Qualitative and Quantitative Analysis of Post-translational Modifications Using CESI-MS
Herbert Lindner, Leopold Kremser, Klaus Faserl and Bettina Sarg
Innsbruck Medical University, Biocenter, Division of Clinical Biochemistry, Innsbruck, Austria

to extensive post-translational modifications, e.g.

B

Yeast Proteome

MS are the analytical techniques of choice

acetylation, phosphorylation, methylation andfor the analysis and quantification of post-deamidation. Both the complexity and uniquetranslationallyandphysical and chemical property of this protein

peptides. However, it is well documented

family is a challenging task requiring the mostthat

modifiedanalysisofproteinsespeciallyphospho-advanced

separation

techniques

sophisticated instrumentation available. Althoughchallenges. Poor ionization efficiency, low

CESI-MS detection of histone modifications isseparation efficiency and neutral losses

limited by the mass loading capacity of theetc.

andsystem, it has the advantage of providing a rapid

characterization. Thus, we evaluated theoverview of the modification status of intact

often

impedes

detection

Yeast proteome: CESI-MS was used tocharacterizeandquantify

phospho-proteins and phosphopeptides present in
thishighlylabeling

andpeptides using LC-ESI-MS is not without

700

complex

wasusedsample.

SILAC

forrelative

quantification of two yeast proteomes.
Protein extracts of these two strains weremixed and enzymatically digested using
Lys-C. The resulting peptides were preseparated by RP-HPLC and analyzed by
Figure1:(A)Basepeakelectro-proteins,reproducible

pherogram of intact H2A.1/H2A.X. (B)and analysis of various common protein

comparisons between treated and untreated

Mass deconvoluted spectra of individualof 1244 phosphorylation sites could bemodifications.

cells or different cell phases [2,3].

H2A.1 peaks (1-8). Average mass ofassigned

unmodified histone H2A.1: 14046.34 Da

peptides quantified. 50 phosphopeptides

Methods

allowing

fastand

and

1371

400

H2A.1H:

13861.11

Da

were found to be differently regulated(Q8CGP6);

H2A.X:

15053.44

Da

relative to the non-phosphorylated protein.

60

phosporylatedpeptides

tri-50tetra40

300
30
200

20100

1000HPLC Fraction no.
Figure 3: Phosphorylated and non-phosphorylated peptides quantified in 182 HPLC yeast fractions.
Peptides were separated by RP-HPLC using a semi-preparative column (4.6 mm i.d. x 25 cm length)
packed with 5 µm reversed-phase C18 material (Nucleosil), 300 Å pore size. HPLC fractions were(P27661); ac0, ac1, ac2: non-, mono-,

For the analysis of protein modifications various

di-500phosphorylated(P22752).

70

mono-

CESI-MS using a neutral capillary. A total

capability of CESI-MS for the separation80
non-phosphorylated peptides

600

17131925313743495561677379859197103
109
115
121
127
133
139
145
151
157
163
169
175
181

Traditionally, LC-ESI-MS and/or MALDI-

Phosphorylated peptides

AThese small highly basic proteins are subjected

Non-phosphorylated peptides

Histones

Introduction

analyzed by CESI-MS using a neutrally coated capillary.

diacetylated forms.

samples were investigated: Core histones which areknown to be extensively modified, as well as
phosphoproteins and phosphopeptides present in aFigure

2:

(A)

Basepeakelectro-highly complex sample, the proteome of yeast. The

pherogramofintact

H4. (B)

Mass

different samples were analysed by CESI-MS using adeconvoluted spectra of individual H4electrophoresis

systemsoldthroughSCIEX

Separations, a part of AB SCIEX, coupled via asheathless high sensitive porous sprayer interface toan LTQ Orbitrap XL ETD (ThermoScientific) mass
spectrometer [1].

References[1] Moini, M. Anal. Chem. 2007, 79: 4241-4246[2] Faserl, K. et al., Anal. Chem. 2011, 83: 7297-305[3] Sarg, B. et al., MCP. 2013, 12: 2640-2656

Acknowledgment
The authors thank SCIEX Separations fortheir support.

B
Light/Heavy ratio ofphosphopeptides

16peaks (1-5). Average mass of histone H4C

Light/Heavy ratio ofcorresponding proteins

16

Change inphosphorylation level

16L/H ratio of phosphopeptide /ratio of corresponding protein

calculated for ac0me2: 11330.27 Da(P62806);

me0-3:

methylated forms.

8non- up to tri-

Light/Heavy ratio

prototype Beckman Coulter CESI 8000 capillaryA

43,1884

4Less than a factorof +/- 22,4222

1,311151015202530354045

11510152025303540

45

10,50,50,50,250,250,2551015202530354045Figure 4: (A) Light/Heavy ratio of 45 quantified phosphopeptides which were differently regulated by a factor of +/- 2 in the yeast proteome. (B) L/H ratio of proteins
corresponding to the phosphopeptides shown in Fig. 4A. (C) Change in phosphorylation level calculated on the basis of Light/Heavy ratios of phosphopeptides andtheir corresponding proteins.