Download Proteomics studies of post-translational modifications in plants

Journal of Experimental Botany, Vol. 57, No. 7, pp. 1547–1551, 2006
Plant Proteomics Special Issue
doi:10.1093/jxb/erj137 Advance Access publication 21 March, 2006
Proteomics studies of post-translational modifications in
plants
Sun Jae Kwon1, Eun Young Choi1, Yoon Jung Choi2, Ji Hoon Ahn1 and Ohkmae K. Park1,*
1
School of Life Sciences and Biotechnology, Korea University, Anam-dong, Seongbuk-gu, Seoul 136-701, Korea
2
Department of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju 500-712, Korea
Received 31 August 2005; Accepted 25 January 2006
Abstract
Post-translational modifications of proteins greatly increase protein complexity and dynamics, co-ordinating
the intricate regulation of biological events. The global
identification of post-translational modifications is a
difficult task that is currently accelerated by advances
in proteomics techniques. There has been significant development in sample preparation methods
and mass spectrometry instrumentation. To reduce
the complexity and to increase the amount of modified
proteins available for analysis, proteins are usually
subjected to prefractionation such as chromatographic purification and affinity enrichment. In this review,
the post-translational modification studies in plants
are summarized. The sample preparation strategies
applied to each study are also described. These include affinity-based enrichment methods, immobilized
metal affinity chromatography and immunoprecipitation
used for phosphorylation and ubiquitination studies,
respectively, and the phase partitioning approach for
glycosylphosphatidylinositol modification studies.
Key words: Glycosylphosphatidylinositol, phosphorylation, plant,
post-translational modification, proteomics, ubiquitination.
Introduction
With the completion of genome sequencing projects and
the development in analytical methods for protein characterization, proteomics has become a major field of functional genomics. The initial objective of proteomics was
the large-scale identification of all protein species in a cell
or tissue. The applications are currently complicated by the
need to analyse the various functional aspects of proteins that undergo post-translational modifications (PTMs).
PTMs are covalent processes modifying the primary
structure of proteins in a sequence-specific way that includes the reversible addition and removal of functional
groups by phosphorylation, acylation, glycosylation, nitration, and ubiquitination (Mann and Jensen, 2003; Seo and
Lee, 2004). These modifications induce structural changes
in proteins, and modulate the activities, subcellular localization, stability, and interactions with proteins and other
molecules. PTMs of proteins thus largely increase protein
complexity and dynamics, resulting in the intricate regulation of biological events.
Despite the pivotal roles of PTMs in cellular functions, studies on PTMs have not really been feasible until
recently. The identification of PTMs requires large amounts
of proteins and a highly sensitive method for their detection. More than 300 different types of PTMs have
been identified and new ones are regularly added to the
list (Jensen, 2004). A single protein frequently presents a
heterogeneous population of proteins with different PTMs
at multiple sites that are transient and dynamic in nature and
thus provide a very small amount of proteins in a certain
modification state when isolated. The complexity can be
reduced by using strategies such as affinity enrichment and
chromatographic fractionation.
Two-dimensional gel electrophoresis (2DE) has been
widely applied to proteomic analysis of PTMs. Once the
proteins have been prefractionated, based on the PTMs to
be identified, it frequently provides sufficient amounts so
that protein species in different modification states can be
resolved in the gel and subjected to an analysis of the
modifications. The modified proteins can be visualized in
the gel by specific methods suitable for different PTMs
(Patton, 2002). For phosphorylated proteins, autoradiography of incorporated 32P or 33P and western analysis with
antiphosphoamino acid antibodies are generally used for
* To whom correspondence should be addressed. E-mail: [email protected]
ª The Author [2006]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
For Permissions, please e-mail: [email protected]
1548 Kwon et al.
detection (Luo and Wirth, 1993; Gronborg et al., 2002;
Astoul et al., 2003). Phosphoproteins may also be specifically recognized in gels through the alkaline hydrolysis
of phosphate esters or phosphatase treatment (Debruyne,
1983; Yamagata et al., 2002). Similarly, radioactive or
non-radioactive staining methods have been devised for
the detection of glycoproteins, proteolytic modifications,
nitrosylation, and methylation (Patton, 2002).
Once modified proteins have been identified, PTMs of
the proteins are subsequently characterized by mass spectrometry (MS) analysis including matrix-assisted laser
desorption/ionization time-of-flight (MALDI-TOF) MS,
electrospray ionization (ESI) tandem mass spectrometry
(MS/MS), and liquid chromatography (LC) MS/MS (Jensen,
2004). Enzymatic or chemical digestion of the isolated
proteins releases peptides, and the modified peptides can be
collected by chromatographic methods or affinity purification. Isotope labelling is helpful for the detection of the
modified peptides. It is conceptually simple to localize
the modifications since modifications either increase or
decrease the molecular mass of the modified amino acid
residue. However, there are many technical challenges such
as ion suppression, purity and instability of the modified
peptides, sequence coverage, and quantification that together make the mapping of PTMs difficult (Mann and
Jensen, 2003). The development of chemical tagging and
enrichment methods, in combination with an advance in
MS instrumentation, would improve the sensitivity and
accuracy for the determination of PTMs.
In contrast to the progress made in animal proteomics
on PTMs, there have been relatively few studies in
plants, limited only to such fields of PTMs as phosphorylation, GPI modification, and ubiquitination. In this
review, the PTM studies in plants are summarized. The
sample preparation methods applied to each study is also
described.
Phosphorylation
Protein phosphorylation is one of the most important and
best characterized PTMs regulating cellular signalling
processes. In studies of the plant PTMs, phosphoproteins
in chloroplast thylakoid and plasma membranes have been
the major targets. Commonly, the surface-exposed regions
of the thylakoid and plasma membrane proteins were
first shaved for analysis and immobilized metal affinity
chromatography (IMAC) was used for enrichment of phosphoproteins (Posewitz and Tempst, 1999). The positively
charged metal ions (Fe3+, Ga3+) of IMAC bind the negatively charged phosphate groups with high affinity.
Light- and redox-controlled protein phosphorylation is
critical for the regulation of the photosynthetic activities in
plants. In order to analyse phosphoproteins in the chloroplast thylakoids, tryptic peptides were released from the sur-
face of Arabidopsis thylakoids, and phosphopeptides were
enriched by IMAC and identified by MALDI-TOF MS and
electrospray ionization (ESI) MS/MS (Vener et al., 2001).
A number of phosphorylated proteins including D1, D2,
and CP43 of photosystem II (PSII), the peripheral protein
PsbH, and the light-harvesting polypeptides LCHII were
detected and successfully mapped for the phosphorylation
sites. These studies were extended to the characterization
of a previously unknown 12 kDa phosphoprotein (TPS9)
isolated from spinach thylakoid membranes (Carlberg
et al., 2003). Extensive MS studies on TSP9 revealed
that three threonine residues are phosphorylated (Fig. 1).
In later studies, the surface-exposed peptides were cleaved
from the Arabidopsis thylakoid membranes by trypsin and
methylated on acidic residues to improve specific binding
of phosphopeptides to IMAC. The enriched phosphopeptides were then sequenced using ESI MS/MS (Hansson
and Vener, 2003). In addition to the five known phosphorylation sites in PSII proteins, three previously unknown phosphorylation sites were identified and then
assigned to three proteins, PsaD, CP29, and a novel protein named TMP14 in PSI. The same approach was applied to thylakoid membranes isolated from the green
algae Chlamydomonas reinhardtii and it revealed the
phosphorylation and acetylation sites on the unprocessed
transit peptide (Turkina et al., 2004).
Protein phosphorylation plays an essential role in pathogen response, for example, plant–pathogen interactions,
gene expression, and defence signalling, in plants (Xing
et al., 2002). To understand the early events induced by
pathogen treatment, a suspension culture of Arabidopsis was
pulse-labelled with 32P and the phosphorylated proteins were
visualized by 2DE. One of these proteins, AtPhos43, that
was phosphorylated within minutes after treatment with
flagellin or chitin fragments was identified by nanoESI
MS/MS (Peck et al., 2001). They found that AtPhos43 is a
novel protein containing ankyrin repeats and its phosphorylation after flagellin treatment is dependent on FLS2, a
receptor-like kinase involved in flagellin perception.
Plasma membrane proteins are involved in the regulation
of cell–cell interactions in developmental processes and
responses to the environment, and the initiation and modulation of diverse signalling pathways. In this regard, the
identification of signalling processes and phosphoproteins
at the plasma membrane is of great interest. A strategy for
large-scale phosphoproteomics of the plasma membrane
was established where the ‘shaving’ approach was extended for membrane proteomics (Nühse et al., 2003).
Trypsin digestion of cytoplasmic face-out vesicles was
combined with IMAC and LC-MS/MS, and strong anion
exchange chromatography was included prior to IMAC
that decreased the complexity of IMAC-purified phosphopeptides and yielded a far greater coverage of monophosphorylated peptides. These methods identified more
than 300 phosphorylation sites from ;200 plasma
Proteomics of post-translational modifications
1549
Fig. 1. Identification of three phosphorylated forms of TSP9 and the corresponding phosphorylation sites. (A) The MALDI-TOF spectrum of the intact
TSP9. The masses of non-, mono-, di-, and triphosphorylated forms of TSP9 are indicated. (B, C) The product-ion spectra obtained by positive mode
electrospray ionization and collision-induced dissociation of phosphorylated peptides 8 (B) and 9 (C). The peptide sequences are shown in respective
spectrum with the lower case t designating phosphorylated threonine residues. The parent molecular ions (M+H)+ with m/z 687.3 (B) and (M+2H)2+ with
m/z 498.7 (C) are indicated along with the b- (N-terminal) and y- (C-terminal) fragment ions. The fragment ions produced by neutral loss of H3PO4
(mass 98) are marked with asterisks. Only fragment ions that help to localize the phosphorylation sites are labelled in the spectra. (D) The product-ion
spectrum of the doubly charged negative ion [(M–2H)2–, m/z 393.7] of peptide 10. The characteristic fragments –79 m/z ðPO
3 Þ; –97 m/z ðH2 PO4 Þ; and
]
are
indicated.
The
ion
with
m/z
–180.0
corresponds
to
a
fragment
of
phospho-threonine,
designated
by
lowercase
t in
–708.4 m/z [(M–2H)2– –PO
3
the peptide sequence shown. (Adapted and reprinted with permission from Carlberg et al., 2003; copyright 2003 National Academy of Sciences, USA.)
membrane proteins in Arabidopsis and very significantly
more than 50 sites mapped on receptor-like kinases (Nühse
et al., 2004). The characteristics of phosphorylation sites
and their conservation in sequence were analysed to determine common motifs surrounding the sites. These analyses
provide the principles for predicting phosphorylation sites
and substrate specificity for kinases in plants. The enormous
amount of data is a valuable resource, enabling prediction
methods of phosphorylation sites to be developed.
Glycosylphosphatidylinositol modification
Many cell surface proteins are tethered to the extracellular
membrane via GPI anchoring, that is one of the PTMs. GPI
membrane anchors provide a mechanism for asymmetric
distribution of membrane proteins that establishes and
maintains cell polarity (Chatterjee and Mayor, 2001).
GPI-anchored proteins (GAPs) have been identified by
genomic and proteomic analyses and have been shown
to be involved in diverse cellular functions such as cell
signalling, adhesion, matrix remodelling, and pathogen
response in plants (Borner et al., 2002).
Arabinogalactan proteins (AGPs) are proteoglycans of
the extracellular matrix that function in plant growth and
development (Schultz et al., 2000). AGPs are predicted
to be, at least transiently, attached to the outer surface of
the plasma membrane via a GPI anchor. AGPs were predeglycosylated and a subgroup of AGPs, known as AGpeptides, were separated from the larger classical AGPs
(Schultz et al., 2004). The deglycosylated AG-peptides are
of 10–17 residues in length and thus subjected to direct
analysis without tryptic digestion. Using MALDI-TOF
MS and tandem MS/MS, the precise cleavage sites for
both the N-terminal endoplasmic reticulum secretion signal
and the C-terminal GPI anchor signal were determined
for eight AG-peptides.
From a database analysis using a computer-based
algorithm developed for the identification of GAPs (Eisenhaber et al., 1999), 167 putative GAPs were identified in
addition to the 43 previously described candidates (Borner
et al., 2002). The predicted GAP included homologues of
b-1,3-glucanases, metallo- and aspartyl proteases, glycerophosphodiesterases, phytocyanins, multi-copper oxidases,
extensins, plasma membrane receptors, and lipid-transferproteins. Classical AG proteins, AG peptides, fasciclin-like
proteins, and many proteins of unknown function were predicted to be GPI anchored. Large-scale proteomic studies
were followed to identify plant GAPs (Borner et al., 2003).
Triton X-114 phase partitioning and treatment with phosphatidylinositol-specific phospholipase C (Pi-PLC) were
used to prepare GAP-rich fractions from Arabidopsis callus
cells (Hooper et al., 1987). GAPs were identified by a
characteristic shift from the hydrophobic detergent-rich
1550 Kwon et al.
Fig. 2. Modification-specific proteomic strategy for isolation and
identification of GPI-anchored proteins by mass spectrometry. LC, liquid
chromatography. (Adapted and reprinted with permission from Elortza
et al., 2003; copyright 2003 American Society for Biochemistry and
Molecular Biology.)
phase to the hydrophilic aqueous phase upon cleavage
of the GPI anchor with Pi-PLC (Fig. 2). Since this is a
well-established method to fractionate GPI anchoring of
proteins, it is generally accepted that the isolated proteins
are GAPs that do not contain GPI anchors. Using LC
MS/MS, 30 GAPs were identified, including b-1,3 glucanases, phytocyanins, fasciclin-like arabinogalactan proteins,
receptor-like proteins, Hedgehog-interacting-like proteins,
putative glycerophosphodiesterases, lipid transfer-like protein, COBRA-like protein, SKU5, and SKS1. These results
validated their previous bioinformatic analysis for the
identification of GAPs in the Arabidopsis protein database.
The search algorithm and genomic annotation were refined
by using the confirmed GAPs from the proteomic analysis
and thus an updated in silico screen revealed 64 new
candidates, raising the total to 248 predicted GAPs in
Arabidopsis. In other proteomic studies, 44 GAPs were
identified in an Arabidopsis membrane preparation using
the same experimental procedure (Elortza et al., 2003).
Ubiquitination
The functional role of ubiquitination is to target proteins
for degradation by the proteasome through the covalent
conjugation of ubiquitin (Ub) to Lys residues (Glickman
and Ciechanover, 2002). It has become increasingly
apparent that ubiquitination is widely involved in the
regulation of diverse cellular processes (Welchman et al.,
2005). The regulatory roles of the Ub-proteasome pathway
in plants have been emphasized in recent years (Smalle
and Vierstra, 2004). The regulated protein degradation
provides a major regulatory mechanism that controls many
of the cellular activities including homeostasis, growth,
development, hormone response, and stress response in
plants (Devoto et al., 2003; Moon et al., 2004).
The Ub-proteasome pathway has been the target for the
proteomic analysis to identify components of Ub- and Ublike proteins (Ubls)-systems in yeast and animals (Denison
et al., 2005). So far, most studies have used affinity
purification methods combined with MS/MS techniques.
Using epitope tags fused to Ub and Ubls for purification,
Ub and Ubl substrates have been successfully identified
(Peng et al., 2003; Wohlschlegel et al., 2004). This method
also leads to the isolation of other interacting proteins in
the Ub-proteasomal complexes (Verma et al., 2000).
The size and complexity of the Ub-26S proteasome
system in plants have been defined through bioinformatic
analysis (Vierstra, 2003; Smalle and Vierstra, 2004). It
was estimated that the Arabidopsis genome encodes more
than 1400 (>5% of the total proteome) components in
the pathway, implying the Ub-26S protesome pathway is
associated with most aspects of cellular processes in plants.
Future work, combined with comprehensive proteomic
studies, would enhance the identification of substrates
and other interacting molecules and our understanding of
their specific functions in the system.
Conclusion
The initial objective of proteomics was the large-scale
identification of proteins expressed in a cell or tissue. The
goal has been extended to include studies of the functional
properties of proteins, which are often regulated by PTMs
of proteins. Numerous techniques have rapidly been developed and applied to the global identification of PTMs
and their modification sites. The long-term challenge
remains to profile the dynamic changes of PTMs, which
would ultimately provide the basis of a comprehensive
understanding of signalling networks regulating complex
biological processes. This area possesses great promise as
it is supported by advanced proteomics technologies, in
particular, developments in the strategies for detection and
selective isolation, and in MS instrumentation.
Acknowledgements
We acknowledge support from the Plant Signaling Network Research Center funded by the Korea Science and Engineering
Foundation and the BioGreen 21 Program funded by the Rural
Development Administration, Republic of Korea.
Proteomics of post-translational modifications
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