International Gluten Workshop, 11th; Beijing (China); 12-15 Aug 2012

1. Daily Changes in the Phosphoproteome
of the Dinoflagellate Lingulodinium
Bolin Liu
IRBV, University of Montreal, Canada
Present address: China National Seed Group Co., Ltd.

2. Dinoflagellate
Lingulodinium
polyedra
A unicellular protist whose
biochemistry and physiology is
modulated over the daily cycle by
the action of an endogenous
circadian clock. This organism
serves as a good model system in
which to understand the
biochemical basis of the observed
rythms
-Lingulodinium polyedra is also
called Gonyaulax polyedra. An
algal bloom of dinoflagellates can
result in red tide
Life history of dinoflagellate

3. Red tide caused by dinoflagellates
Day Night
Light Dark cycle 0-12 (LD0-12); Light Dark cycle 12-24 (LD12-24)

4. Observed rythms
Two examples in Lingulodinium polyedra where
changes in protein amount correlate with the
observed rhythm
• Bioluminescence rhythm, where nightly light
production is correlated with the amount of two key
proteins: luciferase and luciferin binding protein.
Both proteins are synthesized at the start of night
phase
• Daily rhythm in nitrate reduction which correlates
with the amount of a Nitrate Reductase

5. Protein phosphorylation in arabidopsis
Phosphoralated proteins
proteins
Kinase
30%
Phosphotase
70%
0 200 400 600 800 1000 1200
In plant, arabidopsis has about 1100 kinases and 150 phosphatases. And about 30% of total
proteins are phosphoprylated. They are correlated with biochemical processes such as:
DNA repairation, signal transduction, and transcriptional regulation

6. Protein phosphorylation and
dephosphorylation modification
Kinase
Phosphotransphorase
By adding ATP
Dephosphoprotein Phosphoprotein
Phosphatase
By removing phosphate group
Phosphorylation is a key reversible modification that regulates protein
function

7. Methods for phosphoprotein detection
• Proteins autoradiography (hot ATP labelling in
vivo)
• Immunodetection using antibodies
• Staining gel with a phosphoprotein specific
fluorophore (Pro-Q Diamond dye)
• Affinity chromatography (Titanium dioxide)

8. Methods used in this study
• 1st, Two Dimentional Gel Electrophoresis
protocol
2D gel following ProQ Diamond, Coomassie
Blue and MS identification
• 2nd, Tryptic Phosphopeptide enrichment
protocol
Followed by MS identification

9. Phosphorylation
patterns change
over an LD cycle
To assess the possibility that
Lingulodinium proteins might be
differentially phosphorylated at
different times under the daily
light dark cycle. Samples were
taken every four hours, and were
resolved by SDS-PAGE
Protein phosphorylation patterns
change over an LD cycle.
Protein samples (~50 ug) were
prepared for SDS-PAGE from cells
isolated at the indicated times (in
hours) in a light-dark cycle. The
protein samples were resolved by
electrophoresis before
visualization using ProQ Diamond
then Coomassie Blue stains.
A representative gel is shown.
Apparent molecular weights are
shown at left

10. Phosphorylation
patterns change
in 2D gel
Protein samples were prepared for 2D-
GE from cells taken either at midday
(LD6; left hand panels) or midnight
(LD18; right hand panels),
electrophoresed, and stained
sequentially with ProQ Diamond
(upper panels) then Coomassie Blue
(lower panels). Seven proteins (1-7)
that showed at least a 2-fold change in
ProQ Diamond staining in three
independent experiments and that
could be unambiguously assigned to a
particular protein after Coomassie
Blue staining were excised for protein
microsequencing. An additional three
highly phosphorylated proteins that
did not vary between day and night (a-
c) as well as two proteins acting as
technical controls (Rub and PCP) were
also excised

11. Identification of
Rad24 protein
-Shared identity with a Rad24
protein from the dinoflagellate
Alexandrium fundyense (in a
tBLASTx search of the NCBInr
database)
-Suppoted by a good agreement
between predicted and measured
protein size and isoelectric point
-Rad24 protein is 2.4 times higher
in midnight (LD18) than in midday
(LD6)
-Rad24 functions in the DNA
damage checkpoint pathway
LD18/LD24 kD pI
Linguldinium 2.4 28 4.8
Alexandrium - 27 4.9

12. Identification of
HLCP
Shared identity with a chloroplast
light harvesting complex protein
(LHCP) from the dinoflagellate
Heterocapsa triquetra (in a
tBLASTx search of the NCBInr
database)
-The predicted molecular weight
of the Heterocapsa LHCP is higher
than that observed on our gels
because the predicted protein
contains a plastid-directed signal
peptide
-The amount of phophorylation of
LHCP is 4.3 times higher in
midnight (LD18) than in midday
(LD6) LD18/LD24 kD pI
-HLCP functions in photosystem I
and II Linguldinium 4.3 23 4.5
Heterocapsa - 28 7.7

13. Methods used in this study
• 1st, Two Dimentional Gel Electrophoresis
protocol
2D gel following ProQ Diamond, Coomassie
Blue and MS identification
• 2nd, Tryptic Phosphopeptide enrichment
protocol
Followed by MS identification

14. Protein samples
Samples taken at midday (LD6)
and midnight (LD18) were
prepared for titanium dioxide
(TiO2) chromatography analysis
Results
-422 peptides were identified
from LC-MS/MS
-54 phosphopeptides were
analyzed
-45 different phosphoproteins
were identified

15. Types and
motifs of
phosphorylation
Within a protein, phosphorylation
can occur on several amino acids.
Phosphorylation on serine (S) or
threonine (T) is the most
common
The amino acid context
surrounding all phosphorylated
serine or threonine residues was
used to group the phosphosites
into 4 Groups. They are:
Group 1, uncharged/hydrophobic
Group 2, acidic;
Group 3, basic;
Group 4 , S/T followed by proline

16. Discussion
• 1st, Rad24, identified phosphoprotein from this study, phosphorylated
more highly at night is of greatest interest. It is thought to be involved in
DNA repair as well as in the DNA damage checkpoint. Phosphorylation of
Rad24 is consistent with the observation that S-phase onset occurs at
midnight (LD18)
• 2nd, LHCP (light harvesting complex protein) acts to feed energy from light
into both photosystem (PS) I and PSII. The phosphorylation of LHCP results
in a change in the affinity of the protein for PSI and PSII. The
phosphorylated form of LHCP associates preferentially with PSI while the
dephosphorylated form has greater affinity for binding to PSII. Here
phosphorylation serves as a mechanism to balance the amount of light
entering each of the photosystems ensuring the same rate of electron
flow through both. In arabidopsis, phosphorylated LHCP dissociate from
PSII and associate with PSI during the later part of the night period and
decreased throughout the day

17. Discussion (ctn.)
• 3rd, The phosphopeptide enrichment approach identified three proteins
that contain RNA binding domains. That means synthesis of a number of
different proteins is controlled translationally
• 4th, A polyketide synthase with at least a 13-fold day/night difference in
the amount of phosphopeptide. Polyketides are a diverse family of
secondary metabolites and constitute the majority of the dinoflagellate
toxins. Toxin production was found to be restricted to the light phase, and
the post-translational regulation of polyketide synthase activity may be
responsible
• 5th, Two approaches did not identify any of the same phosphoproteins.
This presumably reflects the small number of proteins identified using the
2DGE approach

18. Conclusion
This study provides a proof in principle that an
expanded analysis of the phosphoproteome can contribute
insight into the daily biochemical changes in Lingulodinium
by means of two approaches following MALDI-TOF Mass
Spectrometry. Changes in protein phosphorylation may
underlie some of rhythmic behavior of Lingludinium.

19. Acknowledgements
• Dr. David Morse, Institut de Recherche en Biologie Végétale, Département
de Sciences Biologiques, Université de Montréal, Canada
• Dr. Samuel Chun-Lap Lo, Department of Applied Biology and Chemical
Technology, The Hong Kong Polytechnic University, Hong Kong SAR, China
• Dr. Daniel P. Matton, IRBV, Université de Montréal
• Dr. B. Franz Lang, Centre Robert Cedergren, Départment de Biochimie,
• Mr. Eric Bonneil at the Institut de Recherche en Immunologie et en
Cancérologie (IRIC) for the MS/MS analysis of the TiO2 purified
phosphopeptides
• National Science and Engineering Research Council (NSERC) of Canada to
DM and BFL (Grant numbers 171382-03 and 194560)
• Reference: Bolin Liu, Samule Lo, et al., Protist, Vol. 163, 746–754,
September 2012, Published online date 14 December 2011

20. Thanks