L.B. Areces, V. Matafora and A. Bachi, Eur. J. Mass Spectrom. 10, 383–392 (2004)
383
Analysis of Protein Phosphorylation by Mass Spectrometry
L.B. Areces, V. Matafora and A. Bachi, Eur. J. Mass Spectrom. 10, 383–392 (2004)
Review
Analysis of protein phosphorylation by mass
spectrometry
Liliana B. Areces
European Institute of Oncology, Via Ripamonti 435, 20141 Milan, Italy
Vittoria Matafora and Angela Bachi*
Dibit, San Raffaele Scientific Institute. Via Olgettina 58, 20132 Milan, Italy. E-mail: [email protected]
Phosphorylation is one of the most frequently occurring post-translational modifications in proteins. In eukaryotic cells, protein
phosphorylation on serine, threonine and tyrosine residues plays a crucial role as a modulator of protein function. A comprehensive
analysis of protein phosphorylation involves the identification of the phosphoproteins, the exact localization of the residues that are
phosphorylated and the quantitation of phosphorylation. In this short review we will summarize and discuss the methodologies currently available for the analysis and full characterization of phosphoproteins with emphasis on mass spectrometry-based techniques.
In particular, we will discuss affinity-based purification of phosphopeptides coupled to matrix-assisted laser desorption/ionization
time-of-flight mass spectrometry (MALDI ToF-MS) analysis, their detection using mass mapping and precursor-ion scans, identification of modified sites by tandem mass spectrometry (MS/MS) and quantitative analysis.
Keywords: phosphoprotein, post-translational modifications, mass spectrometry, proteomics, quantitative analysis
Introduction
Characterization of protein modifications is an important aspect of the analysis of protein structure, localization
and function. In this context, protein phosphorylation is of
major relevance as it is estimated that approximately one
third of all proteins in eukaryotic cells is, at any one time,
phosphorylated.1
Protein phosphorylation has been shown to play an
essential role in a variety of fundamental cellular functions
such as gene transcription, cell-cycle progression, cell division and proliferation, cell differentiation, energy storage
and apoptosis.2–4 Therefore, the identification of phosphoproteins, the exact localization of the phosphorylated residues and the quantitation of the modification level are essential in the understanding, at the molecular level, of a
biological pathway. In these last years, mass spectrometry
has become the most powerful technique for protein identification and analysis of post-translational modifications.5,6
However, the comprehensive analysis of phosphoproteins
has encountered some biological and analytical limitations.
DOI: 10.1255/ejms.601
Phosphorylation is often sub-stoichiometric and highly
dynamic, meaning that phosphoproteins are highly heterogeneous and only a small fraction of the protein is modified.
Moreover, many phosphoproteins are physiologically
expressed in low abundance and they require a specific
enrichment. From the analytical point of view, several factors make the analysis of phosphorylated proteins nonstraightforward. First, negatively-charged modifications can
hinder proteolytic digestion by trypsin; second, the sequence
near the phosphorylation site is usually very hydrophilic,
leading to a selective loss of phosphopeptides during the
standard methods used for sample preparation; third, the
mass spectrometric response of a phosphopeptide may be
suppressed by the presence of other peptides in a complex
mixture and, finally, phosphoserine and phosphothreonine
are labile and may decompose during the analysis.
As a result of this, specific strategies for the analysis of
protein phosphorylation have been proposed and will be
discussed in this review. In general, they can be divided into
two different approaches, one aimed at the direct analysis of
the modified proteins by using a specific method for the
ISSN 1356-1049
© IM Publications 2004
384
detection of the phosphorylation site, the other involving a
purification or enrichment of the phosphopeptides before
the analysis.
Mass spectrometric detection of phosphorylation
Once a protein has been isolated, several techniques can
be used to identify and localize the modified amino acids. In
some cases, the precise measurement of the molecular
weight of the intact protein, using mass spectrometry, can
give an idea of the average number of modified residues
present on the protein, just by looking at the increment of the
protein mass in comparison with the unmodified one. To
better characterize the phosphorylated residues of the
protein, however, it is necessary to analyze the peptides
generated by enzymatic or chemical degradation of the
protein. Ideally, every phosphopeptide should be detected.
In practice, however, it is difficult to obtain a 100%
sequence coverage of the protein, and this may limit identification of the region of interest. To overcome these limitations, different specific biochemical and mass spectrometric
techniques have been developed.
Phosphopeptide analysis by matrix-assisted laser
desorption/ionization time-of-flight mass spectrometry
(MALDI ToF-MS)
The utility of MALDI ToF-MS for protein modifications analysis relies on its ability to perform accurate mass
determination of peptide mixtures, its high sensitivity and
resolving power and its relative tolerance to salt-containing
buffers.7–10 The study of phosphopeptides' behavior in
MALDI ToF-MS analysis is a key step for their recognition.
Phosphopeptide mapping requires the proteolytic digestion
of a protein of interest combined with database search.11
The localization of the phosphopeptides can be carried
out by searching for peptides whose mass is shifted by multiples of 80 Da (corresponding to the presence of a phosphate
group) from the predicted one. In the MALDI ToF reflectron
spectrum, the non-phosphorylated form is present, as well as
the phosphorylated form, as result of post-source decay
(PSD).12 PSD is a process where specific ions, called
metastable ions, decompose in the flight tube because they
are not sufficiently stable. In a ToF mass spectrometer operated in the linear mode, metastable ions and precursor ions
move with the same velocity, maintaining the same initial
energy, and arrive simultaneously at the detector, therefore
showing the same m/z value in the spectrum.13 In a ToF mass
spectrometer operated in the reflectron mode, the ions are
instead energy-focused towards the detector by the ion mirror; thus, metastable ions have a decreased flight time relative to the precursor ion and are detected at a lower apparent
mass. This leads to a lower sensitivity for the phosphorylated peptides. In the case of serine and threonine
phosphorylated peptides, the most common fragmentations
are due to the loss of H3PO4 and HPO3. The fragment ions
+
+
(MH – H3PO4) and (MH – HPO3) do not appear in the spectrum at the exact masses because they are not properly
Analysis of Protein Phosphorylation by Mass Spectrometry
focused by the mirror, since the reflectron is calibrated for
the precursor ions. Our experience shows that, in the case of
serine and threonine phosphorylation, the more abundant
+
ion observed is (MH – H3PO4) . Conversely, the loss of
H3PO4 is not favored in the case of phosphotyrosine because
the phosphate moiety is stabilized by the aromatic ring. As a
result of these considerations, the reflectron spectra of
phosphotyrosine rarely display this ion.
As mentioned before, the study of phosphopeptide
behavior in MALDI ToF-MS analysis is crucial for their
investigation; in fact, the presence of the metastable ion can
be used as a trace for the presence of phosphorylated
peptides. In addition, looking at the spectra acquired in both
linear and reflectron modes, the disappearance of the
metastable ion in the spectrum acquired in the linear mode,
and the increase of the relative intensity of the phosphorylated peak, confirm the presence of the phosphate moiety on the peptide (Figure 1). Moreover, comparing the
spectra before and after treatment of the sample with
alkaline phosphatase can further help in the analysis.14–16 As
is clear in Figure 1, after alkaline phosphatase treatment the
peaks belonging to the phosphorylated peptide and to the
metastable ion disappear, whereas the peak belonging to the
dephosphorylated form shows an increase in intensity. A
comparison of the spectra recorded in both linear and
reflectron modes, followed by phosphatase treatment can,
therefore, be a useful tool to confirm that the observed peptide is indeed phosphorylated. A major limitation of MALDI
ToF-MS is the analysis of complex peptide mixtures where
phosphorylated peptides are poorly detected, due mainly to
the suppression effect and to their instability.
Analysis of phosphorylation sites by tandem mass spectrometry (MS/MS)
Tandem mass spectrometry (MS/MS) is based on the
fact that, under particular experimental conditions, phosphopeptides give rise to trace fragments that can be
specifically detected. In fact, the side chains on phosphoserine, phosphothreonine and phosphotyrosine can fragment easily on both sides of the phosphoester bond under
collision-induced dissociation (CID) conditions, producing
phosphate-specific fragment ions, also called “reporter
ions”. In this way, peptides carrying a phosphate group can
be identified by the neutral loss of H3PO4 (– 98 Da) in the
positive-ion mode, or because they produce a 79 Da ion
–
(PO3 ), easily detectable in the negative-ion mode.
Precursor-ion scanning
During a precursor-ion scanning experiment, the mass
spectrometer is set to detect only those peptides that produce
a specific ion, diagnostic of a modified residue, thus allowing the selective identification of the peptide carrying the
modification in a complex peptide mixture. The precursorion scanning for ions producing the 79 Da fragment in the
negative-ion mode has proven to be the best method for the
specific detection of native phosphorylated peptides.17,18 It
L.B. Areces, V. Matafora and A. Bachi, Eur. J. Mass Spectrom. 10, 383–392 (2004)
385
Figure 1. Phosphopeptide identification by MALDI ToF-MS mapping combined with alkaline phosphatase treatment. Panel (a)
shows the MALDI ToF spectra of a tryptic digest of MEF2C (myocite enhancer factor) protein. The phosphorylated peptide, indicated with the symbol ˜, is shifted by 80 Da relative to the predicted non-phosphorylated peptide, indicated with ¡, and increases
its relative intensity in the spectrum acquired in linear mode. The metastable ion, indicated with *, clearly evident in the spectrum
acquired in reflectron mode, disappears. The peak T is a tryptic peptide. Panel (b) shows the MALDI ToF spectra of the same sample
treated with alkaline phosphatase: the disappearance of the phosphorylated peptide and the relative increase of the intensity of
the non-phosphorylated peak confirm the identity of the phosphorylation.
combines high selectivity and good sensitivity and is applicable to serine, threonine and tyrosine phosphorylated peptides. This method has, for example, been successfully used
by Annan et al., in their multi-dimensional phosphopeptide
analysis to map the in vivo and in vitro phosphorylation sites
of Sic1p, a regulator of G1/S transition in budding yeast.18
The multi-dimensional phosphopeptide mapping strategy
consists of three different steps. In the first run, phosphopeptides present in a proteolytic digest are selectively
detected by monitoring the specific marker ion (m/z 79) in
the negative-ion mode and are collected into fractions. The
collected fractions are then analyzed off-line by nanoelectrospray ionization (ESI) mass spectrometry and, in this
simplified mixture, phosphopeptides are detected by precursor-ion scanning. Finally, the peptides are sequenced by
MS/MS and the phosphorylation site is localized. This strategy was further modified to improve its sensitivity, allowing
routine phosphopeptide analysis of in-gel digested proteins.19 The weak point is the necessity to work in negativeion mode which makes it incompatible with on-line separation methods, based on reversed-phase liquid chromatography, and makes it very complicated to get sequence information from the MS/MS analysis.
Recently, a new precursor-ion scanning technique that
can be performed in positive-ion mode, useful for the specific detection of phosphotyrosine-containing peptides, has
20,21
This method is based on the ability to
been developed.
selectively detect the immonium ion of phosphotyrosine that
has an m/z value of 216.043. Using newer high-resolution
instruments, such as those operating on the quadrupole (Q)ToF principle (with a Q2-pulsing function in the case of a QStar instrument), this immonium ion can be easily distinguished from other peptide fragment ions. Once the phosphotyrosine-containing peptides are identified, they can be
selected, fragmented and sequenced in the product-ion
MS/MS mode without any need for switching polarity of the
ion source. This scanning method is able to work with subpicomole amounts of gel-separated proteins but is applicable
only to tyrosine phosphorylation sites. This method was successfully used by Steen et al. as a proteomics strategy to
study the proteins involved in the EGF signalling pathway
and to localize the phosphorylation sites.22 Because of its
high sensitivity and selectivity, this approach is likely to
become one of the most powerful tools in proteomics to
study tyrosine phosphorylation in signal transduction pathways.
386
Neutral-loss scanning
This method, which uses MS/MS to detect the neutral
loss of 98 Da (H3PO4) after CID, has been applied by
Huddleston et al. to mixtures of peptides and phosphopeptide containing the three commonly phosphorylated
amino acids (serine, threonine and, with lower efficiency,
tyrosine).23
The limitations are the low sensitivity and the fact that
the charge state of the phosphopeptide has to be known in
advance; therefore, it has not seen much use for the analysis
of unknown samples.
Recently, MacCoss and co-workers in Yates’
laboratory24,25 described a combination of on-line multidimensional liquid chromatography and new software
algorithms that enable identification of the charge state of
multiply-charged peptides, deletion of spectra of poor
quality and identification of spectra containing a prominent
98 Da (–H3PO4) ion from neutral loss from the precursor.
With this new algorithm, the identification and localization
of protein phosphorylation in highly complex mixtures
derived from a tissue total digest was automatically
achieved.
Phosphorylation site analysis by nano-liquid chromatography
(LC)-MS/MS
One of the best ways to decrease the complexity of samples is to perform a liquid chromatography (LC) separation
26
prior to mass spectrometry analysis. Typically, the peptide
mixture is loaded on a nano-column containing reversedphase C18 material, where separation occurs and the separated components are eluted directly into a tandem mass
spectrometer that works in data-dependent acquisition
mode. Using a variation of classical LC, the 2D chromatographic (MudPIT)27 separation, coupled to MS/MS analysis
and database search by a specific SEQUEST algorithm,
Washburn et al. were able to identify 1,848 proteins, including low abundance proteins like transcription factors and
protein kinases.28 This method, which uses in-house fabricated nano-columns [with reversed-phase C18 and strong cation-exchange (SCX) materials], where peptides are
separated and eluted at very low flow, has been successfully
used by MacCoss et al.24 for the analysis of post-translational
modifications, including phosphorylation, in highly complex mixtures, as we have already described.
All these methods take advantage of the reduced suppression effect observed for phosphopeptides when present
in a complex mixture and of the possibility of a full automation of the nano-LC system, therefore seeming more appealing in a large-scale phosphoproteome analysis.
Electron capture dissociation (ECD) mass spectrometry for the
direct characterization of phosphorylation sites
One of the latest developments in the analysis of proteins and their post-translational modifications is electron
capture dissociation (ECD) coupled with Fourier transform
ion cyclotron resonance (FT-ICR) mass spectrometry. This
Analysis of Protein Phosphorylation by Mass Spectrometry
technology has been successfully applied to the analysis of
phosphorylated peptides and has the advantage of giving
less complex spectra in comparison with the conventional
29
CID spectra. No loss of water, phosphoric acid or phosphate from the parent peptide or its fragment ions is
observed, giving rise to a complete or near-complete amino
acid sequence of the phosphopeptides analyzed. Recently,
Shi et al. reported the use of ECD for the direct characterization of phosphorylation sites in a heterogeneous phosphoprotein, -casein, without any prior degradation, such as
proteolysis.30 This possibility, to directly analyze and
sequence intact proteins, is one of the most promising techniques for the future, as it is likely to provide a real picture of
the phosphorylation status of the protein.
Enrichment of phosphorylated proteins
As mentioned before, only a fraction of the proteins is
phosphorylated at any given time; therefore, major
challenges in phosphoprotein analysis are first the isolation
of the modified protein, and then the isolation or enrichment
of the phosphopeptides from the overwhelming amount of
nonphosphorylated peptides present in a protein total digest.
Phosphospecific antibodies
Although tyrosine phosphorylation in mammalian cells
is far less frequent than serine/threonine phosphorylation, it
has been studied quite intensively, mainly because of the
availability of excellent antibodies that recognize phosphorylated tyrosine residues in immunoprecipitation as well
as in Western blotting experiments.31–34
The major limitation for studying serine- and threoninephosphorylated proteins (i.e. the lack of availability of
immuno-precipitating antibodies) has been recently overcome by Grønborg et al.35 Working with a new set of antibodies directed against phosphoserine and phosphothreonine residues, they have shown that these antibodies
can be used specifically for enrichment of phosphoserineand phosphothreonine-containing proteins in a global
approach, as has already been done in the case of tyrosine
phosphorylation involved in signaling pathways.36
Chemical modification of phosphorylation sites
Since the phosphate moiety on serine or threonine
amino acids is labile under alkaline conditions (-elimination), it can be replaced by an alternative chemical group (by
Michael addition) that can be used as an affinity handle or as
a reporter ion for the recognition of phosphorylated peptides
(Figure 2).
In the classical Oda protocol, the phosphopeptides are
exposed to high pH in the presence of ethanedithiol.37 The
loss of H3PO4 by -elimination produces an , -unsaturated
residue that is a Michael acceptor, which can readily react
with the nucleophile ethanedithiol. In the next step, biotin is
attached to the thiol group via a sulphydryl-reactive group,
and, finally, the biotin tagged peptides are isolated by affinity chromatography on avidin resin. To avoid side reactions
L.B. Areces, V. Matafora and A. Bachi, Eur. J. Mass Spectrom. 10, 383–392 (2004)
387
Figure 2. Scheme for chemical conversion of a phosphoserine residue to a biotinylated residue based on -elimination. In basic conditions, the phosphate moiety on phosphoserine or phosphothreonine undergoes -elimination and can be replaced, by Michael
addition, with an alternative chemical group. The specific fragment of m/z 446 produced under CID conditions can be used as a
reporter ion for the MS/MS spectra.
on the cysteine thiol groups, alternative methods have been
proposed to block them. Performic acid oxidation is preferred over alkylation because the alkylated Cys may
undergo -elimination in a fashion similar to phosphoserine
38
and phosphothreonine. The main disadvantages of this
chemical modification method are that it is not applicable to
tyrosine phosphorylation, and that the yield from -elimination tends to be sub-stoichiometric.
Using an alternative chemistry, Zhou et al. modified
phosphopeptides by attachment of a cystamine group to the
phosphate moiety using a carbodiimide condensation reaction.39 The modified peptides are then covalently attached to
iodoacetyl resin and their elution is performed by treatment
with trifluoroacetic acid. The new aspect of this chemically
laborious methodology is that it can be applied to phosphotyrosine-containing peptides and not only to those containing phosphoserine and phosphothreonine residues;
however, it requires several chemical reactions and purification steps before mass spectrometry analysis, which can
lead, as usual, to substantial losses.
As is generally the case, an approach based on chemical
modification methods requires large amounts of sample,
with the result that only abundant proteins can be analysed.
In an effort to improve the sensitivity and the specificity of
these approaches, Steen and Mann described a new strategy,
where they applied -elimination/Michael addition reactions to introduce a functional group at the original site of
phosphorylation, giving rise to a dimethylamine-containing
sulfenic acid derivative with a unique m/z value of 122.06
upon low-energy CID.40 This enables the detection of
phosphorylated species within peptide mixtures by sensitive
and specific precursor-ion scanning in the positive-ion
mode. Working under acidic conditions in the positive-ion
mode has the added advantages that peptide sequencing, for
the exact localization of the phosphorylated residues, can be
performed directly, it can be combined with LC-MS/MS
experiments, and it allows the use of isotopically-labeled
derivatization agents for relative quantitation of phosphorylation states.
Chromatographic enrichment of phosphopeptides
As discussed before, in order to reduce the suppression
effects and enhance phosphopeptide detectability, several
methods have been investigated to specifically isolate
phosphopeptides from crude mixtures. Among these, the use
of immobilized metal ion affinity chromatography (IMAC)
has been shown to be useful in both MALDI and ESI mass
spectrometry and can also be combined with nanoelectrospray MS/MS for phosphopeptide purification and
characterization at picomole and sub-picomole levels.41–44
3+
3+
Immobilized metal ions, usually Fe or Ga , bind with
high specificity to phosphoproteins and phosphopeptides,
because of the affinity of the metal ion for the phosphate
moiety.45,46 Recently, Tempst et al. reported that Ga3+ shows a
47
better selectivity for phosphopeptides. These metal ions are
immobilized on a chelating resin: iminodiacetic acid (IDA)
or nitrilotriacetic acid (NTA). Some groups reported that
IDA resin is less specific than NTA,48 others reported little
differences in the discrimination and selective binding of
47
singly- or multiply-phosphorylated peptides. The elution
of the phosphopeptides from the column is then performed
using high pH or phosphate buffer, which requires a further
step of desalting before mass spectrometry analysis.
Even though this technique can be used successfully
with both on-line and off-line mass spectrometry analysis,
there are several limitations that include possible losses of
phosphopeptides during the elution from the column and the
presence of interfering peptides deriving from acidic nonphosphorylated peptides that show affinity for the immobilized metal ions. Several studies were carried out to minimize the loss of sample from the elution step. Zhou et al.48
reported direct MALDI analysis of phosphopeptides bound
to the IMAC support. Other methods use IMAC coupled to
49
HPLC on line to mass spectrometry analysis. Chin et al.
investigated a strategy to reduce loss of small hydrophilic
peptides that are recovered in the flow-through fraction
using standard HPLC methods. They used a porous graphitic
carbon (PGC) column for desalting peptides and demonstrated that this column can be an alternative approach for
the separation of hydrophilic, phosphorylated peptides from
their non-phosphorylated counterparts.50 For the same purpose, Larsen et al. explored the use of graphite powder
packed in GELoader tips. They showed the usefulness of
this microcolumn for desalting and concentration of peptide
mixtures and its enhanced capability in retaining small
and/or hydrophilic peptides, including phosphorylated pep-
388
Analysis of Protein Phosphorylation by Mass Spectrometry
51
tides. Ficarro et al. developed a method for phosphopeptide
isolation, which is based on the esterification of the
carboxylic acid group of the peptides from the tryptic mixture. This peptide derivatization seems to improve the IMAC
selectivity for phosphopeptides and to eliminate the coenrichment of acidic peptides, leading to a more generally
efficient method.52–54
Quantitation of protein phosphorylation
As discussed before, phosphorylation is very dynamic
so that changes in the level of modification, more than
changes in protein expression, are often responsible for the
biological effect. To gather this kind of information, several
efforts have been made to develop reliable, quantitative
methods for the study of phosphorylation. The techniques
that, typically, have been used for quantitation of phosphorylation are phosphoamino acid analysis or Edman degradation after 32P incorporation.55,56 These are not highthroughput procedures and often require handling of high
Figure 3. Analysis of phosphorylated proteins.
amounts of radioactivity. More recently, Oda et al.
described a mass spectrometry-based method for determining the relative changes in the levels of phosphorylation at
specific sites on individual proteins. Accurate quantitation is
achieved through the use of whole-cell stable isotope label15
ing, by growing two different cell populations, one in N14
labeled and the other in N-labeled media. The cell pools are
combined and the proteins of interest are extracted and separated by SDS-PAGE. Gel spots of interest are excised and
digested as usual, and the resulting peptide fragments are
analyzed by mass spectrometry.57 Peptides from the two cell
pools that either remain unmodified, or do not undergo a
change in the level of modification, yield pairs of peaks with
a fixed ratio of intensities, whereas peptides that undergo a
change in their level of modification yield pairs of mass
spectrometry peaks with intensity ratios that are used to
quantitate these changes.58 A further development of this
strategy uses phosphoprotein-specific isotope-coded affin37,59,60
introduced by a -elimination reaction
ity tags (PhIAT),
L.B. Areces, V. Matafora and A. Bachi, Eur. J. Mass Spectrom. 10, 383–392 (2004)
and allowing the simultaneous purification and quantitation
of peptides containing phosphoserine and phosphothreonine
residues. As an alternative, Weckwerth et al. reported a similar approach where they replaced the phosphate group on
serine and threonine residues by -elimination and Michael
addition of ethanethiol or d5-ethanethiol.61 The relative
quantitation of phosphorylation of proteins under two different conditions is, once again, achieved by comparison of the
peak intensities from the hydrogen- and deuterium-containing peptides. As pointed out, however, these methods are
suitable for phosphoserine- and phosphothreonine-containing peptides only and require several chemical reactions and
purification steps, which might at the end lead to poor yields.
They are, therefore, unlikely to be used in a large-scale
phosphoproteome analysis. In an alternative approach,
Bonenfant et al. devised a method which consists of a combination of three simple steps: a proteolytic digestion in
16
18
H2 O or H2 O to tag two different phosphoprotein pools, an
affinity selection of the phosphopeptides from the combined
pools by IMAC, and a dephosphorylation with alkaline
phosphatase to allow for quantitation by MALDI ToF mass
spectrometry of the changes of modification occurring in a
given phosphopeptide.62
Conclusion
As it is clear from the above discussion, there is no
single analytical method for phosphoprotein characterization.63 The choice of the most appropriate technique strongly
depends on the nature of the sample. The abundance of the
protein of interest, the nature of the phosphorylated sites
(whether phosphoserine, phosphothreonine or phosphotyrosine) and the degree of the sample purification are all
relevant factors for the success of the analysis (Figure 3).
Although with some limitations, mass spectrometry has
been shown to be the technique of choice for this purpose.
The combination of mass spectrometry techniques with
enzyme treatment and affinity-based purification is central
to achieve robust and reliable phosphopeptide mapping. As
described in this short review, MALDI ToF-MS enables
protein identification and phosphopeptide investigation with
high sensitivity, whereas MS/MS provides specificity of
phosphopeptide detection and localization of the modified
sites using precursor- and product-ion analysis, respectively. Even if these techniques have given encouraging
results in the characterization of protein phosphorylation,
further developments are still needed to fully characterize all
phosphorylated proteins in a proteome, especially if they are
present in small quantities. Challenges for the future include
developing a method to ensure that all sites of
phosphorylation are detected and accurately quantitated. In
this context, improvement of enrichment strategies and
quantitation of phosphorylation are the main goals for the
future. Going towards this direction, more and more information will be available for the full characterization of the
389
biological processes in which phosphoproteins are functionally involved.
Acknowledgement
This work is supported by Italian MIUR FIRB Grant
(project RBNE01JFFA-002) and Italian Health Ministry RF
Grant (No. 185 2001).
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Received: 3 April 2003
Revised: 26 August 2003
Accepted: 2 September 2003
Web Publication: 3 December 2003
392
Analysis of Protein Phosphorylation by Mass Spectrometry
Biographies
Liliana B. Areces
1983 Degree in Biochemistry, National University of Rosario (Argentina). Clinical trial in the
Hematololy-Oncology section (Department of Clinical Biochemistry). 1990 PhD in Biological Chemistry at the University of Buenos Aires, Argentina. From 1991 to 1992 post-doctoral fellow in Feldman’s
laboratory at Maryland University, Baltimore (USA). From 1992 to 1995 Fogarty Visiting Fellowship
awarded by National Cancer Institute – National Institute of Health (NIH), Bethesda (USA) in
Blumberg’s laboratory. In 1995 senior research fellow in Di Fiore’s laboratory at the European Institute
of Oncology (IEO), Milan (Italy). In 1998 Supervisor of Peptides Synthesis and Protein Identification
Unit at the Department of Experimental Oncology, (IEO). From 2000 to June 2003 ,worked in a collaboration program at the Proteomics Facility at DIBIT – Scientific Park San Raffaele, Milan, Italy.
Vittoria Matafora
1999 Degree in Chemistry at Federico II University of Naples, Italy. Research activity at the
Organic and Biological Chemistry Department, (prof. Gennaro Marino), Federico II University of
Naples. 2000 Fellow in the Cell Adhesion Unit (Prof. Ivan De Curtis) in department of Molecular
Biology and Functional Genomics, DIBIT, S.Raffaele Sscientific Institute, Milan. From 2001 fellow in
the Biological Mass Spectrometry Unit (Dr. Angela Bachi) DIBIT, S.Raffaele Scientific Institute,
Milan, IItaly.
Angela Bachi
1990 Degree in Pharmaceutical Chemistry & Technology, University of Genoa, Italy. 1991
Degree in Pharmacy, University of Genoa, Italy. 1996 PhD in Pharmacology at the Mario Negri Institute for Pharmacological Research, Milan, Italy. 1997-1998 Postdoctoral fellow in Matthias Mann
group, at the EMBL, Heidelberg, Germany. 1998-1999 Postdoctoral fellow in Matthias Wilm group, at
the EMBL, Heidelberg, Germany. From 2000 group leader of the Biological Mass Spectrometry Group
a DIBIT, S.Raffaele Scientific Institute, Milan, Italy.