Beruflich Dokumente
Kultur Dokumente
pubs.acs.org/jpr
S Supporting Information
*
INTRODUCTION
Protein phosphorylation is one of the most important posttranslational modications (PTM) with roles in regulating cell
growth, adhesion, proliferation, and dierentiation via a variety
of signaling pathways.13 In living cells, phosphorylation
precisely controls spatiotemporal forms of cytoskeletal
components and temporal activities of protein kinases.
Therefore, it is important to comprehensively investigate
activated or basal phosphorylation states of these proteins to
understand the biological processes and/or mechanisms of
diseases.
Over the past decade, accompanying progress in liquid
chromatography (LC) combined with mass spectrometry
(MS),4 many researchers have reported comprehensive
analyses focusing on phosphorylated proteins such as drug
kinase interactions,5,6 mechanisms of drug side eects,7 and
analyses of signaling pathways.810 In phosphoproteomic
analysis, enrichment of phosphopeptides prior to MS is the
key factor to success because it is dicult to detect
phosphopeptides that have a low degree of ionization by
their own negatively charged phosphate groups in the presence
2013 American Chemical Society
Article
Reagents
Article
MALDI-TOF MS Analysis
For analysis of phosphopeptides enriched by TiO2 chromatography with each additive, we utilized an Ultraex II MALDITOF/TOF mass spectrometer (Bruker Daltonics). After the
TiO2-enriched samples were dissolved in 10 L of matrix
solution (10 mg/mL DHB, 0.1% TFA, 30% ACN, 1% H3PO4),
1 L was spotted onto an AnchorChip target plate (Bruker
Daltonics), followed by drying prior to MALDI-TOF MS
analysis. MS spectrum acquisition was carried out with the
following parameters: ion mode, positive-reectron; laser
power, 15% constant; accumulated sample positions, 20
positions; total laser shot number, 1000 shots; mass range,
m/z 8005000. To evaluate the selectivity of the TiO2 column
with each additive, the obtained MS spectra were cut o below
both the 5.0% maximal peak area intensity and a signal-to-noise
ratio of 5.0. All measurements were performed in triplicate.
LC-MS/MS Analysis
Article
MS Data Analysis
additive
phosphopeptides
nonphosphopeptides
input (before
purication)
no additive
lactic acid
pyruvic acid
glycerol
propylene glycol
trimethylene glycol
isopropanol
NDc
38.3 1.5d
1.3
6.7
2.0
6.3
6.3
6.3
0.7
0.6
0.6
0.0
0.6
0.6
0.6
0.6
38.0
8.3
14.0
6.0
15.7
21.7
30.0
2.6
3.1
1.0
3.6
2.1
1.5
3.6
eciencyb
(%)
1.1
76.1
7.4
80.6
34.4
19.6
0.6
0.2
1.7
0.5
4.7
1.5
2.3
0.6
Article
elution buer
pH
5% NH4OH
5% TEA
1 M Tris
1 M bis-Tris propane
1 M bis-Tris
1 M NH4HCO3
1 M NaOH
12.1
12.3
10.9
11.3
9.5
8.6
13.7
identied phosphopeptides
1005.0
661.3
549.0
1162.3
80.0
259.0
495.0
113.9a
20.2
207.9
117.6
14.1
87.7
166.3
Mean SD (n = 3).
Article
Figure 1. The property distributions of phosphopeptides obtained from PC3 cell lysate digest by TiO2 chromatography with each elution buer. (A)
Molecular weight, (B) GRAVY score, and (C) relative ratio of the number of acidic amino acids (Asp and Glu) to the full-length of phosphopeptide.
Elution buers are represented as follows: () bis-Tris propane, () NH4OH, () TEA, () Tris, () NaOH, () NH4HCO3, and () bis-Tris.
Article
Figure 3. Comparison of the number of phosphopeptides and phosphoproteins obtained from PC3 cell lysate digest by the conventional method
(lactic acid additive/NH4OH elution) and the optimized method (glycerol additive/two-step elution with NH4OH and bis-Tris propane). (A)
Phosphopeptides and (B) phosphoproteins.
5593
Article
Figure 4. Comparison of the number of phosphopeptides and phosphoproteins obtained from PC3 cell lysate digest in the presence or absence of
300 nM dasatinib. (A) Phosphopeptides and (B) phosphoproteins.
Figure 5. Ion chromatograms and Western blotting patterns of representative phosphorylation sites in PC3 cells suppressed by treatment with
dasatinib. (A) LIEDNEpY 419 TAR of SRC, (B) VLEDDPEATpY 772 TTSGGK of EPHA2, (C) YMEDSTYpY 577 K of FAK, (D)
pY827ATPQVIQAPGPR of ACK1. The red line and blue line on the ion chromatogram indicate the peak area intensity of treatment with 0 nM
dasatinib (DMSO) and that of treatment with 300 nM dasatinib, respectively. Western blotting analysis was performed using 20 g of PC3 cell lysate
per lane cultured in the presence of 0, 100, or 300 nM dasatinib for 1 h. Each band was detected by specic antibody against total or phosphorylated
protein.
5594
Article
CONCLUSIONS
We investigated the conditions of TiO2 chromatography for the
phosphopeptide enrichment and established a method for
comprehensive phosphoproteomic analysis. In this study,
glycerol, one of the most eective additives, showed marked
improvement of phosphopeptide selectivity. In particular,
glycerol as an additive played a characteristic role in the
enrichment of singly phosphorylated peptides compared to the
conventional conditions using lactic acid as an additive, but
there were no dierences in the numbers of multiply
phosphorylated peptides. In addition, the elution conditions,
such as a combination of NH4OH and bis-Tris propane, made
it possible to recover phosphopeptides of a wide range of
peptide lengths including more hydrophilic and/or more acidic
residues. These optimized additive and elution conditions are
expected to achieve the maximal phosphopeptide recovery
compared to conventional methods.
For the feasibility study of our optimized method, we tried to
search for target kinases of dasatinib in PC3 prostate cancer
cells. As a result, a total 8296 phosphopeptides were identied,
and Western blotting analysis revealed that the phosphorylation
changes of phosphopeptides, such as SRC and EPHA2 tyrosine
kinases, were correlated to their MS peak area intensities. These
results indicate that our optimized method could be applicable
for a large phosphoproteomic analysis. In summary, the
preparation of highly pure phosphopeptides obtained by our
TiO2 enrichment technique could facilitate subsequent MS
analysis. Our method is a powerful tool for phosphoproteomics
and will be useful for observations of a variety of complicated
cellular activations via intersecting signaling pathways in the
future.
ASSOCIATED CONTENT
S Supporting Information
*
Article
(16) Engholm-Keller, K.; Larsen, M. R. Titanium dioxide as chemoaffinity chromatographic sorbent of biomolecular compounds
applications in acidic modification-specific proteomics. J. Proteomics
2011, 75 (2), 31728.
(17) Fila, J.; Honys, D. Enrichment techniques employed in
phosphoproteomics. Amino Acids 2011, 43 (3), 102547.
(18) Batalha, I. L.; Lowe, C. R.; Roque, A. C. Platforms for
enrichment of phosphorylated proteins and peptides in proteomics.
Trends Biotechnol. 2012, 30 (2), 10010.
(19) Jensen, S. S.; Larsen, M. R. Evaluation of the impact of some
experimental procedures on different phosphopeptide enrichment
techniques. Rapid Commun. Mass Spectrom. 2007, 21 (22), 363545.
(20) Aryal, U. K.; Ross, A. R. Enrichment and analysis of
phosphopeptides under different experimental conditions using
titanium dioxide affinity chromatography and mass spectrometry.
Rapid Commun. Mass Spectrom. 2010, 24 (2), 21931.
(21) Mazanek, M.; Roitinger, E.; Hudecz, O.; Hutchins, J. R.;
Hegemann, B.; Mitulovic, G.; Taus, T.; Stingl, C.; Peters, J. M.;
Mechtler, K. A new acid mix enhances phosphopeptide enrichment on
titanium- and zirconium dioxide for mapping of phosphorylation sites
on protein complexes. J. Chromatogr., B: Anal. Technol. Biomed. Life Sci.
2010, 878 (56), 51524.
(22) Hsieh, H. C.; Sheu, C.; Shi, F. K.; Li, D. T. Development of a
titanium dioxide nanoparticle pipette-tip for the selective enrichment
of phosphorylated peptides. J. Chromatogr., A 2007, 1165 (12), 128
35.
(23) Kyono, Y.; Sugiyama, N.; Imami, K.; Tomita, M.; Ishihama, Y.
Successive and selective release of phosphorylated peptides captured
by hydroxy acid-modified metal oxide chromatography. J. Proteome Res.
2008, 7 (10), 458593.
(24) Larsen, M. R.; Thingholm, T. E.; Jensen, O. N.; Roepstorff, P.;
Jorgensen, T. J. Highly selective enrichment of phosphorylated
peptides from peptide mixtures using titanium dioxide microcolumns.
Mol. Cell Proteomics 2005, 4 (7), 87386.
(25) Thingholm, T. E.; Jorgensen, T. J.; Jensen, O. N.; Larsen, M. R.
Highly selective enrichment of phosphorylated peptides using titanium
dioxide. Nat. Protoc. 2006, 1 (4), 192935.
(26) Mazanek, M.; Mituloviae, G.; Herzog, F.; Stingl, C.; Hutchins, J.
R.; Peters, J. M.; Mechtler, K. Titanium dioxide as a chemo-affinity
solid phase in offline phosphopeptide chromatography prior to HPLCMS/MS analysis. Nat. Protoc. 2007, 2 (5), 105969.
(27) Yu, L. R.; Zhu, Z.; Chan, K. C.; Issaq, H. J.; Dimitrov, D. S.;
Veenstra, T. D. Improved titanium dioxide enrichment of
phosphopeptides from HeLa cells and high confident phosphopeptide
identification by cross-validation of MS/MS and MS/MS/MS spectra.
J. Proteome Res. 2007, 6 (11), 415062.
(28) Li, Q. R.; Ning, Z. B.; Tang, J. S.; Nie, S.; Zeng, R. Effect of
peptide-to-TiO2 beads ratio on phosphopeptide enrichment selectivity.
J. Proteome Res. 2009, 8 (11), 537581.
(29) Sugiyama, N.; Masuda, T.; Shinoda, K.; Nakamura, A.; Tomita,
M.; Ishihama, Y. Phosphopeptide enrichment by aliphatic hydroxy
acid-modified metal oxide chromatography for nano-LC-MS/MS in
proteomics applications. Mol. Cell. Proteomics 2007, 6 (6), 11039.
(30) Imami, K.; Sugiyama, N.; Kyono, Y.; Tomita, M.; Ishihama, Y.
Automated phosphoproteome analysis for cultured cancer cells by
two-dimensional nanoLC-MS using a calcined titania/C18 biphasic
column. Anal. Sci. 2008, 24 (1), 1616.
(31) Sugiyama, N.; Nakagami, H.; Mochida, K.; Daudi, A.; Tomita,
M.; Shirasu, K.; Ishihama, Y. Large-scale phosphorylation mapping
reveals the extent of tyrosine phosphorylation in Arabidopsis. Mol. Syst.
Biol. 2008, 4, 193.
(32) Nakagami, H.; Sugiyama, N.; Mochida, K.; Daudi, A.; Yoshida,
Y.; Toyoda, T.; Tomita, M.; Ishihama, Y.; Shirasu, K. Large-scale
comparative phosphoproteomics identifies conserved phosphorylation
sites in plants. Plant Physiol. 2010, 153 (3), 116174.
(33) Mayer, E. L.; Krop, I. E. Advances in targeting SRC in the
treatment of breast cancer and other solid malignancies. Clin. Cancer
Res. 2010, 16 (14), 352632.
AUTHOR INFORMATION
Corresponding Author
REFERENCES
(1) Hunter, T. Signaling2000 and beyond. Cell 2000, 100 (1), 113
27.
(2) Adams, J. A. Kinetic and catalytic mechanisms of protein kinases.
Chem. Rev. 2001, 101 (8), 227190.
(3) Manning, G.; Whyte, D. B.; Martinez, R.; Hunter, T.;
Sudarsanam, S. The protein kinase complement of the human
genome. Science 2002, 298 (5600), 191234.
(4) Cravatt, B. F.; Simon, G. M.; Yates, J. R., 3rd. The biological
impact of mass-spectrometry-based proteomics. Nature 2007, 450
(7172), 9911000.
(5) Sharma, K.; Weber, C.; Bairlein, M.; Greff, Z.; Keri, G.; Cox, J.;
Olsen, J. V.; Daub, H. Proteomics strategy for quantitative protein
interaction profiling in cell extracts. Nat. Methods 2009, 6 (10), 7414.
(6) Li, J.; Rix, U.; Fang, B.; Bai, Y.; Edwards, A.; Colinge, J.; Bennett,
K. L.; Gao, J.; Song, L.; Eschrich, S.; Superti-Furga, G.; Koomen, J.;
Haura, E. B. A chemical and phosphoproteomic characterization of
dasatinib action in lung cancer. Nat. Chem. Biol. 2010, 6 (4), 2919.
(7) Yu, Y.; Yoon, S. O.; Poulogiannis, G.; Yang, Q.; Ma, X. M.; Villen,
J.; Kubica, N.; Hoffman, G. R.; Cantley, L. C.; Gygi, S. P.; Blenis, J.
Phosphoproteomic analysis identifies Grb10 as an mTORC1 substrate
that negatively regulates insulin signaling. Science 2011, 332 (6035),
13226.
(8) Ficarro, S. B.; McCleland, M. L.; Stukenberg, P. T.; Burke, D. J.;
Ross, M. M.; Shabanowitz, J.; Hunt, D. F.; White, F. M.
Phosphoproteome analysis by mass spectrometry and its application
to Saccharomyces cerevisiae. Nat. Biotechnol. 2002, 20 (3), 3015.
(9) Oberprieler, N. G.; Lemeer, S.; Kalland, M. E.; Torgersen, K. M.;
Heck, A. J.; Tasken, K. High-resolution mapping of prostaglandin E2dependent signaling networks identifies a constitutively active PKA
signaling node in CD8+CD45RO+ T cells. Blood 2010, 116 (13),
225365.
(10) Chevrier, N.; Mertins, P.; Artyomov, M. N.; Shalek, A. K.;
Iannacone, M.; Ciaccio, M. F.; Gat-Viks, I.; Tonti, E.; DeGrace, M. M.;
Clauser, K. R.; Garber, M.; Eisenhaure, T. M.; Yosef, N.; Robinson, J.;
Sutton, A.; Andersen, M. S.; Root, D. E.; von Andrian, U.; Jones, R. B.;
Park, H.; Carr, S. A.; Regev, A.; Amit, I.; Hacohen, N. Systematic
discovery of TLR signaling components delineates viral-sensing
circuits. Cell 2011, 147 (4), 85367.
(11) Schroeder, M. J.; Shabanowitz, J.; Schwartz, J. C.; Hunt, D. F.;
Coon, J. J. A neutral loss activation method for improved
phosphopeptide sequence analysis by quadrupole ion trap mass
spectrometry. Anal. Chem. 2004, 76 (13), 35908.
(12) Gruhler, A.; Olsen, J. V.; Mohammed, S.; Mortensen, P.;
Faergeman, N. J.; Mann, M.; Jensen, O. N. Quantitative
phosphoproteomics applied to the yeast pheromone signaling
pathway. Mol. Cell Proteomics 2005, 4 (3), 31027.
(13) Beausoleil, S. A.; Villen, J.; Gerber, S. A.; Rush, J.; Gygi, S. P. A
probability-based approach for high-throughput protein phosphorylation analysis and site localization. Nat. Biotechnol. 2006, 24 (10),
128592.
(14) Thaler, F.; Valsasina, B.; Baldi, R.; Xie, J.; Stewart, A.; Isacchi, A.;
Kalisz, H. M.; Rusconi, L. A new approach to phosphoserine and
phosphothreonine analysis in peptides and proteins: chemical
modification, enrichment via solid-phase reversible binding, and
analysis by mass spectrometry. Anal. Bioanal. Chem. 2003, 376 (3),
36673.
(15) Dunn, J. D.; Reid, G. E.; Bruening, M. L. Techniques for
phosphopeptide enrichment prior to analysis by mass spectrometry.
Mass Spectrom. Rev. 2010, 29 (1), 2954.
5596
Article
5597