Specificity of MOAC Enrichment Applied for Mature Pollen

Specificity of MOAC Enrichment Applied for Mature Pollen

2012 International Conference on Biomedical Engineering and Biotechnology
Specificity of MOAC enrichment applied for mature
pollen phosphoproteomics studies
Jan Fíla, Vra apková, David Honys
Andrea Matros, Hans-Peter Mock
Laboratory of Pollen Biology
Institute of Experimental Botany ASCR
Prague, Czech Republic
[email protected]
Applied Biochemistry Group
IPK Gatersleben
Gatersleben, Germany
Due to these facts, the application of various enrichment
techniques is of key importance. For our phosphoproteomic
studies, we have applied metal-oxide affinity chromatography
(MOAC) using aluminium hydroxide as chromatography
matrix [7]. Similarly to other phoshoprotein enriching
strategies, it is advantageous in obtaining information about
protein isoelectric point and molecular weight since the
enriched fraction is usually fractionated by 2D-GE. Unlike
phosphopeptide enrichment, the phosphoprotein enrichment
does not rely on identification according to only one peptide
since the un-modified peptides contribute to protein
identification as well. On the contrary, lipophilic, tiny as well
as proteins with an extreme pI value are likely to be
handicapped by this approach.
Abstract—Androgenesis represents one of the most important
and fast-developing biotechnological approaches for the
production of haploid and di-haploid crop plants with great
potential for plant breeding. Tobacco serves as a suitable model
plant with available protocols for androgenesis. However, it is
necessary to understand the molecular regulatory mechanisms
underlying this process.
Phosphorylation is the most dynamic posttranslational
modification. In a time, the cell contains only few percent of
phosphoproteins that can furthermore be accompanied by their
native forms. Thus, the enrichment techniques have to be applied
in order to concentrate the phosphorylated forms. The aim of our
studies was to compare the phosphoproteins extracted by
TCA/acetone out of mature pollen and 30-min-activated pollen.
The crude protein extracts were enriched by the metaloxide/hydroxide affinity chromatography (MOAC) with
aluminium hydroxide matrix. The efficiency of the enrichment
was verified by 1D SDS-PAGE of the fractions coming out of the
enrichment. The separated phosphoproteins were detected by
phospho-specific ProQ Diamond stain (Invitrogen) whilst total
proteins were stained by colloidal CBB G250.
Although several studies applied MOAC with aluminium
hydroxide matrix for studies of protein phosphorylation [7-11],
the method specificity was not widely tested. Wolschin and
colleagues were enriching a mixture of standard proteins, part
of which were phosphoproteins whilst the others were nonphosphorylated [7]. This is a good pre-requisite for proving the
method specificity but it remains a possibility that some
proteins from complex protein extracts will exhibit nonspecific
binding to the matrix. Röhrig and coworkers showed the
presence of phosphoproteins in the enriched fraction by its
dephosphorylation by alkaline phosphatase. There was
observed only a decrease of phosphate-specific staining
intensity after dephosphorylation so still any unspecific
proteins could be masked by the background staining [9].
Another proof of higher phosphate content in the enriched
fraction was shown by higher phosphate/sulfur ratio
determined by liquid chromatography (Kruger 2007). Although
the results showed 40-100% proteins from the eluate to be
phosphorylated, it was not shown which proteins were the nonspecific ones, if any.
In this short paper, we present our aims in revealing the
MOAC specificity for phosphoprotein enrichment. The
enrichment of dephosphorylated mature pollen crude extract and
the original mature pollen crude extract were compared. Since
alkaline phosphatase did not accomplish the dephosphorylation
step properly, we decided to apply cerium dioxide for sample
dephosphorylation. Since cerium dioxide captured the
phosphoproteins rather than dephosphorylated them, the proof
of MOAC specificity was impossible to be achieved by these
experiments.
Keywords—androgenesis; tobacco; male gametophyte; pollen;
phosphoproteins, sample dephosphorylation.
I.
INTRODUCTION
To our knowledge, nobody tried to dephosphorylate the
crude extract and enrich it under the same conditions as the
conventional protein extract. If the dephosphorylation was to
be fully accomplished, only non-specific interactions would be
possible. At first, we tried to dephosphorylate the sample by
alkaline phosphatase but the reaction was not complete. Then,
cerium dioxide was applied instead but this compound captured
phosphoproteins rather than dephosphorylated them.
Protein phosphorylation is one of the most dynamic posttranslational modifications, role of which during cell cycle
regulation,
transcription
and
translation
regulation,
cytoskeleton dynamics, protein targeting, metabolism
regulation, and signal transduction has been reported [1-5].
Unfortunately, the importance of phosphoproteins is usually
not mirrored in their abundance. Furthermore, one protein can
coexist as both phosphorylated and un-hosphorylated form in a
cell. Last but not least, the detection of phosphoproteins by MS
in the positive ion scan mode is quite challenging due to ion
supression effects [6].
978-0-7695-4706-0/12 $26.00 © 2012 IEEE
DOI 10.1109/iCBEB.2012.362
523
II.
On Fig. 1, there is visible high signal on phospho-specific
ProQ stain in case of negative control and crude extract
revealing that (1) the crude extract was formerly
MATERIALS AND METHODS
A. Biological material and TCA/acetone protein extraction
Mature tobacco pollen was collected every day from
flowers shortly before anthesis and the anthers were let to
dehisce at room temperature for overnight [12]. The sieved
pollen was stored at -20°C. Protein extraction was performed
by TCA/acetone as described previously [13].
B. Sample dephosphorylation by alkaline phosphatase
Protein pellet was resuspended in 6 M urea buffer (6 M
urea, 1mM Tris-Cl, 0.1mM MgCl2, pH 9.5). After sample
resuspension, it was 30× diluted with phosphatase buffer (1mM
Tris-Cl, 0.1mM MgCl2, pH 9.5). The sample was incubated at
37°C for 3 h.
C. SDS-PAGE and gel staining
The desalted samples [14] were fractionated by SDSPAGE. Resolving gel was 11.25 0%, and stacking gel was 5 %
acrylamide. The run was: 75V/stacking gel, 150V/resolving
gel. After the run was finished, the gels were stained by ProQ
Diamond Phosphoprotein Gel Stain (Invitrogen) accrding to
the modified protocol [15]. The scan was performed on Fuji
FLA 5500, laser 492nm, green filter. Afterwards, the gel was
stained by CBB G250 [16].
Figure 1. SDS-PAGE of tobacco pollen crude extract dephosphorylated
by alkaline phosphatase. Staining: left – ProQ Diamond; right – CBB
G250. Lane labelling: CE – crude extract; Ph+ dephosphorylated sample;
Ph- negative control..
phosphorylated and (2) incubation at 37 °C itself did not lead
to sample dephosphorylation. After all, the dephosphorylation
by alkaline phosphatase seemed to be incomplete since the
phospho-specific dye showed quite high signal for this sample
(although it was lower than the signal of negative control and
crude extract). It is not likely that the difference in signal
intensities came from different protein amounts since the
protein amount was proven to be the same by CBB G250
staining.
D. Sample dephosphorylation by cerium dioxide and MOAC
enrichment
TCA/acetone pellet was resuspended in MOAC incubation
buffer (30 mM MES, 20 mM imidazole, 0.2 M L-aspartic acid
potassium salt, 0.2 M L-glutamic acid sodium salt hyd., 8 M
urea, 2.5 % w/v CHAPS, phosphatase inhibitors; pH 6.1) and
1 g cerium dioxide was added per 1 mg proteins (if not
otherwise mentioned). The sample was incubated 15 min at
37 °C if not otherwise mentioned. The CeO2 particles were
pelleted by centrifugation (15 min; 22,000×g; 4 °C) and the
supernatant was transferred into a fresh tube. MOAC
phosphoprotein enrichment was performed according to the
standard protocol [7].
III.
Incomplete protein dephosphorylation was insufficient for
further studies since there could not be distinguished the
binding of non-dephosphorylated proteins and the nonspecifically bound proteins. The incomplete dephosphorylation
was probably caused by the presence of sites with phosphoamino acids that were unavailable and/or not recognized by
alkaline phosphatase. It is noteworthy that in natural
conditions, there are plenty of phosphatases differing in their
substrate specificity [17].
RESULTS AND DISCUSSION
A. Sample dephosphorylation by alkaline phosphatase
The mature pollen proteins were extracted by TCA/acetone
[13]. It was necessary to find out conditions enabling
phosphatase activity on one hand and effective sample
resuspension on the other hand. The dephosphorylation could
not be achieved directly in MOAC incubation buffer since (1)
this buffer lacked magnesium ions that are necessary cofactor
of alkaline phosphatase, (2) the pH was weakly acidic (alkaline
phosphatase has its optimum at pH 9.5) and (3) presence of
CHAPS caused protein denaturation and consequently reduced
the activity of alkaline phosphatase.
B. Sample dephosphorylation by cerium dioxide and MOAC
enrichment
Due to only partial dephosphorylation by alkaline
phosphatase, we decided to employ more rapid
dephosphorylation protocol relying on cerium dioxide [18].
This non-enzymatic method was originally employed for
phosphopeptide dephosphorylation in 0.2 M ammonia solution
(pH 8.0). In this study, we wanted to perform cerium dioxidemediated dephosphorylation of phosphoproteins.
So the protein pellet was resuspended in 6 M urea buffer
(6 M urea, 1 mM Tris-Cl, 0.1 mM MgCl2, pH 9.5). Six-molar
urea (being a chaotropic agens) helped to resuspend proteins
but in such high concentration inhibited the activity of alkaline
phosphatase (data not shown). It was necessary to dilute the
sample 30-fold with phosphatase buffer (1mM Tris-Cl, 0.1mM
MgCl2, pH 9.5) in order to reduce urea concentration.
Subsequently, the sample was incubated at 37°C for 3 h.
MOAC incubation buffer was chosen as the reaction buffer
since after cerium-dioxide was removed by centrifugation, the
MOAC enrichment could be done directly without any
precipitation and buffer exchange. To exclude any possibility
of enzymatic dephosphorylation by phosphatases present in the
sample itself, phosphatase inhibitors were added to the buffer.
524
Finally, the concentration of cerium dioxide was taken into
account. 1 g of cerium dioxide per 1 mg proteins led to
complete dephosphorylation whilst lower concentrations ended
up with only partial reaction. Even as high concentration as
750 mg cerium dioxide per 1 mg proteins did not lead to
successful dephosphorylation. After all, the optimal conditions
for all subsequent experiments were as follows: 1 g cerium
dioxide per 1 mg proteins; incubation at 37 °C for 15 min.
Three sets of conditions had to be optimized: incubation
time, incubation temperature and CeO2-amount. Sample
resuspended in MOAC incubation buffer was mixed with
desired amount of cerium dioxide. The shortest reliable
incubation time showed out to be 10 min. Since also 2-hour
incubation at 37 °C did not lead to sample dephosphorylation
(data not shown), we decided for 15-min incubation to exclude
the possibility being under the time threshold necessary for
successful dephosphorylation.
The dephosphorylation by cerium dioxide seemed to be
complete (Fig. 2, lane Ex+) compared to dephosphorylation
achieved by alkaline phosphatase (Fig. 1).
Incubation temperature of 37 °C turned to be the best,
slightly worse was the dephosphorylation at room temperature
(~22 °C). Dephosphorylation, if any, was very slow at 4 °C and
0 °C. Moreover, incubation on ice was controversial, since
Until now, the way how to achieve dephosphorylation was
discussed. However, the main reason for crude-extract
dephosphorylation was to go for MOAC enrichment of
dephosphorylated sample. The dephosphorylated sample could
only reach the eluate-fraction by non-specific binding to the
chromatography matrix since phosphoamino acids were not
present in the starting crude extract.
Various MOAC phases were compared by phosphatespecific gel staining. These phases were crude extracts, S0s,
S1s and eluates (Fig. 2). Crude extract before
dephosphorylation (i.e. the same extract as starting material for
MOAC of negative control) showed high ProQ signal revealing
sample phosphorylation. On the contrary dephosphorylation of
crude extract led to very low ProQ-signal (probably
background staining only). The eluate of negative control
showed high ProQ signal revealing phosphoproteins present in
the eluate. The eluate coming from dephosphorylated sample
contained very low amount of proteins. The background
staining of the ProQ gel is likely to be caused by very high
ProQ sensitivity [19].
C. Cerium dioxide and its real function
Finally, we tested, whether cerium dioxide caused really
protein dephosphorylation. Phosphorylated casein was
resuspended in MOAC incubation buffer and treated by cerium
dioxide. The CBB G250 staining of the 1D SDS-PAGE gel
revealed that the protein was lost during the procedure, since
was probably captured. To test whether exclusively
phosphoproteins were lost during this procedure, we
dephosphorylated casein by alkaline phosphatase (casein
dephosphorylation was fully accomplished) and treated this
sample with cerium dioxide. The sample was dephosphorylated
(by alkaline phosphatase) as shown by ProQ Diamond staining
but the sample was not lost. It is likely that cerium phosphate
captured the phosphoproteins under the experimental
conditions rather than dephosphorylated them.
IV.
CONCLUDING REMARKS
Dephosphorylation by alkaline phosphatase was not
completed due probably to its substrate specificity. The
incompletely dephosphorylated sample could subsequently
bind to chromatography matrix so we searched for an
alternative dephosphorylation protocol that would complete its
task. Cerium dioxide was originally used for peptide
dephosphorylation [3] so the protocol had to be adapted for
protein dephosphorylation. The first results of enriching
dephosphorylated sample by MOAC seemed very promising
since almost no proteins were present in the enriched fraction.
Figure 2. SDS-PAGE of several MOAC fractions. Lane labelling: Ex crude extract; S0 - flow-through containing almost no phosphoproteins;
El - eluate enriched for phosphoproteins. Conditions labelling: - negative
control; + dephosphorylated sample.
sometimes crystallization of urea occurred and consequently its
concentration could be reduced after centrifugation (the
crystals could be present in the pellet and discarded).
525
However, the verification of cerium dioxide activity by
casein dephosphorylation led to surprising observation. Casein
was captured by cerium dioxide rather than dephosphorylated.
Furthermore, this entrapment was shown to be phosphoproteinspecific rather than random. Due to lack of rapid
dephosphorylation protocol our objective could not be realized
and alternative experiments have to be performed in order to
test MOAC specificity for phosphoprotein enrichment.
ACKNOWLEDGEMENT
The authors gratefully acknowledge the financial support
from ESF (COST Action 0603; COST-STSM-FA0603-04559
and COST-STSM-FA0603-05564), Czech Science Foundation
(grant no. P501/11/1462) and Ministry of Education, Youth
and Sports of the Czech Republic (grants no. OC08011).
REFERENCES
[1] Maekawa, T., Kosuge, S., Sakamoto, S., Funayama, S., et al.,
Biochemical characterization of 60S acidic ribosomal P proteins associated
with CK-II from bamboo shoots and potent inhibitors of their
phosphorylation in vitro. Biological & Pharmaceutical Bulletin 1999, 22,
667-673.
[2] Kameyama, K., Kishi, Y., Yoshimura, M., Kanzawa, N., et al., Tyrosine
phosphorylation in plant bending - Puckering in a ticklish plant is
controlled by dephosphorylation of its actin. Nature 2000, 407, 37-37.
[3] Dephoure, N., Zhou, C., Villen, J., Beausoleil, S. A., et al., A
quantitative atlas of mitotic phosphorylation. Proceedings of the National
Academy of Sciences of the United States of America 2008, 105, 1076210767.
[4] Mishra, N. S., Tuteja, R., Tuteja, N., Signaling through MAP kinase
networks in plants. Archives of Biochemistry and Biophysics 2006, 452, 5568.
[5] op den Camp, R. G. L., Kuhlemeier, C., Phosphorylation of tobacco
eukaryotic translation initiation factor 4A upon pollen tube germination.
Nucleic Acids Research 1998, 26, 2058-2062.
[6] Marcantonio, M., Trost, M., Courcelles, M., Desjardins, M., Thibault,
P., Combined enzymatic and data mining approaches for comprehensive
phosphoproteome analyses. Molecular & Cellular Proteomics 2008, 7,
645-660.
[7] Wolschin, F., Wienkoop, S., Weckwerth, W., Enrichment of
phosphorylated proteins and peptides from complex mixtures using metal
oxide/hydroxide affinity chromatography (MOAC). Proteomics 2005, 5,
4389-4397.
[8] Wolschin, F., Weckwerth, W., Combining metal oxide affinity
chromatography (MOAC) and selective mass spectrometry for robust
identification of in vivo protein phosphorylation sites. Plant Methods 2005,
1.
[9] Röhrig, H., Colby, T., Schmidt, J., Harzen, A., et al., Analysis of
desiccation-induced candidate phosphoproteins from Craterostigma
plantagineum isolated with a modified metal oxide affinity chromatography
procedure. Proteomics 2008, 8, 3548-3560.
[10] Petrova, O. E., Sauer, K., A Novel Signaling Network Essential for
Regulating Pseudomonas aeruginosa Biofilm Development. Plos
Pathogens 2009, 5, 16.
[11] Ito, J., Taylor, N. L., Castleden, I., Weckwerth, W., et al., A survey of
the Arabidopsis thaliana mitochondrial phosphoproteome. Proteomics
2009, 9, 4229-4240.
[12] Petr, E., Hrabtová, E., Tupý, J., The technique of obtaining
germinating pollen without microbial contamination. Biologia Plantarum
1964, 6, 68-69.
[13] Méchin, V., Damerval, C., Zivy, M., in: Thiellement, H., Zivy, M.,
Damerval, C., Méchin, V. (Eds.), Methods in Molecular Biology, Springer
2006, pp. 1-8.
[14] Wessel, D., Flügge, U. I., A method for the quantitative recovery of
protein in dilute solution in the presence of detergents and lipids. Analytical
Biochemistry 1984, 138, 141-143.
Figure 3. SDS-PAGE of casein after various treatments. 1 – resuspended
casein before any treatment; 2 – dephosphorylation by alkaline
phosphatase; 3 – dephosphorylation by alkaline phosphatase and
subsequent treatment by cerium dioxide; 4 – negative control incubated
at 37 °C for 2h; 5 – direct treatment by cerium dioxide.
[15] Agrawal, G. K., Thelen, J. J., Development of a simplified,
economical polyacrylamide gel staining protocol for phosphoproteins.
Proteomics 2005, 5, 4684-4688.
[16] Kang, D. H., Gho, Y. S., Suh, M. K., Kang, C. H., Highly sensitive
and fast protein detection with coomassie brilliant blue in sodium dodecyl
sulfate-polyacrylamide gel electrophoresis. Bulletin of the Korean
Chemical Society 2002, 23, 1511-1512.
[17] Morton, R. K., Substrate specificity and inhibition of alkaline
phosphatases of cows milk and calf intestinal mucosa. Biochemical Journal
1955, 61, 232-240.
[18] Tan, F., Zhang, Y. J., Wang, J. L., Wei, J. Y., et al., An efficient
method for dephosphorylation of phosphopeptides by cerium oxide.
Journal of Mass Spectrometry 2008, 43, 628-632.
[19] Steinberg, T. H., Agnew, B. J., Gee, K. R., Leung, W. Y., et al.,
Global quantitative phosphoprotein analysis using multiplexed proteomics
technology. Proteomics 2003, 3, 1128-1144.
526