Analytica Chimica Acta 703 (2011) 1930

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Analytica Chimica Acta

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Review

Tools for analyzing the phosphoproteome and other phosphorylated

biomolecules: A review
Alexander Leitner ,1 , Martin Sturm 1,2 , Wolfgang Lindner
Department of Analytical Chemistry, University of Vienna, Vienna, Austria

a r t i c l e

i n f o

Article history:
Received 9 May 2011
Received in revised form 7 July 2011
Accepted 10 July 2011
Available online 19 July 2011
Keywords:
Mass spectrometry
Protein phosphorylation
Proteomics
Sample preparation
Enrichment

a b s t r a c t
Enrichment, separation and mass spectrometric analysis of biomolecules carrying a phosphate group
plays an important role in current analytical chemistry. Application areas range from the preparative
enrichment of phospholipids for biotechnological purposes and the separation and purication of plasmid
DNA or mRNA to the specic preconcentration of phosphoproteins and -peptides to facilitate their later
identication and characterization by mass spectrometry. Most of the recent improvements in this eld
were triggered by the need for phosphopeptide enrichment technology for the analysis of cellular protein
phosphorylation events with the help of liquid chromatographymass spectrometry. The high sensitivity
of mass spectrometry and the possibility to combine this technique with different separation modes in
liquid chromatography have made it the method of choice for proteome analysis. However, in the case
of phosphoprotein analysis, the low abundance of the resulting phosphopeptides and their low quality fragment spectra interfere with the identication of phosphorylation events. Recent developments
in phosphopeptide enrichment and fragmentation technologies successfully helped to overcome these
limitations. In this review, we will focus on sample preparation techniques in the eld of phosphoproteomics, but also highlight recent advancements for the analysis of other phosphorylated biomolecules.
2011 Elsevier B.V. All rights reserved.

Alexander Leitner studied Chemistry in Vienna and

obtained his PhD in Analytical Chemistry under Prof.
Lindner in 2004. After continuing his research in
Vienna, he joined the group of Prof. Ruedi Aebersold at the Institute of Molecular Systems Biology
at ETH Zurich as a postdoctoral researcher in 2008.
His general interest is the development of analytical
methodology for applications in proteomics, with a
strong focus on mass spectrometry. Currently, he is
working on the development of new techniques for
the structural analysis of proteins and protein complexes using chemical cross-linking and advanced MS.

Martin Sturm studied Biochemistry at the University of Vienna and obtained his Master degree at the
Medical University of Vienna in 2006. After one year
research work at the Vienna Biocenter in the lab of
Prof. Ammerer he started his doctoral thesis in Analytical Chemistry under Prof. Lindner, obtaining his
PhD 2010. Currently he is employed at the Austrian
Research Center OFI as an expert for LCMS. There
he is working on establishing multicomponent analysis methods for extractables and leachables studies
by LCMS.

Corresponding author. Current address: Institute of Molecular Systems Biology, ETH Zurich HPT E 53, Wolfgang-Pauli-Str. 16, 8093 Zurich, Switzerland.
E-mail address: leitner@imsb.biol.ethz.ch (A. Leitner).
1
These authors contributed equally.
2
Current address: o - Austrian Research Institute for Chemistry and Technology, Vienna, Austria.
0003-2670/$ see front matter 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.aca.2011.07.012

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A. Leitner et al. / Analytica Chimica Acta 703 (2011) 1930

Wolfgang Lindner holds since 1996 a Chair of Analytical Chemistry at the University of Vienna, Austria.
Throughout his career he got strongly inuenced by
life science themes spanning from pharmaceutical
analysis, metabolomics, proteomics, etc. and separation science methodologies as of HPLC, GC, CE/CEC and
LCMS. In this context particular interest developed
also towards non-covalent interactions and molecular
recognition phenomena with focus on stereochemistry leading to the development of novel synthetic
selectors useful for enantioselective separation techniques. Working on the interface of organic, analytical
and biological chemistry characterizes best his scientic credo supported by a rich portfolio of publications.

1. Introduction
1.1. The biological role of phosphates
Phosphate groups are a very common functional group in a variety of different biological compounds. They form the hydrophilic
backbone of DNA or RNA poly- and oligomers and represent
the hydrophilic group in many amphiphilic membrane lipids.
Reversible protein phosphorylation works as a molecular switch,
which allows the regulation of metabolism and signal transduction in cells. Phosphorylation marks the activation of sugars for
metabolism, and the triphosphate nucleotide ATP serves as the
main energy carrier and phosphate group donor in all organisms.
Westheimer summarized some of the unique chemical properties of the phosphate group in a seminal paper [1]. Phosphates
are capable to carry two negative charges depending on the pH
of the solvent. Typical pKa values are 2.2 for the singly charged
and 7.2 for the doubly charged free phosphate [2], although these
values can vary depending on the specic chemical environment.
This ensures that phosphates remain ionized at physiological pH.
Another important structural feature is that phosphate groups are
basically tetrahedral, with a symmetry depending on the number of substituents on the O-atoms [3]. This allows the formation
of geometrically restrained hydrogen bond networks. Phosphates
also exhibit chemical reactivity that may be particularly attractive
for carrying out and/or preventing biologically relevant transformations; for example the slow rate of phosphate ester hydrolysis
in DNA ensures its long-term stability. Although phosphorous has
been deemed an essential element in living organisms, the recent
(and controversial) discovery of a microorganism that is able to
replace phosphorous with arsenic [4] represents an important nding that this may not necessarily be the case.
1.2. The phosphate group as a target for enrichment methods
(Fig. 1)
Many enrichment methods take advantage of the ionic and
Lewis base (electron-pair donating) character of the phosphate
group for interaction. Therefore, methods based on ion exchange
mechanisms are generally suitable for the analysis of phosphates.
Additionally, the Lewis base properties allow coordinative binding
to positively charged iron or gallium central atoms in a chelating
matrix, a concept called immobilized metal afnity chromatography (IMAC). Recently, new metal oxide materials like TiO2 , ZrO2
or SnO2 , which possess Lewis acid and ion exchange properties,
are frequently used for selective phosphopeptide enrichment. In
addition to the methods mentioned above, there are different
chemical tagging techniques that take advantage of the reactivity
of the phosphate group. Finally, antibodies may be raised against
phosphate group-containing motifs and are frequently used for
immunoprecipitation of phosphoproteins.

Fig. 1. The phosphate group as enrichment target. The selective recognition of

the phosphate group by various approaches is illustrated: Structure (antibodies);
charge and polarity (ion exchange chromatography, partially also antibodies and
metal afnity techniques); metal afnity (IMAC and MOAC) and chemical reactivity (chemoselective reactions, see also Fig. 3). IMAC, immobilized metal afnity
chromatography; MOAC, metal oxide afnity chromatography.

Some of the techniques that were primarily developed for phosphoproteomic analysis may also be applied to other classes of
phosphorylated biomolecules. Due to the fact that there is an
increased interest of academic researchers, but also from the pharmaceutical industry for the selective enrichment and separation
of phospholipids, DNA (plasmids), or phosphorylated metabolites,
we will also cover selected recent applications and developments
in these elds at the end of this review.
1.3. Phosphoproteomics
The majority of enrichment and separation strategies were
developed as a result of the need for the selective enrichment of phosphopeptides to facilitate their identication by
mass spectrometry. Reversible protein phosphorylation is the
most widespread post-translational protein modication (PTM) in
(eukaryotic) cells, and it is the main chemical protein modication
involved in cellular signaling, metabolism, protein transport or cell
division and apoptosis, when proteins interact with each other [5].
Recent proteomic research revealed that the onset of many severe
diseases, especially many types of cancers, is inuenced by the
activity of tyrosine kinases in certain regulation/signal transduction
pathways [6,7]. Recently developed anticancer drugs like imatinib,
dasatinib or bosutinib act as direct inhibitors of the respective protein tyrosine kinases in cancer specic signaling networks [8,9].
New proteins in protein interaction pathways could successfully be
inferred by LCMS/MS identication coupled with phosphopeptide
enrichment methods [1013]. These achievements show that phosphoprotein identication and phosphorylation site determination
is of high clinical and research interest.
O-phosphorylation is the most common type of protein phosphorylation. It occurs mainly on the hydroxyl group-containing
amino acids serine, threonine and tyrosine. Although widely differing ratios have been reported, phosphotyrosines are clearly the
least abundant residues among the three. Far less frequently, N-, Sand acyl-phosphorylation occurs on histidine, lysine, cysteine and
aspartic or glutamic acid residues [14]. The covalent attachment
of the phosphate group to the respective amino acid residues is

A. Leitner et al. / Analytica Chimica Acta 703 (2011) 1930

catalyzed by a class of enzymes called kinases which use the energy

of adenosine triphosphate (ATP) or guanine triphosphate (GTP)
hydrolysis for the transfer of the phosphate group onto the substrate protein [15,16]. This may cause a conformational change and
thus alteration of the activity of the respective substrate proteins
or attracts phosphospecic binding domains from other proteins
leading to a proteinprotein interaction [17].
Although some proteins remain permanently phosphorylated,
the larger fraction, especially those involved in signaling pathways show a highly dynamic and regulated pattern resulting from
interactions between kinases and phosphatases, which rapidly
dephosphorylate the proteins after a phosphorylation event,
resembling an on/off mechanism. Even though phosphoproteins
account for at least 30% of the eukaryotic proteome, based on
recent comprehensive studies, the ratio of the phosphorylated to
unphosphorylated form of the protein is rather small, so that the
phosphoproteins will usually be present in substoichiometric concentrations.

2. The role of mass spectrometry in phosphoproteomics

Mass spectrometric analysis of phosphopeptides poses some
additional challenges compared to the characterization of mixtures
of unmodied peptides. The widely held opinion of reduced ionization sensitivity of phosphopeptides is mostly related to the fact that
phosphorylation is a substoichiometric event and phosphopeptides
are generally less abundant [18]. At similar concentrations, the
detection sensitivity of singly and doubly phosphorylated peptides
is not necessarily dramatically different. Similarly, the retention
behavior of peptides is not signicantly altered upon phosphorylation for the majority of peptides; in fact, some phosphopeptides
may be even more retained in reversed-phase chromatography
than their unmodied counterparts depending on the mobile
phase composition. In contrast, the situation is different for highly
phosphorylated peptides that are usually detectable only with considerably reduced sensitivity in positive ion mode while negative
ionization is commonly avoided for practical reasons (reduced stability). Therefore, the actual presence of highly modied peptides
may be severely underestimated.
Such a bias could also result from a bias introduced during
enrichment steps (see below), but also as a result of suboptimal fragmentation properties. In fact, the latter constitutes the
most signicant obstacle in mass spectrometric analysis of phosphopeptides [19,20]. During collision-induced dissociation, many
pSer- and pThr-peptides exhibit a neutral loss of phosphoric acid
(H3 PO4 , 98 Da) as the dominant fragmentation pathwayin contrast to pTyr peptides where the modication remains stable.
Consequently, informative fragments stemming from backbone
fragmentation may be present only at low intensities or some cleavages may be entirely suppressed. A substantial amount of research
has therefore been contributed to improving phosphopeptide fragmentation. To this end, chemical removal of the phosphate group by
beta-elimination has been proposed [21,22], which is however not
practical for complex samples due to possible side reactions. Similarly, altering the general ionization properties of a phosphopeptide
by reducing the basicity of the protonation site upon arginine
derivatization has been shown to reduce neutral loss, although only
for model peptides [23].
More attention has been directed to the development of rened
and new fragmentation mechanisms [19,20]. Neutral loss driven
MS3 is one way to deal with the loss of the phosphate group, where
the loss of H3 PO4 from a precursor is detected in real time and
another stage of fragmentation is induced. Although this feature
is now routinely implemented on many tandem mass spectrometers containing an ion trap analyzer and can be used for improving

21

the statistical certainty at the data analysis stage, this comes at a

price of reduced scan speeds and more complex bioinformatic processing. Alternatively, multi-stage activation has been introduced
on linear ion trap systems, where instead of two separate isolation and fragmentation steps, the precursor and the neutral loss
reaction product are activated subsequently without an intermittent isolation step. Therefore, a hybrid spectrum containing MS2
and MS3 information is obtained, this facilitates data analysis as
only one level of fragmentation data needs to be analyzed [24].
Both approaches, however, fail in the case of highly phosphorylated peptides characterized by multiple successive losses of the
phosphates.
CID can be performed either in ion traps or in dedicated collision cells, and the differences in energy deposition (slow heating
in ion traps and fast heating in collision cells, reviewed in [25])
result in differences in the appearance of mass spectra, especially
for modied peptides such as phosphopeptides. Therefore, beamtype collision cell CID is not only used in quadrupole-time-of-ight
instruments, but has also been introduced on the highly popular
Orbitrap hybrid instruments (termed HCD for higher-energy CID
[26]). As a result of the faster energy deposition and the occurrence
of consecutive dissociation, such collision cell CID spectra may
contain less abundant neutral loss signals and more interpretable
sequence information [19]. For example, Mann and co-workers
used HCD on a new Orbitrap instrument to identify more than
16,000 phosphorylation sites in HeLa cells following multidimensional enrichment/fractionation of phosphopeptides [27].
The introduction of the complementary electron transfer dissociation technique [28] has also had enormous impact on large-scale
phosphopeptide analysis. In contrast to CID, ETD does not cause
neutral loss of phosphoric acid, thereby dramatically improving
spectral quality in certain cases. While it was initially thought that
ETD would to some extent replace CID, especially for the characterization of modied peptides, several years of research have made
apparent some limitations of the technique: Slower scan speeds
(due to longer reaction times), lower fragmentation efciencies and
considerable m/z and charge state dependence of spectral quality.
Coon and co-workers therefore introduced a decision tree method
[29] that switches between CID and ETD according to precursor m/z
and charge state. Furthermore, most widely used search engines
perform suboptimal for ETD data as they do not take into account
peculiar properties of ETD spectra, e.g. the formation of hydrogen rearrangement products [30]. Spectral postprocessing and the
development of new search algorithms [31,32] attempt to overcome these issues, although there is no universal gold standard
yet.
Finally, quantitation of phosphopeptides or, more general, the
phosphorylation state of a protein or even a proteome remains an
area of intense research. Due to the enormous biological signicance of phosphorylation dynamics in signaling events, a number
of approaches have been used to study quantitative changes in
phosphosite occupancy between different biological conditions.
For details, the reader is referred to recent reviews on this topic
[3335]. While quantitative data can be obtained by various
means of stable isotope labeling, e.g. metabolic or chemical labeling using established techniques, it has to be considered that
changes in the relative abundance of phosphopeptides may not
only result from up- or down-regulation of a particular phosphorylation site. Additionally, phosphorylation on additional sites within
the same peptide may lead to the peptide evading detection in
its multiphosphorylated form. Furthermore, in some experimental set-ups changes in absolute protein amounts may skew the
results, as recently demonstrated by Gygi and co-workers [36].
Ideally, both the unmodied form of the peptide and all possible
phosphoisoforms would need to be evaluated to obtain denitive
information.

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Fig. 2. Commonly used enrichment workows in phosphoproteomics. Only the most commonly used approaches relying on peptide-level enrichment are illustrated:
Immunoprecipitation and IMAC/MOAC enrichment with or without additional prefractionation (ion exchange chromatography or HILIC). For additional details, see text.
HILIC, hydrophilic interaction chromatography; IMAC, immobilized metal afnity chromatography; MOAC, metal oxide afnity chromatography.

3. Enrichment techniques in phosphoproteomics

To facilitate the identication of phosphopeptides, many different enrichment strategies were developed and are in permanent
improvement. Fig. 2 gives an overview about frequently used
workows that are discussed in detail in the following chapters. Enrichment techniques in phosphoproteomics are usually
embedded in elaborate workows that involve numerous sample
preparation steps, i.e. protein extraction, enzymatic cleavage, etc.
In the context of analyzing protein phosphorylation it is essential
although not necessarily straightforward to minimize the activity of phosphatases that are released during cell lysis, to avoid
the generation of artifacts during workup [37]. Similarly, protease inhibitors should be added to avoid degradation of the
sample. Most enrichment techniques (with immunoprecipitation
as a notable exception) are used at the peptide level, not only
because these methods work more efciently but also because of
the more substantial depletion of unphosphorylated compounds
that is achievable. Proteolytic digestion, however, leads to an
unavoidable loss of contextual information on the interdependence
of modications on the protein level, for example, whether a given
phosphorylation site is only occupied when another one is already
present. Techniques that study the interplay of different types of
post-translational modication are still in their infancy, but of high
biological signicance [38].
3.1. Immunoafnity chromatography
Immunoafnity chromatography or immunoprecipitation is a
widely used enrichment strategy in biochemistry. Antibodies are
known to have a high afnity and selectivity for their target epitopes. Commonly used for trapping whole proteins, they are also

used for enrichment of smaller molecules like peptides or amino

acids, although the generation of good working antibodies for
smaller molecules is more elaborate.
In a traditional experiment to analyze protein phosphorylation the setup focused on a single phosphoprotein of interest. For
detection and purication specic antibodies directed against the
phospho-epitopes had to be generated. The production and validation of proper antibodies is a time consuming and expensive
process taking up to one year to generate and validate an effective
antibody [39]. Antibodies used for phosphopeptide or phosphoprotein enrichment on a proteomic scale should therefore be directed
solely against the phosphorylated amino acid, i.e., they should have
the same or similar afnity and selectivity for all the phosphopeptides or -proteins in a biological sample to obtain a balanced
and unbiased enrichment of all phosphorylated analytes. However, the recognition of phosphate groups in peptides is affected
by the surrounding amino acids so it is difcult to generate adequate antibodies. Although there are antibodies against the three
most common phosphoamino acids, phosphoserine (pS), phosphothreonine (pT) and phosphotyrosine (pY), their use is frequently
limited by the specicity [40,41]. Highly specic pY antibodies exist
and are predominantly used. Tyrosine kinases play an important
role in human cancer, taking part in oncogenic signaling for cellular proliferation and survival. Therefore an unregulated tyrosine
kinase activity can lead to malignancy and tumor formation [42].
In a global phosphoproteomic approach the number of identications of phosphotyrosine containing peptides would be rather small
because of its low abundance, therefore pTyr-selective methods are
essential.
As an example, a comprehensive proteomic study of tyrosine
phosphorylation in Jurkat cells was done by Rush et al. [42]. Phosphotyrosine containing peptides from a cell digest of pervanadate

A. Leitner et al. / Analytica Chimica Acta 703 (2011) 1930

(a tyrosine phosphatase inhibitor) treated Jurkat cells were

immunoprecipitated with P-Tyr-100, a phosphotyrosine specic antibody non-covalently coupled to protein G agarose. The
enriched peptides were later on analyzed by conventional reversed
phase chromatographytandem mass spectrometry. Using this
strategy, 688 pTyr-containing peptides and 628 pTyr-sites could be
identied. The same strategy was used by Rikova et al. for a large
scale analysis of tyrosine kinase activity in non-small cell lung cancer (NSCLC) cell lines [43]. Known tyrosine kinases such as EGFR
and c-Met as well as new oncogenic tyrosine kinases not known
to play a role in lung cancer were identied. In summary over 50
different tyrosine kinases and over 2500 downstream substrates
were identied by this approach. White and co-workers combined
immunoprecipitation with relative quantitation by using isobaric
iTRAQ tags to elucidate time-resolved phosphorylation events in
EGFR signaling [44].
3.2. Immobilized metal afnity chromatography (IMAC)
Cysteines and histidines are known to form stable complexes
with zinc and copper ions in an aqueous solution [45]. This effect
was used for selective enrichment of proteins via transition metals trapped in a chelating matrix. The concept, termed IMAC, may
be seen as a sort of pseudo-afnity chromatography and is widely
used for the enrichment of recombinant histidine-tagged proteins
for biotechnological purposes. A string of six His residues is genetically attached to the C- or N-terminus of proteins of interest and
the high afnity of histidine to covalently xed Ni2+ is used for
enrichment of the tagged protein. The concept was expanded for
enrichment of phosphorylated proteins by Andersson and Porath
in the year 1986 [46]. In their experiments they used Fe3+ bound
via iminodiacetic acid (IDA) to a sepharose matrix. Phosphorylated
amino acids like phosphoserine, phosphothreonine or phosphotyrosine were retained by the chromatographic material whereas
non-phosphorylated amino acids were not, or in some cases, like
aspartic acid or glutamic acid, only weakly bound. A separation of
ovalbumin phosphoisoforms, carrying different numbers of phosphates, succeeded using this technology. An advantage of the
method was that all steps could be carried out in water or buffer
and no protein denaturing components were needed.
Over the years, a variety of different supports have been introduced for IMAC-based enrichment, and the technique has long been
the most frequently used method for enrichment of phosphopeptides, also due to the fact that commercial kits are available from
different suppliers. However, the high level of unspecic binding of
acidic peptides via their carboxylic residues remains a challenging
problem. A method to circumvent unspecic binding of acidic peptides was introduced by Ficarro et al. [47]. In their approach a tryptic
digest of a yeast whole cell lysate was analyzed and carboxylic
residues on peptides were chemically derivatized by O-methyl
esterication with methanolic HCl before Fe3+ -IMAC enrichment.
The esterication led to an elimination of unspecically bound peptides without loss in sensitivity. Using this method the authors
could identify 200 phosphopeptides from S. cerevisiae which was a
substantial achievement at that time.
Although this technology increased the selectivity of IMAC phosphopeptide enrichment considerably and has been used frequently,
the esterication step might be incomplete and cause sample loss.
Therefore, additional ways to improve the specicity of the enrichment were evaluated, such as adjusting the pH of the loading
buffer or using different chelated ions. Kokubu et al. used 50%
acetonitrile, 0.1% triuoroacetic acid (TFA) as loading buffer for
Fe3+ -IMAC enrichment when analyzing a tryptic digest of a mouse
brain protein extract [48]. With TFA, selective protonation of carboxylic residues on peptides was reported; additionally, the high
acetonitrile content reduced hydrophobic interactions between

23

peptides and the IMAC resin. Using this loading buffer a reduction of nonspecic binding of acidic peptides was obtained. Just 96
nonphosphopeptides and 1654 phosphopeptides were assigned by
Mascot from a mouse brain sample. After manual validation 166
phosphosites on 135 different proteins were identied using this
approach.
Although iron is used as central ion in most IMAC methods,
other metal ions have been evaluated for selective phosphate
afnity. Posewitz tested different metal ions like Ga, Sn, Ge, Fe,
and others for their applicability in IMAC phosphopeptide enrichment [49]. With Ga3+ a better selectivity compared to conventional
Fe3+ -IMAC was reported when analyzing a tryptic digest of phosphoproteins. An interesting approach was reported from the group
of Zou [50,51]. They used a phosphate polymer to coordinatively
bind Ti4+ or Zr4+ ions, and the resulting IMAC resin was used for
phosphopeptide enrichment and compared to Fe3+ -IMAC, TiO2 and
ZrO2 enrichment methods. For the preparation of the IMAC resin
they used a special chemistry to attach the phosphate groups to
the matrix via a linker to improve the accessibility of the interaction sites and to provide a benecial structural orientation for
the selective binding of phosphorylated biomolecules. Additionally,
phosphates form a stable MO6 octahedron structure with Ti and Zr
ions. So the metal ions that are stably bound to the phosphonate
groups form an interface on the surface of the resin on which phosphate ions may form a stable double layer. To illustrate the potential
of this material, a mix of a standard phosphoprotein digest and a
BSA digest as control in a ratio of 1 to 500 was analyzed. Additionally, the method was applied to the phosphoproteome analysis of
mouse liver. The method was reported to outperform conventional
metal oxide enrichment (see following chapter) and Fe3+ -IMAC in
terms of efcacy and selectivity, even when using optimized conditions for the respective methods. A bias of Ti4+ -IMAC towards
monophosphorylated peptides and of Zr4+ -IMAC towards multiply phosphorylated peptides was reported. The results of the new
methods were ascribed on one hand to the highly specic interaction between the Ti and Zr ions and the phosphate groups on
peptides and on the other hand to the novel resin design.
Notable IMAC-based studies on complex biological samples
were able to identify several hundred to thousands of phosphorylation sites [5254]. A way to further increase the specicity
and efcacy of the IMAC technology is to combine it with other
enrichment or fractionation methods. In most cases ion exchange
chromatography or hydrophilic interaction liquid chromatography
(HILIC) chromatography is used for prefractionation before specic
enrichment for phosphopeptides with IMAC or metal oxides is performed. A detailed discussion is given in the chapter on combined
enrichment methods.
3.3. Metal oxide afnity chromatography (MOAC)
Since the beginning of the 1990s, titanium dioxide (titania,
TiO2 ) and zirconium dioxide (zirconia, ZrO2 ) were increasingly used
as new chromatographic materials for high performance liquid
chromatography in normal phase mode. The advantages of these
metal oxides included large adsorption capacities, chemical stability when used under extreme pH ranges, mechanical stability and
unique amphoteric ion exchange properties [5559]. Phosphates
are known to bind to metal oxide materials [3] and in 1990 Matsuda
et al. [56] reported the selective adsorption of organic phosphates
to ceramic TiO2 material. Henceforward several studies concerning
the enrichment of phosphate group-containing biomolecules with
metal oxide materials emerged [58,59], but not a lot was known
about the surface chemistry and binding properties of these materials. Connor and McQuillan [3] investigated phosphate adsorption
onto TiO2 from aqueous solutions with infrared spectroscopy. They
proposed a pH-dependent bidentate binding of monosubstituted

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phosphate groups to TiO2 solgel lms. In another work, the surface chemistry of metal oxide materials was characterized by their
acidity and basicity which are of Lewis and Bronsted base type
[60]. Alumina (Al(OH)3 ), TiO2 and ZrO2 possess strong Lewis acid
properties with increasing Lewis acidity from alumina to zirconia. In addition, the high coordination numbers of TiO and ZrO
are responsible for strong complexation properties of these oxides.
Both the Lewis acid and the strong complexation properties may
be a possible explanation for the afnity of some metal oxides for
phosphate molecules.
Tani and Miyamoto [61] characterized the chromatographic
properties in terms of ion exchange and ligand exchange behavior of different calcinated titania materials. TiO2 was calcinated in
an oven at 200800 C and a strong temperature dependence of the
ion and ligand exchange behavior could be observed. At 700 C TiO2
is completely converted from anatase to rutile form and no ionexchange or ligand-exchange behavior could be detected anymore.
The authors ascribed the absence of these properties to the loss of
the surface hydroxyl groups and coordinatively bound water by
calcination at high temperatures. In another experiment Tani and
Ozawa [62] compared the chromatographic behavior of TiO2 and
ZrO2 with hydroxy acids and other substituted aliphatic carboxylic
acids. The highest retention times were achieved with -hydroxy
carboxylic acids because they can form a stable ve-membered ring
between metal ion and acid. -hydroxy acids were later introduced
as additives to avoid unspecic binding of acidic unphosphorylated peptides to TiO2 to increase selectivity in phosphopeptide
enrichment ([63], see further discussion below). The stable ring
formation can be a possible explanation why -hydroxy acids are
able to displace acidic peptides from TiO2 or ZrO2 . A work with a
focus to screen differently treated metal oxide materials for their
phosphate afnity was performed by Leitner et al. [64]. They also
compared different surface treated tin dioxide microspheres [65],
an alternative metal oxide used for phosphopeptide enrichment,
for phosphopeptide afnity and selectivity purposes. The results
gave evidence that parameters like calcination temperature and
surface treatment with acid or base can differently affect the surface chemistry and morphology of metal oxide materials, which
in turn will affect the afnity and selectivity of the materials to
phosphates.
The rst proteomic application of TiO2 for phosphopeptide
enrichment was reported by Pinkse et al. [66]. In this work a novel
automated method for the enrichment of phosphopeptides from
complex mixtures with TiO2 was developed. A two dimensional
chromatographic setup with titanium dioxide-based solid-phase
material (Titansphere) as the rst dimension and reversed-phase
material as the second dimension was employed. Phosphorylated
peptides were separated from non-phosphorylated peptides in the
rst dimension by trapping them under acidic conditions (0.1 M
acetic acid) on the TiO2 precolumn. Nonphosphopeptides were not
retained in the rst dimension but trapped in the second dimension
precolumn before they were analyzed by nanoow LCESIMS/MS.
The phosphopeptides were eluted from the column under alkaline conditions (ammonium bicarbonate pH 9.0), concentrated on
the second dimension and analyzed by nanoow LCESIMS/MS.
125 fmol of a phosphopeptide in a 1:1 mixture of the phosphorylated and unphosphorylated form could be successfully identied
with a recovery rate of above 90%. Additionally a novel autophosphorylation site could be identied from a digest of the cGMP
dependent protein kinase. A drawback of this strategy was the high
level of unspecic binding of acidic non-phosphorylated peptides
to TiO2 under these conditions which were reported to be on a
comparable level as for IMAC. The authors initially recommended
O-methyl esterication [47] to reduce unspecic binding. However,
in the following years numerous rened protocols were reported
in the literature.

Larsen et al. [67] used 2,5-dihydroxybenzoic acid (DHB), a

matrix compound widely used in MALDI mass spectrometry, to
increase selectivity without a reduction in phosphopeptide recovery. In this procedure, peptides from a tryptic digest of the
phosphoproteins ovalbumin and -and -casein were used for
phosphopeptide enrichment with TiO2 columns. Peptides were
loaded onto TiO2 columns with different concentrations of DHB
(0200 mg mL1 in 80% acetonitrile, 0.1% TFA) in the loading buffer.
The column was washed with 20 mg mL1 DHB in 50% acetonitrile and subsequently with an 80% acetonitrile/0.1% TFA solution.
Bound peptides were eluted with NH4 OH, pH 10.5. With increasing concentrations of DHB in the loading buffer the number of
non-phosphorylated peptides bound to the column decreased
dramatically with no unspecically bound peptides observed at
200 mg mL1 DHB. When using very complex samples the author
recommended the use of even higher concentrations of DHB to
exclude unspecic binding of non-phosphorylated peptides. A possible reason why DHB is able to avoid unspecic binding of acidic
non-phosphorylated peptides without reducing phosphopeptide
binding was explained by different binding properties of these
molecules to TiO2 . DHB was proposed to bind to TiO2 via a chelating bidentate geometry whereas phosphates bind in a bridging
bidentate way. The assumption was that the two molecules target different binding sites on the TiO2 surface and DHB directly
competes with carboxylic residues from acidic peptides. A big disadvantage of this protocol is that complete removal of DHB before
LCMS analysis is necessary to avoid interference with the chromatographic separation and ionization in the mass spectrometer.
In the mean time, several attempts to increase selectivity without a decrease in sensitivity were carried out by screening different
loading buffer additives to suppress unspecic binding [63,68].
Mazanek et al. [68] suggested using a mix of DHB and octanesulfonic acid (OSA), an ion pairing agent used for improved peptide
separation in reversed phase chromatography, to reduce unspecic binding. The additives were used in lower concentration and
should therefore be less problematic for the following analysis.
Additionally, OSA should improve the quality of the reversed phase
separation by acting as an ion pairing agent. The protocol was tested
with a mix of phosphorylated and non-phosphorylated peptides
and the applicability of the protocol to more complex samples
like a tryptic HeLa digest was demonstrated. More recently, this
method was further optimized in terms of selectivity using slightly
increased concentrations of DHB and OSA, and with the addition
of heptauorobutyric acid [69]. The optimized protocol allowed
improved recovery of phosphorylated peptides and a reduction of
unspecic binding from a trypsinized bovine serum albumin matrix
when compared to a previous protocol [68]. The new method was
applicable to both titania and zirconia enrichment. The usefulness
for more complex samples was demonstrated with the identication of in vivo phosphorylation sites of the afnity enriched
anaphase promoting complex (APC/C) and the mitotic complex,
condensin-I. Sugiyama et al. screened different hydroxy acids as
alternative displacement additives in TiO2 and ZrO2 enrichment
[63]. The best results in terms of selectivity were achieved when
using lactic acid in a concentration of 300 mg mL1 in 80% acetonitrile/0.1% TFA for TiO2 and 100 mg mL1 -hydroxypropanoic acid
in 80% ACN/0.1% TFA for ZrO2 enrichment. Again the applicability
to complex samples was successfully demonstrated by analyzing a
tryptic HeLa sample [63]. Lactic acid modied TiO2 enrichment gave
the highest selectivity as well the largest number of identied phosphopeptides. In total, 1100 phosphopeptides could be identied by
four replicate experiments using TiO2 and ZrO2 enrichment.
An alternative to optimizing the loading buffer composition for
TiO2 enrichment is the use of other metal oxide materials for phosphopeptide enrichment purposes [70]. Soon after the introduction
of TiO2 -MOAC, ZrO2 was also evaluated due to its structural,

A. Leitner et al. / Analytica Chimica Acta 703 (2011) 1930

chemical and physical similarities [71]. Initially, a different bias

towards the enrichment of mono- and polyphosphorylated peptides was reported for titania and zirconia. Subsequent studies
failed to reproduce these tendencies which may be attributed to
differences in other experimental parameters. The Ishihama group
has published a series of studies where ZrO2 was used alongside
TiO2 to provide a more comprehensive coverage of the phosphoproteome [63,72]. Porous tin dioxide microspheres, a new material
for phosphopeptide enrichment, were tested in our group as novel
phosphoafnity chromatographic material [64,65]. The spheres
were manufactured by the nanocasting process [73] which allows
the ne tuning of material properties like particle size, morphology and pore size of different metal oxide materials. Using a tryptic
digest of 10 proteins with ovalbumin as the only phosphorylated
protein, phosphopeptide enrichment with SnO2 and a simple loading buffer composition without additives performed on the same
level to TiO2 with the use of 300 mg mL1 lactic acid as additive
[65] which was used as benchmark protocol. Later, a tryptic digest
of a HeLa cell lysate was used for a comparison of SnO2 with two
different types of titania materials [74].
In addition to the three materials, a number of other metal
oxides have now been shown to possess some phosphopeptide
afnity (reviewed in ref. [70]). These include Al(OH)3 [75], Nb2 O5
[76], Ta2 O5 [77], Fe2 O3 [78] and ZnO [79], among others. In addition to conventional micrometer-sized particles, several groups
reported the preparation of magnetic metal oxide nanoparticles for
MOAC enrichment. Although these materials offer potential advantages in terms of capacity (due to the high surface-to-volume-ratio)
and rapid binding kinetics (due to short diffusion paths), applications to more complex biological samples remain limited up to
now.
Summing up, metal oxide materials possess a high afnity
towards phosphates, exhibit chemical and physical stability and
can be manufactured in different morphologies and sizes. These
properties make it the materials of choice in many current applications. Additionally, MOAC has shown to be more tolerant against
buffer additives like salts, detergents and denaturing components
than IMAC enrichment. These unique properties make metal oxides
the ideal materials for automated online enrichment/LCMS [66].
In this regard, Agilent Technologies has introduced a chip-based
device with integrated TiO2 enrichment and RP-LC separation that
is now commercially available [80,81].
3.4. Ion exchange chromatography
Different forms of ion exchange chromatography are commonly used in a two-dimensional chromatography set-up in
proteomics. Because of the strong negative charge of the phosphate
group, most phosphopeptides show particular retention behavior
in ion exchange chromatography in comparison to the majority of
unmodied peptides.
Beausoleil et al. were the rst to exploit this in a phosphoproteomic approach [10] when analyzing a tryptic digest of a HeLa
cell lysate using strong cation exchange (SCX) chromatography.
For loading of peptides on the SCX material, peptides were acidied to a pH of 2.7 and bound peptides were eluted with increasing
salt and pH. At the pH 2.7 Lys, Arg, His and the N-terminus of
peptides are positively charged. Tryptic proteolysis produces peptides with an Arg or Lys on the C-terminus, consequently, tryptic
peptides should carry a solution charge of +2 and because phosphate groups retain their negative charge at this pH, the net charge
state of phosphopeptides should be +1. Thus the phosphopeptides
tend to elute earlier from the SCX column. The phosphopeptidecontaining fractions were analyzed by LCMS2 and additional
data-dependent MS3 for improved phosphopeptide identication
was performed. This chromatographic setup combined with elab-

25

orate mass spectrometric identication allowed the determination

of 2002 phosphorylation sites on 967 phosphoproteins. But the
combination of SCX with RP chromatography also revealed some
difculties, especially when both columns were coupled on-line.
For example, the interaction of peptides with the SCX resin is proposed to be (mainly) of electrostatic and (partially) of hydrophobic
character, due to some residual hydrophobicity of the polymeric
stationary phase, so that structurally similar peptides with the same
net charge may be separated to some degree. For the increase of
peptide recovery from the SCX column, the use of organic modier has been recommended [82], which can on the other hand
interfere with the following RP separation. As a consequence both
dimensions cannot be optimized independently [83].
In contrast, when using strong anion exchange chromatography (SAX) instead of SCX, phosphopeptides are strongly retained.
Zhang et al. demonstrated the applicability of anion exchange chromatography (AEC) for selective phosphopeptide enrichment when
analyzing a tryptic digest of the standard phosphoprotein -casein
[84]. Later, SAX was used for the enrichment and separation of
phosphopeptides from a digest of human liver cancer tissue by
Gu and co-workers [85]. In contrast to SCX chromatography, the
authors claimed that not only phosphopeptide enrichment, but also
fractionation according to the number of phosphate groups was
possible. This was demonstrated when analyzing a tryptic digest of
- and -casein. For separation of bound phosphopeptides a salt
gradient of NH4 Cl was used and the pH of the solvent was set to a
value of 4. At this pH phosphopeptides remain ionized and hence
are able to interact with the SAX resin via their phosphate groups.
Consequently, multiply phosphorylated peptides should show a
higher afnity to the SAX resin than singly phosphorylated ones.
The performance of the technology was compared to a Fe3+ -IMAC
enrichment with a commonly used protocol. For comparison of
the two methods only one fraction (528 min retention time) of
retained peptides was collected from the SCX column due to the
assumption that nonphosphopeptides would not be retained by
the SCX column and will therefore elute with the ow through.
A higher number of unique phosphopeptides, 47 compared to 24
with IMAC, could be identied using the SAX method and an overlap
of 12 unique phosphopeptides was identied with both methods.
Although both methods suffer from unspecic binding, the nonphosphopeptides identied with SAX were mainly acidic peptides
whereas IMAC enriched peptides were reported to be more heterogeneous as a result of histidine binding to IMAC. Additionally, the
ability for fractionating phosphopeptides was investigated. Twominute fractions from the liver digest were taken and analyzed by
MALDIMS. This led to the identication of 274 phosphorylation
sites from 305 unique phosphopeptides corresponding to 168 proteins at a false discovery rate of less than 1%. A disadvantage arising
from this method is that the solvents used in SAX chromatography
are not optimal for online LCMS/MS coupling because the weakly
acidic to neutral pH and the aqueous buffer lower the ionization
efciency when LCESIMS is used.
3.5. Hydrophilic interaction chromatography and related
techniques
Beyond partitioning by charge state, hydrophilic interaction
chromatography (HILIC) provides an additional orthogonal separation tool to reversed phase chromatography. In HILIC, analytes
are separated according to their polarity. HILIC is a special form
of normal phase chromatography using mobile phases that are
540% buffered aqueous solutions. In HILIC, the dominant retention
mechanism is claimed to be a partitioning of the analytes between
a polar, water-rich stationary phase layer and an organic modierrich mobile phase. In addition, adsorption phenomena with the
modied silica surface may occur, leading to a mixed-mode

26

A. Leitner et al. / Analytica Chimica Acta 703 (2011) 1930

Fig. 3. Chemoselective approaches targeting the phosphate group. (a) Beta-elimination and Michael addition, (b) phosphoramidate chemistry.

retention mechanism. In general, the more polar the eluent

becomes the more elution strength it has. Typical starting conditions for HILIC separations are up to 95% organic solvent
(acetonitrile). HILIC as part of a multidimensional peptide separation strategy was used for the phosphoproteomic analysis of a
tryptic HeLa cell digest [86]. Phosphopeptides with the highly polar
phosphate group should therefore be strongly retained on the HILIC
stationary phase. IMAC phosphopeptide enrichment was added
before or after the HILIC step to increase selectivity of the setup.
Nearly 100% selectivity for phosphopeptides was achieved when
IMAC enrichment was performed after HILIC chromatography, and
over 1000 phosphorylation sites on 914 peptides were identied,
demonstrating the use of HILIC as a powerful prefractionation tool
before selective phosphopeptide enrichment is performed. Additionally phosphopeptides elute at a buffer composition of 7050%
ACN containing 0.1% TFA which is asserted to be the optimal loading buffer composition for IMAC phosphopeptide loading. Other
groups have recently adopted HILIC in their protocols [87,88].
Somewhat similar to HILIC, electrostatic repulsion-hydrophilic
interaction liquid chromatography (ERLIC), introduced by Alpert
[89], uses electrostatic repulsion as an additional chromatographic
stationary phase property to adjust selectivity in HILIC chromatography. By superimposing the properties of ion exchange and
HILIC chromatography, selectivity can be adjusted by changing
the organic content or the pH of the solvent and additionally by
using a salt gradient. In an elaborate comparative study, ERLIC was
compared to SCX and SCX-IMAC for phosphopeptide enrichment
purposes by Gan et al. [90]. In their approach human epithelial carcinoma A431 cells were analyzed leading to a total identication
of 2058 unique phosphopeptides of which 1801 were identied by
only one of the methods. ERLIC accounted for 38%, SCX-IMAC for
57% and SCX alone for 5% of the phosphopeptide pool. The overlap between ERLIC and SCX-IMAC identied phosphopeptides was
12% showing that for a comprehensive phosphoproteome analysis
a combination of different enrichment strategies is sometimes nec-

essary. Recently, a similar approach was applied to the analysis of

rat kidney tissue [91].
3.6. Chemical modication (Fig. 3)
In addition to chromatography-based methods, tagging phosphate species with certain compounds using a specic chemical
derivatization reaction is another strategy for phosphopeptide
enrichment (reviewed in ref. [92]). Site-specic modication of
phosphoseryl and phosphothreonyl residues [21] using a combination of -elimination and Michael addition is a way to introduce
phosphosite specic tagging. Chait and co-workers used this technology to introduce a biotin tag for biotin/avidin enrichment [93].
Under strongly alkaline conditions the phosphate moiety from
phosphoserine or phosphothreonine undergoes -elimination to
form a dehydroalanyl or -methyldehydroalanyl residue, respectively. These ,-unsaturated residues are Michael acceptors,
which can react with nucleophiles like ethandithiole, which is further coupled to biotin. The benets for this method are that one
can selectively enrich via different types of tags available, and the
tags can be isotope labeled for quantication purposes [93] or carry
functional groups to increase the ionization efciency or to facilitate phosphorylation site determination [22,94,95]. However, some
drawbacks are associated with this procedure. The high pH necessary for efcient -elimination can cause some protein or peptide
degradation. In addition, O-glycosylated serines can be converted
to dehydroalanine as well. These drawbacks can be avoided by
additional protecting reactions or method modications [96] but
this may lead to a more complicated workow and potential sample loss. In addition, only phosphoserine and phosphothreonine
containing peptides can be enriched using -elimination/Michael
addition because tyrosine is not able to form an ,-unsaturated
bond.
Aebersold and co-workers used a carbodiimide-catalyzed
reaction for the reversible capture of phosphate groups on

A. Leitner et al. / Analytica Chimica Acta 703 (2011) 1930

phosphopeptides using phosphoramidate chemistry [97]. Initially,

a laborious six step protocol was developed that only yielded a
moderate number of identications. Later, this method was further
rened and relative quantitation was implemented by the introduction of isotope labeling [98]. The resulting three step strategy
includes the protecting of free carboxylate groups by methylation
with methanolic HCl (which also offers the possibility for the introduction of deuterium labeled methyl groups), the coupling of the
methylated peptides to a dendrimer via their phosphate groups,
catalyzed by imidazole and carbodiimide in a one pot reaction, and
nally the release of the peptides by acidic hydrolysis with 10% TFA.
Using a tandem purication protocol with anti-phosphotyrosine
immunoprecipitation followed by phosphoserine and -threonine
enrichment by implementing the above stated protocol, 80 serine
and threonine phosphorylation sites on 97 tyrosine phosphoproteins from Jurkat T cells could be identied. A later application of a
similar protocol was reported by Bodenmiller et al. when performing an elaborate phosphosite assignment of D. melanogaster KC167
cells [99].
3.7. Ca and Ba precipitation
In 1994 Reynolds et al. used an excess of Ca2+ in 50% ethanol for
precipitating phosphopeptides from a tryptic casein hydrolysate. At
lower pH, only peptides containing multiple phosphoserines were
enriched. At a pH of 8 all phosphopeptides except two monophosphorylated ones could be found in the precipitate [100]. Calcium
phosphate/calcium phosphopeptide coprecipitation in combination with two subsequent IMAC enrichment steps was used when
analyzing the phosphoproteome of rice embryonic cells. Peptides
were mixed with disodium phosphate and ammonia solution followed by the addition of calcium chloride at a pH of 10. In total
242 phosphopeptides representing 125 phosphoproteins could be
identied [101]. Although no tyrosine phosphorylated peptide was
detected in this study, this was attributed to the low abundance of
these in rice, rather than lacking selectivity for it. A similar method
was used for the phosphoproteomic analysis of human brain by Xia
et al. allowing the identication of 551 phosphopeptides with 466
unique phosphorylation sites [102]. Recently, also Ba2+ has been
reported to precipitate phosphopeptides in a study from Yates and
co-workers [103].
3.8. Method comparisons and combined methods
Multiple phosphospecic enrichment techniques can be
employed by combining different methods in parallel to obtain a
more comprehensive view of the phosphoproteome. On the other
hand it is possible to combine them in a serial workow to maximize
selectivity. For example, several groups reported that using TiO2
enrichment preferably singly phosphorylated peptides are isolated,
whereas IMAC enrichment shows a bias towards multiply, predominantly doubly phosphorylated peptides. This may be explained by
the different binding properties and/or some microstructural preferences, but also by the different peptide-to-resin ratios of both
materials [104]. In many recent phosphoproteome studies TiO2 and
IMAC enrichment are carried out in parallel [11,105] utilizing the
different preferences of the methods. A comprehensive comparison of TiO2 , IMAC and chemical (phosphoramidate) enrichment
was carried out by Bodenmiller et al. In their experiments they
obtained good reproducibility within the same method when analyzing repeated isolates but the overlaps of peptide identications
from different methods were rather low (around 30% between all
methods) [105]. In a recent work from our group the phosphoproteome of HeLa cells was screened by combining different metal
oxide protocols (nanocast SnO2 and TiO2 , commercial TiO2 ) in a
parallel setup [74]. In sum, 1595 unique phosphopeptides were

27

identied. The overlap of identications between the methods was

less than one third; almost 140 phosphopeptides were exclusively
identied by SnO2 , but more surprisingly also both titania materials contributed more than 20% unique identications each. These
and other studies indicate that for a complementary coverage of
the phosphoproteome, a combined approach may be necessary.
For two-dimensional fractionation/enrichment schemes, LC
based methods are used in the rst dimension and fractions are collected according to resolving power of the respective method used.
One has to pay attention that the elution conditions used for the rst
dimension do not interfere with the following enrichment step. Primarily high salt concentration or low pH elution conditions, which
may be used to elute peptides from prefractionation columns, can
cause some problems with following enrichment methods. Therefore a desalting step or other sample buffer adjustment may be
required to allow further phosphopeptide enrichment. Trinidad
et al. analyzed the postsynaptic density, a part of the mammalian
central nervous system using SCX or SCX-IMAC phosphopeptide
enrichment [106]. 88 SCX fractions were collected and subjected
either to direct LCMS analysis or to further IMAC enrichment. In
the early fractions the highest numbers of phosphopeptides were
found, but phosphopeptides were identied throughout the whole
fractionation. Thus 311 phosphopeptides were identied with SCX
alone and with the addition of IMAC chromatography a threefold increase of identications could be achieved, resulting in a
total of 998 unique phosphopeptide identications on 287 phosphoproteins out of 1263 proteins which were identied in total
with this approach. Using SILAC peptide labeling in combination
with SCX-IMAC phosphopeptide enrichment and high accuracy
mass spectrometry with MS3 peptide sequencing, double auxotroph yeast strains were analyzed for their pheromone response
by the Jensen group [53]. More than 700 phosphopeptides could be
identied of which 18% were regulated by pheromone response.
Another comprehensive phosphoproteomic approach using SILACquantication, but using SCX-TiO2 enrichment and high resolution
mass spectrometry with multistage activation peptide sequencing
[107] for accurate phosphopeptide identication was performed
by Olsen et al. [13]. In this analysis of EGF-treated HeLa cells, more
than 99% condence of phosphopeptide identications could be
achieved by combining high precursor mass accuracy information
and two stage fragmentation of peptides and 6600 phosphorylation sites on a total of 2244 proteins could be identied. Fourteen
percent of identied phosphorylation sites were found to be modulated more than twofold upon EGF stimulation. SCX fractionation
and a combination of IMAC and TiO2 enrichment with high mass
accuracy peptide identication and SILAC quantication was used
for the analysis of the phosphorylation dependent cell cycle regulation of HeLa cells [11]. Cells were arrested in the G1 or M-phase
of the cell cycle and grown in media supplemented with differently isotope labeled amino acids to be able to distinguish between
the different cell cycle phases. After SCX chromatography, fractions were split into halves and equally enriched with either TiO2
or IMAC. Differently enriched fractions were pooled again and
analyzed by mass spectrometry. The combined setup allowed the
identication of over 14,000 sites of phosphorylation on 3682 proteins with over 1000 proteins showing increased phosphorylation
during mitosis. The authors reported nearly an order of magnitude
increase in the number of sites identied from nocodazole arrested
HeLa cells compared to previous work [10].
Thingholm et al. developed a strategy for the sequential
separation of monophosphorylated peptides and multiply phosphorylated peptides by combining IMAC and MOAC in a sequential
fashion. The strategy, termed sequential elution from IMAC
(SIMAC), uses IMAC in the rst dimension and TiO2 for secondary
enrichment of IMAC ow through and eluates [108]. Peptides were
eluted from IMAC under acidic conditions (1% TFA, pH 1.0)when

28

A. Leitner et al. / Analytica Chimica Acta 703 (2011) 1930

preferably monophosphorylated peptides should elute, followed

by TiO2 enrichment to remove remaining non-phosphorylated
peptides which may coelute from IMAC under these conditions.
Subsequently basic elution with ammonium hydroxide solution
(pH 11.3) is applied to IMAC to recover remaining multiply phosphorylated peptides. Combining the advantages of IMAC and TiO2
it was possible to identify 306 monophosphorylated peptides, 186
multiply phosphorylated peptides and 716 unique phosphosites of
a tryptic digest of human mesenchymal stem cells. These numbers
were in contrast to 232 monophosphorylated and just 54 multiphosphorylated peptides and 350 unique sites when using an
optimized TiO2 protocol alone.
4. Analytical strategies for other classes of phosphorylated
biomolecules
As already mentioned in the Introduction, other phosphorylated
biomolecules such as phospholipids, DNA/RNA, and phosphorylated metabolites play an important role in biological and
pharmaceutical research as well. Several strategies introduced
above have been adapted for the analysis of these analyte classes.
In the following, we will only highlight a few examples.
4.1. The phosphate group as afnity handle for the enrichment of
other PTMs
In a recent interesting application, Parker et al. introduced a phosphate group as an afnity tag for the analysis
of peptides carrying an O-GlcNAc modication (Fig. 4) [109].
Using an established chemoenzymatic labeling technique, the
azidosugar, N-azidoacetylgalactosamine, was attached to the Nacetylglucosamine moiety. The azide group can be reacted with
alkynes in a bioorthogonal 1,3-cycloaddition (click chemistry)
reaction. In this case, cyclohexylammonium pent-4-ynyl phosphate was used as the alkyne probe, enabling the isolation of
O-GlcNAc peptides using titanium dioxide-based MOAC. For a separate enrichment of phosphopeptides and O-GlcNAc peptides, an
enzymatic dephosphorylation step was introduced. It seems feasible to use a similar approach also for other post-translational
modications.
4.2. Phospholipids
Phospholipids (PLs) are the main constituent of the cellular
membrane bilayer but also play an important role in cellular
signaling [110]. Lipidomics provides analytical strategies for the
analysis of lipid metabolism and lipid mediated signaling processes
[111]. Mass spectrometry is currently the method of choice for
straightforward lipidomic analysis [112,113], however, elaborate
separation or specic enrichment is also in this case essential for efcient MS identication. Analytical tools such as gas
chromatographymass spectrometry (GCMS) and thin layer chromatography (TLC) in combination with MALDI-TOFMS can be
used, but HPLCESIMS is currently the most preferred combination for lipidomic analysis [110,111,114].
In general, total lipids are extracted from biological samples like
cells or tissue and extracts are separated by normal phase (on diol
or amino phases) or reversed phase chromatography prior to mass
spectrometric identication [114]. For a more specic enrichment
of phospholipids, MOAC has been employed. This method was rst
introduced by Ikeguchi and Nakamura. In their approach they used
TiO2 for selective enrichment of PLs from egg yolk prior to LC analysis. Recovery rates up to 70% for diverse PLs could be achieved and
the method was also effective for removal of fatty acids and neutral lipids [59]. A further renement of the method was done by
Calvano et al. [115]. TiO2 was packed into frits allowing a fast and

Fig. 4. Enrichment O-GlcNAc-peptides by introduction of a phosphate group as

afnity handle after chemoenzymatic attachment of N-azidoacetylgalactosamine.
Adapted from ref. [109].

uncomplicated SPE enrichment of PLs from dairy products. Similar to the protocol of Larsen for phosphopeptide enrichment they
used DHB to reduce unspecic binding from non-phosphorylated
lipids. Only PLs could be identied in the elution fraction, proving the high selectivity for PLs of the method. In a recent work
from our group, ZrO2 packed into SPE cartridges was used to selectively isolate phosphatidylcholines (PCs) from natural samples like
milk, or human or mouse plasma [116]. Recovery rates up to 100%
were achieved using an optimized extraction, incubation and elution protocol. Using the protocol optimized for ZrO2 , TiO2 and SnO2
were also tested for PC enrichment, however ZrO2 performed superior compared to other materials tested.
4.3. Nucleotides and nucleosides
The negatively charged phosphate diester backbone of DNA can
also serve as a ligand for purication with phosphoafne enrichment materials. Due to the advancements of gene therapy and
genetic vaccines during the last decade, the demand for highly puried plasmid DNA (pDNA) is still increasing [117]. pDNA is mainly
produced by recombinant E. coli fermentation, so for research and
clinical applications pDNA has to be puried and separated from
genomic DNA, RNA, remaining proteins and endotoxins. Secondly,
only the super coiled conformation of pDNA (sc pDNA) is biologically active and hence has to be separated from other isoforms

A. Leitner et al. / Analytica Chimica Acta 703 (2011) 1930

[118]. Therefore chromatographic methods such as size exclusion, hydrophobic interaction, hydroxyapatite, reversed phase,
or thiophilic adsorption and afnity chromatography, mostly in
combination with ion exchange chromatography as the second
dimension are used [119]. To date, no phosphate specic method
for pDNA purication was reported, although Sousa et al. reported
the complete separation of sc pDNA and open circular pDNA by
arginine afnity chromatography. The specic recognition was
described to be the result of multiple interactions between arginine and pDNA, including electrostatic interaction with the pDNA
phosphate backbone and also some degree of biorecognition of
nucleotide bases by the arginine ligand [120]. A similar recognition
principle has been proposed for phosphoproteomic applications as
well, using polyarginine or poylethyleneimine materials [121,122].
RNAprotein interactions have been probed by Urlaub and
co-workers using UV-induced cross-linking and enrichment of
interacting peptide-RNA oligonucleotide fragments using an integrated two-dimensional LC set-up that included a TiO2 column for
afnity enrichment of the conjugates [123].
These examples show that also phosphate group-containing
analytes other than proteins and peptides need to be isolated, identied and quantied in low concentrations in a complex biological
matrix. Therefore, specic enrichment methods for phosphorylated
biomolecules that were initially developed for phosphoproteome
analysis may help to overcome limitations which currently hamper
their effective LCMS analysis.
5. Conclusions and outlook
The biological relevance of the phosphate group has led to the
development of various analytical tools to probe biomolecules carrying this functional group. Mass spectrometry plays a central
role in these analytical workows but researchers need to employ
prefractionation and enrichment methods to deal with the sample complexity in biological systems. In this respect, established
technologies continue to be rened while promising emerging
protocols are quickly adopted. Although impressive results have
already been obtained using available tools, the future will certainly see the development of additional ones. We expect that
the increased emphasis on quantitative workows in phosphoproteomics will put the robustness and reproducibility of the complete
protocol, from sample preparation to nal MS analysis, into the
focus.
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