The potential of fractional diagonal chromatography

e u p a o p e n p r o t e o m i c s 4 ( 2 0 1 4 ) 165–170
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The potential of fractional diagonal
chromatography strategies for the enrichment of
post-translational modiﬁcations
A. Saskia Venne, René P. Zahedi ∗
Leibniz-Institut für Analytische Wissenschaften – ISAS – e.V., Dortmund, Germany
a r t i c l e
i n f o
a b s t r a c t
Article history:
More than 450 post-translational modiﬁcations (PTMs) are known, however, currently only
Available online 24 July 2014
some of those can be enriched and analyzed from complex samples such as cell lysates.
Therefore, we need additional methods and concepts to improve our understanding about
Keywords:
the dynamic crosstalk of PTMs and the highly context-dependent regulation of protein func-
PTM
tion by so-called ‘PTM codes’. The mere focus on afﬁnity-based enrichment techniques may
Enrichment
not be sufﬁcient to achieve this ambitious goal. However, the complementary use of two-
Signaling
dimensional chromatography-based strategies such as COFRADIC and ChaFRADIC might
PTM crosstalk
open new avenues for enriching a variety of so far inaccessible PTMs for large-scale proteome
studies.
© 2014 The Authors. Published by Elsevier B.V. on behalf of European Proteomics
Association (EuPA). This is an open access article under the CC BY-NC-SA license
(http://creativecommons.org/licenses/by-nc-sa/3.0/).
1.
The relevance of post-translational
modiﬁcations
Organisms have to adapt continuously their physiological processes in order to maintain homeostasis under a large range
of environmental changes. Compared to gene expression and
protein translation, post-translational modiﬁcations (PTMs)
and protein degradation enable a faster regulation of cellular processes. Thus, PTMs allow the precise and dynamic
response to internal and external stimuli, modulating for
instance the subcellular localization, activity, stability and
interaction of proteins. Consequently, understanding this
sensitive and complex system is essential for cell biology, disease prevention and development of therapeutic approaches
[1,2].
Currently, over 450 PTMs [3,4] are listed in the UniProt
database including the most prominent members such as
phosphorylation, glycosylation, ubiquitination and acetylation. The number of experimentally observed PTMs literally
exploded in the past 10 years [5,6] mainly owing to the recent
improvements in mass spectrometry (MS) and the availability
of more sensitive and faster mass analyzers.
However, often more than 50% of MS/MS spectra acquired
in an LC–MS/MS run cannot be identiﬁed by database searches.
Such unmatched spectra can arise from e.g. (a) contaminations
Abbreviations: COFRADIC, combined fractional diagonal chromatography; ChaFRADIC, charge-based fractional diagonal chromatography; HPLC, high performance liquid chromatography; MOAC, metal oxide afﬁnity chromatography; LC, liquid chromatography; MS,
mass spectrometry; PTM, post-translational modiﬁcation; SCX, strong cation exchange chromatography; TAILS, terminal amine isotopic
labeling of substrates.
∗
Corresponding author at: Leibniz-Institut für Analytische Wissenschaften – ISAS – e.V., Otto-Hahn-Str. 6b, 44227 Dortmund, Germany.
Tel.: +49 0231 1392 4143.
E-mail address: [email protected] (R.P. Zahedi).
http://dx.doi.org/10.1016/j.euprot.2014.07.001
2212-9685/© 2014 The Authors. Published by Elsevier B.V. on behalf of European Proteomics Association (EuPA). This is an open access
article under the CC BY-NC-SA license (http://creativecommons.org/licenses/by-nc-sa/3.0/).
166
e u p a o p e n p r o t e o m i c s 4 ( 2 0 1 4 ) 165–170
such as polymers or other biomolecules, (b) co-isolation of
peptides (and such contaminations) so that MS/MS spectra
represent fragment ions derived from a mixture of precursor
ions, (c) point mutations or (d) sequences that are incorrectly annotated in databases, (e) degradation products, (f)
enzymatic mis-cleavages and in-source decay [7,8], and (g)
unknown or unanticipated PTMs (and their combinations,
respectively). The possible extent of protein modiﬁcation is
well represented by histones, which are also among the bestcharacterized proteins. All core histones can be modiﬁed by up
to eight different PTMs (acetylation, phosphorylation, mono-,
di- and tri-methylation, butyrylation, crotonylation and propionylation) at the same time, while the individual sites can be
modiﬁed by different PTMs with occupancies varying between
<1% and 100% [9,10]. In histones these PTMs can act in concert so that the highly context-dependent and combinatorial
pattern of different modiﬁcations referred to as ‘PTM code’,
modulates and deﬁnes the ﬁnal cellular output [11]. A similarly
high degree of modiﬁcation was shown for the tumor suppressor p53, with 10 different PTMs that act in concert and thus
can induce different cellular responses. For both examples,
different PTM codes could be mapped to speciﬁc molecular
functions [11], however, this required a large number of elaborate experiments and studies.
Nowadays, large-scale characterization of PTMs is performed routinely [12–17] and improved methods enable PTM
localization and quantiﬁcation with high conﬁdence [18–22].
Nevertheless, this vibrant research ﬁeld is still emerging and
accompanied by incomplete assignment of identiﬁed PTM
sites to speciﬁc cellular functions [23]. Although extensive
databases about PTMs exist, complete ‘PTM catalogs’ [23] summarizing all possible modiﬁcations (or sites) for protein entries
are still far from reality. Such catalogs will represent valuable
resources for both functional/structural studies and modeling.
This holds true for extensively analyzed PTMs such as
phosphorylation or glycosylation, but even more for the huge
proportion of so far known but largely uncharacterized PTMs
[6]. Because many PTMs have a low stoichiometry and thus
are almost inaccessible for the global proteome analysis of
complex samples such as cell lysates, their detection requires
speciﬁc enrichment prior to analysis. For some PTMs antibodyor afﬁnity-based enrichment is routinely used, but most PTMs
remain hidden from large-scale MS-based detection owing
to the lack of dedicated enrichment methods. Hence, there
is a strong need for alternative and versatile strategies for
PTM enrichment that provide high sensitivity and ﬂexibility,
to allow for a more comprehensive analysis of PTMs and PTM
codes in the future.
2.
Strategies for MS-based analysis of PTMs
In general there are three strategies to analyze proteins via MS:
(i) top-down, (ii) middle-down and (iii) bottom-up (see Fig. 1).
In top-down proteomics intact proteins are analyzed,
potentially revealing complete protein PTM patterns such that
the information about the number, type and the localization of PTMs is retained [24]. One substantial advantage is
the capability to determine the relative abundance of different proteoforms and therefore their relative proportion in the
Fig. 1 – Strategies for MS-based analysis of PTMs. (i) In
top-down experiments intact and puriﬁed proteins are
analyzed. As prior digestion is not required, the
comprehensive identiﬁcation and mapping of PTMs is
possible. (ii) Middle-down approaches produce relatively
long peptide-stretches that can potentially contain multiple
PTMs. Like in bottom-up experiments the modiﬁed
peptides can be enriched to obtain speciﬁc sub-proteomes
(e.g. phosphoproteome, acetylome, N-terminome). (iii) In
bottom-up strategies shorter peptides are generated. This
is usually accompanied by a loss of information about
complex PTM patterns of different proteoforms.
sample. Relative site occupancies can be calculated for different PTMs to determine stoichiometries [24]. Furthermore,
top-down allows characterizing structural changes induced
upon PTM of proteins [25]. However, certain limitations still
impede the dissemination of top-down as routinely used
method for high-throughput PTM analysis [26]. So far efﬁcient
fragmentation is mainly achieved for small proteins (<30 kDa)
and the need for elaborate pre-fractionation involves high
amounts of starting material [26–28], while PTM enrichment
is much more challenging on the protein compared to the
peptide level.
In contrast, bottom-up strategies mostly generate shorter
peptides [8,29] (6–30 amino acids [8,29]), which, compared to
proteins, are less heterogeneous and thus can be separated
and detected more efﬁciently. This allows detection down to
the amol range, even for complex samples. The physicochemical properties of peptides can be exploited effectively to enrich
for certain PTMs that can be mapped with high localization
probabilities [30,31]. However, the improved enrichment and
detection capabilities of bottom-up approaches are accompanied by an inherent loss of qualitative and quantitative
information, considerably impeding the differentiation of proteoforms, PTM stoichiometry and consequently also PTM
crosstalk [32]. Despite those inherent limitations, peptidecentric bottom-up proteomics is still the method-of-choice
to screen for PTMs and their dynamics, providing important
information about PTM localization and changes between different cellular states or time points.
More recently, a promising alternative, so-called middledown, was established. Here, proteases that generate peptides
in the range of 3–9 kDa are used, allowing the identiﬁcation of
e u p a o p e n p r o t e o m i c s 4 ( 2 0 1 4 ) 165–170
167
Fig. 2 – Separation principle of two-dimensional re-chromatography strategies. All strategies follow the same three steps to
enrich the PTM of interest: (1) initial separation of a complex sample, (2a + b) changing the chromatographic behavior of a
certain subclass of peptides and (3) re-chromatography – in (A) and (B) under the same conditions – to enrich for speciﬁc
PTM-peptides. During positive selection (A) the PTM-peptide of interest is speciﬁcally derivatized to induce a retention time
shift in the following second dimension. Vice versa, in negative selection (B) all other peptides are derivatized in order to
induce a retention time shift. (C) Changing the conditions between ﬁrst and second dimension chromatography, as depicted
here for the pH can also lead to an altered elution proﬁle as demonstrated by Hennrich et al. [47].
larger peptide-stretches that potentially reveal more complex
PTM patterns. This approach basically combines the strengths
of top-down and bottom-up, however without completely
eliminating their weaknesses. Thus, PTM peptide enrichment
techniques can be applied and the context of contiguous PTMs
may be recovered, potentially providing more information
about global PTM stoichiometry [6,26].
3.
The need for enrichment
Regardless of which approach is pursued, the low abundance
of PTMs renders a dedicated enrichment essential to lower
sample complexity, to enhance the dynamic range for detection and thereby the speciﬁcity of analysis [24,33]. Especially
for top-down strategies, enrichment is rather restricted to
reducing sample complexity by fractionation and puriﬁcation of the proteins of interest, respectively [24,28,34–37]. In
contrast, bottom-up experiments allow a more straightforward separation of the small proportion of PTM-peptides from
the immense excess of unmodiﬁed peptides. The applied
strategies mostly utilize certain structural or chemical characteristics of the respective PTM to achieve a dedicated
enrichment. The general bottleneck here is the ultimate goal
to address as many PTMs as possible, and not only the
most abundant ones. For the latter, efﬁcient methods are
established, but even these are often restricted to subclasses
of the speciﬁc PTM-proteome, due to inherent preferences
and limitations either of the method, or the preceding sample preparation steps. Even widely-established methods such
as metal oxide afﬁnity chromatography (MOAC), used for
enriching phospho- and glycopeptides, show preferences for
speciﬁc subclasses, that may be altered based on the applied
protocol. For other PTMs such as ubiquitination, lysine acetylation, acylation and methylation, speciﬁc enrichment is
achieved using immunoafﬁnity-based strategies, whereas e.g.
168
e u p a o p e n p r o t e o m i c s 4 ( 2 0 1 4 ) 165–170
the enrichment of SUMOylated species was achieved using a
genetically introduced afﬁnity tag [38]. Unfortunately, those
approaches are often expensive and time-consuming, as e.g.
immunoafﬁnity enrichment requires either purchase or generation of speciﬁc antibodies. These, however, often show low
speciﬁcity and demand huge amounts of starting material
(often 10 mg and more), which does not comply with the analysis of clinical samples or primary tissues. For instance, most
large-scale ubiquitin studies used proteasome inhibitors to
increase the endogenous levels of ubiquitination and thus the
number of identiﬁed sites [39].
In order to improve our presently limited knowledge
about PTMs in cells, important steps will be (i) to further improve already established strategies, as has been
successfully demonstrated e.g. for TiO2 [18,40–43] and (ii)
to develop novel strategies for enrichment and separation
of so far inaccessible or rather uncharacterized PTMs. We
think that exploiting techniques that involve a dedicated
chemical derivatization of peptides in combination with speciﬁc enrichment/depletion of certain peptide classes holds a
strong potential to ﬁll the current gap of knowledge. Twodimensional re-chromatography strategies such as ‘combined
fractional diagonal chromatography’ (COFRADIC) [44,45], and
our recently introduced derivative ‘charge-based fractional
diagonal chromatography’ (ChaFRADIC) [46] are highly ﬂexible and offer a variety of alternative applications for the
enrichment of PTMs. The general concept is to (i) separate
and fractionate a complex peptide sample under robust chromatographic conditions, (ii) to employ a dedicated chemical
or enzymatic derivatization step to alter the chromatographic
behavior of a certain peptide class, and (iii) to separate the
derivatized fractions again under the same conditions. As
illustrated in Fig. 2, peptides of interest retain their previous
retention time window, whereas others shift considerably out
of that window – or vice versa.
4.
Fractional diagonal chromatography
approaches
The original COFRADIC concept was published in 2002 by
Gevaert et al. [44]. Here, in the primary LC run a complete cell
lysate was separated via reversed phase chromatography, fractions were collected every minute and methionine-containing
peptides were oxidized using H2 O2 . Afterwards, all fractions
were re-injected and separated under the same conditions.
Thus, due to the oxidation, methionine-containing peptides
shifted to earlier retention times, whereas unmodiﬁed peptides retained their retention times. The same group later
demonstrated further applications of COFRADIC for enrichment of N-terminal peptides, cysteine-containing peptides
[48,49], phosphopeptides [50], glycopeptides [51] and nitrosylated peptides [52]. The ChaFRADIC approach utilizes an
optimized strong cation exchange chromatography (SCX)based separation of peptides, based on their charge states
at pH 2.7, which is mainly deﬁned by the N-terminal amine
group and Lys, Arg and His side chains. We demonstrated the
proof-of-principle by enriching N-terminal peptides from Saccharomyces cerevisiae using only 50 ␮g protein per condition.
After only 10 h of LC–MS/MS measuring time we could identify
1459 non-redundant N-terminal peptides whereby only 40% of
the obtained fractions were measured. Before, Henrich et al.
used a pH shift between two SCX runs to selectively alter the
elution proﬁle of phosphopeptides [47] (Fig. 2).
Many PTMs bear charged or ionizable groups (e.g. GPI
anchors, arginylation, phosphorylation, sulfation, ubiquitination, SUMOylation, certain types of glycosylation) that
may be used to add/remove charges by means of chemical/enzymatic derivatization or by shifting the pH, in order
to induce a change in their chromatographic behavior. SCXbased strategies such as ChaFRADIC or the approach by Henrich et al. therefore offer a new set of possibilities for dedicated
enrichment of so far inaccesible PTMs, and, based on the different separation principle, are complementary to COFRADIC.
In ChaFRADIC less fractions have to be collected (usually only
fractions containing net charges +1, +2, +3, +4 and higher),
converging high sensitivity, robustness and throughput. Once
the reproducible and robust charge-based HPLC separation
is established, the same system can be further adapted to
target other PTM classes, supposing a speciﬁc derivatization step leading to a deﬁned change of net charge can be
applied.
5.
COFRADIC and ChaFRADIC as versatile
tools for future PTM-research
The general concept renders COFRADIC and ChaFRADIC multifunctional, highly ﬂexible and well adjustable for the speciﬁc
context of the user. Moreover, as mentioned earlier, both
methods can be used for positive or negative selection of
peptide classes. Although establishing and maintaining this
technology is more challenging than other methods such as
MOAC and furthermore may require the use of dedicated HPLC
system, the general strategy of using two-dimensional rechromatography setups offers unrivaled possibilities to enrich
for different classes of PTMs. It can be adapted to other
chromatography modes, further expanding the set of possible applications. Notably, for some applications transferring
the system from HPLCs to cartridges or tips may sufﬁce,
thus considerably facilitating the procedure and reducing the
accompanied costs.
Transparency document
The Transparency document associated with this article can
be found in the online version.
Acknowledgements
The ﬁnancial support by the Ministerium für Innovation, Wissenschaft und Forschung des Landes Nordrhein-Westfalen
is gratefully acknowledged. We furthermore would like to
thank Laxmikanth Kollipara for valuable discussions and
the organizers of the EuPA 2013 conference for a great
meeting.
e u p a o p e n p r o t e o m i c s 4 ( 2 0 1 4 ) 165–170
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