides. Such

Arg-C, which preferentially cleaves after arginine residues. Thisa change can be caused by the inclusion of a phosphate residue

highly efficient enzyme allows very short reaction times which inat different positions on the peptide. This was demonstrated byturn prevent generation of artificial deamidation products. The

the identification of a number of phosphopeptide isomers in asample was then analyzed by CESI-MS using a neutral capillary

test of a fraction of metabolically labeled peptides obtained afterand MS/MS spectra were obtained and searched against theLys-C cleavage from 2 yeast strains (Figure 1). In Figure 1,

IPI-human database. Figure 2 shows how CESI-MS is capable

3 different phosphorylated sites were separated and identified

of separating Asp/iso-Asp modifications as well as acetylated

in a digest sample.

and non-acetylated forms of the same N-terminal peptide
TENSTSAPAAKPK. Quantification revealed 85% Asp and only
15% iso-Asp present in this sample.

Figure 2. CESI-MS separation using aneutrally coated capillary (BGE 0.1 M
acetic acid) of human placenta H1 histones
digested with endoproteinase Arg-C.
(A) Base peak electropherogram(B) Extracted ion electropherogram ofdeamidated and acetylated forms of theN-terminal peptide TENSTSAPAAKPK.

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Identification of citrullinated sites
When H4 histones originating from human blood cells wereanalyzed in their intact form, heterogeneity causing massdifferences of just +1 Da were found that could not be assigned
to either acetylation or methylation (Figure 3). To confirm if
this mass shift was caused by deamidation, the proteins werecleaved with endoproteinase Glu-C.4 Using CESI-MS analysis
with a neutral capillary, 8 peptide sequences covering 89.2%
of histone H4 were identified. The reason for the heterogeneitywas found to be located within the first 24 amino acids of H4.
MS/MS analysis identified the peptide 1-24 to be N-terminallyacetylated and di-methylated at Lys-20. A second peak exhibiting
the same isotope distribution for a 6+ charge but with a massdifference of roughly 1 Da was also identified as peptide 1-24,
and was citrullinated at position 3 (Figure 4). As Glu-C wasused as the cleaving enzyme, deamidation could be excluded,4
but as similar shifts in mass and migration time can also occur
when a positively charged arginine residue is post translationallyconverted into the uncharged non-coded amino acid citrulline,
this modification was attributed to citrullination.4 If both
citrullinated and non-citrullinated peptides coelute, the m/z signal
from the citrullinated peptide will fall within the isotopic cluster ofthe un-citrullinated peptide, hindering identification, and so theseparation achieved by CESI-MS is essential for the identificationof this post translational modification in both the intact anddigested sample.

Figure 3. CESI-MS separation using a neutrally coated capillary(BGE, 0.1 M acetic acid) of intact citrullinated and non-citrullinatedcore histone H4. (A) Total ion electropherogram. (B-D) Massdeconvoluted spectra of individual H4 peaks.

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Figure 4. CESI-MS separation using
a neutrally coated capillary with a
BGE of 0.1 M acetic acid of peptides
from human H4 histones extracted
from human blood cells and digested
with endoproteinase Glu-C.
(A) Base peak electropherogram.
(B) Extracted ion electropherogramof peak 1 (m/z 425.09), the 6+
charged peptide 1-24 (N-terminallyacetylated, di-methylated at Lys-20),
and of peak 2 (m/z 425.26; N-terminally acetylated, di-methylated at
Lys-20 and citrullinated at Arg-3).

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Further information

References

For further information on this topic we would like to refer1. H. Lindner, B. Sarg, B. Hoertnagl, W. Helliger. The
microheterogeneity of the mammalian H1(0) histone.
Evidence for an age-dependent deamidation. J.Biol. Chem.
273 (1998) 13324–13330.

readers to the full scientific publication on which this applicationnote is based.4

2. W. Helliger, H. Lindner, O. Grubl-Knosp, B. Puschendorf.
Alteration in proportions of histone H1 variants during thedifferentiation of murine erythroleukaemic cells. Biochem. J288 (Pt 3) (1992) 747–751.
3. K. Faserl, L. Kremser, M. Muller, D. Teis, H.H. Lindner.
Quantitative proteomics using ultralow flow capillaryelectrophoresis-mass spectrometry. Anal. Chem. 87 (2015)
4633–4640.
4. K. Faserl, B. Sarg, V. Maurer, H.H. Lindner. Exploiting charge
differences for the analysis of challenging post-translational
modifications by capillary electrophoresis-mass spectrometry.
J. Chromatogr. A. 1498 (2017) 215–223.

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