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Originally published In Press as doi:10.1074/mcp.T600060-MCP200 on February 23, 2007.
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Molecular & Cellular Proteomics 6:1103-1109, 2007.
© 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

Technology

Phosphopeptide Enrichment by Aliphatic Hydroxy Acid-modified Metal Oxide Chromatography for Nano-LC-MS/MS in Proteomics Applications*,S

Naoyuki Sugiyama{ddagger},§, Takeshi Masuda{ddagger}, Kosaku Shinoda{ddagger},§, Akihiro Nakamura{ddagger}, Masaru Tomita{ddagger},§ and Yasushi Ishihama{ddagger},¶,||

From the {ddagger} Institute for Advanced Biosciences, Keio University, Tsuruoka 997-0017, Japan, § Human Metabolome Technologies, Kakuganji, Tsuruoka 997-0052, Japan, and Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
We developed novel methods for phosphopeptide enrichment using aliphatic hydroxy acid-modified metal oxide chromatography (MOC). Titania and zirconia were successfully applied to enrich phosphopeptides with the aid of aliphatic hydroxy acids, such as lactic acid and ß-hydroxypropanoic acid, to reduce the interaction between acidic non-phosphopeptides and the metal oxides. These methods removed the vast majority of non-phosphopeptides from phosphoprotein standard digests, and large numbers of phosphopeptides could be readily identified. The methods were coupled with nano-LC-MS/MS systems without difficulty. Recovery of phosphopeptides in MOC varied greatly from peptide to peptide, ranging from a few percent to 100%, and the average was almost 50%. Repeatability and linearity were satisfactory. In an examination of the cytoplasmic fraction of HeLa cells, more than 1000 phosphopeptides were identified using lactic acid-modified titania MOC and ß-hydroxypropanoic acid-modified zirconia MOC, respectively. The overlap between phosphopeptides enriched by these two methods was 40%, and the combined results provided 1646 unique phosphopeptides. To our knowledge, this is the first successful application of a single MOC-based approach to phosphopeptide enrichment from complex biological samples such as cell lysates.

Phosphorylation is a key event in cellular signaling networks (1), and an understanding of proteome-wide phosphorylation/dephosphorylation dynamics is important not only in relation to particular signal transduction pathways but also to obtain an overview of the whole network. MS is currently one of the most powerful techniques for proteome-wide experiments (2), but further improvements in protein identification efficiency are still needed (3). One of the key requirements for phosphoproteome analysis is to remove abundant non-phosphopeptides from complex mixtures, such as cell lysates, to detect low abundance phosphopeptides. Several approaches to enrich phosphopeptides prior to MS analysis, coupled with phosphate-specific MS acquisition techniques, have been reported. For phosphotyrosine-containing proteins, antibodies such as 4G10 and pY100 work adequately at both the protein and peptide levels (4, 5). In addition, precursor ion scanning specific to phosphotyrosine has been applied to filter out non-phosphopeptides (4). On the other hand, a similar approach has not been effective for phosphoserine- and phosphothreonine-containing proteins, although it has helped to identify new players in the signaling pathway in some cases (6). As more general approaches for phosphopeptide enrichment, IMAC (7–9), IMAC with methyl esterification (10, 11), strong cation exchange chromatography (12, 13), and metal oxide chromatography (MOC)1 using titania (14–18), zirconia (19), and alumina (20) have been reported. In addition, precursor ion scanning in the negative mode (21), neutral loss-triggered MS3 (22, 23), pseudo-MS3 (24), and electron transfer dissociation (25) have been developed to detect phosphopeptides selectively. By combining these methods, it is currently possible to identify hundreds or thousands of phosphopeptides (8, 10, 12, 13, 22, 23, 26). Nevertheless current phosphopeptide enrichment methods are still not sufficiently specific (9), and samples generally contain large amounts of non-phosphopeptides even after enrichment. This causes ambiguity in peptide identification, although high accuracy measurement of precursor ions helps to reduce false positives (22). The situation at present is that improvement of phosphopeptide enrichment processes will lead directly to higher quality phosphoproteome analysis.

Titania was first applied as a chemoaffinity medium for organophosphates including phosphopeptides by Ikeguchi and Nakamura (14) and was subsequently made commercially available for proteomics applications (15–17). Zirconia also has affinity for phosphate (27) and has been used for phosphopeptide enrichment as well (19). Larsen et al. (18) showed that inclusion of o-hydroxybenzoic acid and its derivatives, such as 2,5-dihydroxybenzoic acid (DHB), in the sample loading buffer was effective to remove acidic non-phosphopeptides during phosphopeptide enrichment with titania for MALDI-MS analysis. However, this protocol is not directly applicable to LC-MS/MS analysis because residual DHB interferes with peptide detection, and the system becomes unstable because of precipitation of DHB around the orifice and in the LC system.

We developed new approaches for phosphopeptide enrichment using MOC modified with aliphatic hydroxy acids. The conditions were optimized for phosphopeptide enrichment from cell lysates followed by LC-MS/MS analysis to identify phosphopeptides.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
Materials—
Titania (titanium dioxide; particle size, 10 µm) was obtained from GL Sciences (Tokyo, Japan). Zirconia (zirconium dioxide; particle size, 10 µm) was from ZirChrom Separations (Anoka, MN). Alumina for TLC (catalog no. 1.01072.0500) was purchased from Merck. Al(OH)3·xH2O, bovine {alpha}-casein, bovine fetuin, chicken phosvitin, and DHB were from Sigma. ß-Hydroxypropanoic acid (HPA) was obtained from Tokyo Kasei (Tokyo, Japan). C8 and C18 Empore disks were from 3M (St. Paul, MN). Modified trypsin was from Promega (Madison, WI). Water was obtained from a Millipore Milli-Q system (Bedford, MA). MS-grade Lys-C, boehmite, and all other chemicals including DL-lactic acid and other hydroxy acids were purchased from Wako (Osaka, Japan).

Digestion of Standard Phosphoproteins—
{alpha}-Casein, fetuin, and phosvitin were individually reduced with DTT, alkylated with iodoacetamide, and digested with Lys-C followed by dilution and trypsin digestion as described previously (28). These digested samples were desalted using StageTips with C18 Empore disk membranes (29). The eluates were mixed, and the peptide concentration was adjusted to 0.5 mg/ml with 0.1% TFA, 80% acetonitrile.

Enrichment of Phosphopeptides from Standard Phosphoprotein Digests—
Custom-made MOC tips were prepared using C8 StageTips and metal oxide bulk beads (3 mg of beads/200-µl pipette tip) as described for strong cation exchange (beads)-C18 tips (30). Prior to loading samples, the MOC tips were equilibrated with 0.1% TFA, 80% acetonitrile with hydroxy acids as selectivity enhancers (solution A). As the enhancers, glycolic acid, lactic acid, malic acid, tartaric acid, and DHB were used at a concentration of 300 mg/ml, and HPA was used at 100 mg/ml. The digested standard phosphoprotein mixture (15 µl) was mixed with 100 µl of solution A and loaded on the MOC tip. After successive washing with solution A and solution B (0.1% TFA, 80% acetonitrile), 0.5% ammonium hydroxide was used for elution. The eluted fraction was acidified with TFA, desalted using C18 StageTips as described above and concentrated in a vacuum evaporator followed by the addition of solution A for subsequent nano-LC-MS/MS analysis.

Analysis of HeLa Cell Cytoplasmic Fraction—
HeLa cells, cultured to 80% confluence in 15-cm diameter dishes, were homogenized with a Dounce homogenizer (10 strokes) after having been spiked with protein phosphatase inhibitor mixtures 1 and 2 (Sigma) and protease inhibitors (Sigma) in 100 mM Tris buffer (pH 8.0). The homogenate was centrifuged at 1500 x g for 10 min, and the supernatant was digested and desalted as shown above. Titania or zirconia MOC tips were preconditioned with 20 µl of solution A. The digested sample from a total of 200 µg of HeLa cytoplasmic proteins was diluted with 100 µl of solution A and loaded to the MOC tip. Washing was done with 20 µl each of solutions A and B, and then phosphopeptides were eluted with 20 µl of 0.5% ammonium hydroxide followed by acidification with 20 µl of 1% TFA. Subsequent procedures were the same as described for the standard phosphoproteins.

Nano-LC-MS System—
Nano-LC-MS/MS analyses were conducted by using a QSTAR system (QSTAR XL (AB/MDS-Sciex, Toronto, Canada), Agilent 1100 nanoflow pump (Waldbron, Germany), and HTC-PAL autosampler (CTC Analytics, Zwingen, Switzerland)) or an Orbitrap system (Finnigan LTQ-Orbitrap (Thermo Electron, Bremen, Germany), Dionex Ultimate3000 pump with FLM-3000 flow manager (Germering, Germany), and HTC-PAL autosampler). ReproSil C18 materials (3 µm, Dr. Maisch, Ammerbuch, Germany) were packed into a self-pulled needle (150-mm length x 100-µm inner diameter, 6-µm opening) with a nitrogen-pressurized column loader cell (Nikkyo Technos, Tokyo, Japan) to prepare an analytical column needle with "stone-arch" frit (31). A polytetrafluoroethylene-coated column holder (Nikkyo Technos) was mounted on an x-y-z nanospray interface (Nikkyo Technos), and a Valco metal connector with a magnet was used to hold the column needle and to set the appropriate spray position. The injection volume was 5 µl, and the flow rate was 500 nl/min. The mobile phases consisted of 0.5% acetic acid (A) and 0.5% acetic acid and 80% acetonitrile (B). A three-step linear gradient of 5–10% B in 5 min, 10–40% B in 60 min, 40–100% B in 5 min, and 100% B for 10 min was used throughout this study. A spray voltage of 2400 V was applied via the metal connector as described previously (31). The MS scan range was m/z 300–1500, and the top three (QSTAR) or top six (LTQ-Orbitrap) precursor ions were selected for subsequent MS/MS scans. A lock mass function was used for the LTQ-Orbitrap to obtain constant mass accuracy during gradient analysis (32).

Database Searching—
Mass Navigator version 1.2 (Mitsui Knowledge Industry, Tokyo, Japan) was used to create peak lists on the basis of the recorded fragmentation spectra. All parameters used in this process are described in Supplemental Table S1. Peptides and proteins were identified by means of automated database searching using Mascot version 2.1 (Matrix Science, London, UK) against UniProt/Swiss-Prot release 9.0 (October 31, 2006) with a precursor mass tolerance of 50 ppm (QSTAR) or 3 ppm (LTQ-Orbitrap), a fragment ion mass tolerance of 0.25 Da (QSTAR) or 0.8 Da (LTQ-Orbitrap), taxonomy of human, and strict trypsin specificity (33) allowing for up to two missed cleavages. Carbamidomethylation of cysteine was set as a fixed modification, and oxidation of methionines, and phosphorylation of serine, threonine, and tyrosine were allowed as variable modifications. Note that neutral loss products from both precursor and fragment ions were considered for Mascot scoring in this phosphorylation modification setting, although no assignment was indicated for precursor-origin neutral loss peaks in the output results according to the supplier.

Peptides were considered identified if the Mascot score was over the 95% confidence limit based on the "identity" score of each peptide and at least three successive y- or b-ions with two and more y-, b-, and/or precursor-origin neutral loss ions were observed, based on an error-tolerant peptide sequence tag concept (34). A randomized decoy database created by a Mascot Perl program estimated a 4.01% false-positive rate for identified peptides within the criteria.

Phosphorylated sites were determined based on the difference in Mascot scores of peptides with different phosphorylated sites (delta score). If the delta score was more than 5, the top ranked phosphorylated site was considered determined. All MS/MS spectra with delta score ≤5 were manually inspected as to whether the phosphorylation sites were unambiguously determined or not. Note that phosphorylation can be discriminated from sulfation by LTQ-Orbitrap with the lock mass function because the error distribution is within 2 ppm (32), which is less than the mass difference between sulfation and phosphorylation (9.516 mDa) for most of detectable peptides.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
Effect of Hydroxy Acids on Phosphopeptide Enrichment—
A digested phosphoprotein standard mixture of {alpha}-casein, fetuin, and phosvitin was used to evaluate the effect of hydroxy acids in the loading buffer on selective enrichment of phosphopeptides with titania, zirconia, alumina, Al(OH)3·xH2O, and boehmite MOC tips (Table I). All experiments were performed under acidic conditions in the presence of 0.1% trifluoroacetic acid and 80% acetonitrile because it has been reported that acidic conditions with 50–80% acetonitrile were effective to reduce the co-purification of acidic peptides (8, 10, 18, 19). Without hydroxy acids, eight or nine phosphopeptides were identified with titania, zirconia, and Al(OH)3·xH2O MOC tips, and the amounts of non-phosphorylated peptides were not negligible.


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  		TABLE I Enrichment of phosphopeptides derived from standard proteins

Tryptic digests of {alpha}-casein, fetuin, and phosvitin (started from 2.5 µg of each protein) were purified using MOC tips with/without hydroxy acids, and the eluted fractions were analyzed by means of nano-LC-MS/MS with QSTAR and Mascot search.

 
Larsen et al. (18) reported that DHB, which is commonly used as a matrix in MALDI-MS analysis, reduced the number of non-phosphorylated peptides in phosphopeptide enrichment with titania. In our study, DHB was also effective to decrease non-phosphorylated peptides in the cases of zirconia and Al(OH)3·xH2O as well as titania. The number of phosphopeptides, however, also decreased as DHB was added to the loading buffer. This is because we used LC-ESI-MS in which the residual DHB was eluted in the same retention time range as phosphopeptides, and significant suppression of phosphopeptide ionization occurred. Furthermore DHB caused various problems with the LC-MS system, including column clogging, precipitation around the orifice, and decrease of the sensitivity after several runs. Therefore we decided to look for an alternative. Aliphatic {alpha}-hydroxycarboxylic acids are well known to interact specifically with metal oxides, such as titania and zirconia (35), probably through chelation to form a five-membered ring. As summarized in Table I, most of the hydroxy acids improved the selectivity of phosphopeptide enrichment by titania and zirconia MOC tips, although they were not effective for Al(OH)3·xH2O MOC tips.

Among the hydroxy acids used, lactic acid and HPA were most effective with titania and zirconia MOC tips, respectively, efficiently suppressing the identification of non-phosphorylated peptides. Fig. 1 shows that no peaks from non-phosphopeptides were observed in the total ion chromatogram of LC-MS/MS analysis after phosphopeptide enrichment with titania MOC tips modified with lactic acid. Because these aliphatic hydroxy acids are hydrophilic enough to pass through the C18 columns under the conditions used, they can be easily removed during the desalting step using C18 StageTips. These aliphatic acids did not appear to impair the stability or the sensitivity of our LC-MS systems.


Figure 1
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  		FIG. 1. Total ion chromatograms of digested standard phosphoproteins. Digested peptides from {alpha}-casein, fetuin, and phosvitin were analyzed by nano-LC-MS/MS with QSTAR before (a) or after the enrichment step (b) using lactic acid-modified titania MOC tips. Asterisk- labeled peaks in b were identified as phosphorylated peptides by Mascot search. All unlabeled peaks in b are derived from singly charged ions, and the peaks with retention time >36 min were also observed in the blank. The MS spectrum at 33.6 min and MS/MS spectrum for the precursor ion of m/z 830.7 are shown in c and d, respectively. cps, counts/s.

 
Hydroxy acid binds to metal oxide by forming a cyclic chelate (35, 36), whereas phosphate anions do so by bridging two metal ions (37). Thus, hydroxy acids such as DHB and aliphatic hydroxycarboxylic acids should bind to metal oxides more weakly than a phosphate group but more strongly than the carboxylic groups of acidic non-phosphopeptides. Interestingly HPA was more effective than glycolic acid with both titania and zirconia in this study, whereas Tani and Ozawa (35) reported the opposite results in terms of affinity strength. This may be because there are various solid states of titania, and the retention properties depend strongly on the calcination temperature of the beads not only for hydroxy acids (38) but also for mono- and disaccharides (39). We also evaluated a mixture of glutamate, aspartate, and imidazole as additives in the loading buffer (20). However, we found that several non-phosphorylated peptides were always identified in the same standard sample loaded on titania and zirconia and Al(OH)3·xH2O MOC tips, and no improvement was seen compared with aliphatic hydroxy acid-modified MOC tips.

Recovery of Phosphopeptides after Enrichment with Titania MOC Tip—
Recovery of phosphopeptides after enrichment with lactic acid-modified titania MOC tips was examined using the peak areas in extracted ion current chromatograms of phosphopeptide-enriched and non-enriched samples (Table II). Recovery of individual phosphopeptides ranged from a few percent to around 100% (average, 49% for 13 phosphopeptides). On the other hand, recovery of all non-phosphorylated peptides was below 0.5% (data not shown). Similar results were obtained with HPA-modified zirconia MOC tips. These results indicated that this enrichment procedure is highly specific for phosphopeptides. Variable recovery was also observed in IMAC enrichment, and the average recovery was very low (less than 3% for 20 phosphopeptides and less than 10% for 10 phosphopeptides after methyl esterification) (9). A more efficient elution procedure from MOC tips, that would be independent of peptide amount and the sequence, is clearly needed. So far we have found that the recovery was slightly improved by successive elution with ammonium hydroxide followed by 80% acetonitrile. Further study is needed on this issue.


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  		TABLE II Recovery rates and reproducibility of detection of selected phosphopeptides in the enrichment procedure using lactic acid-modified titania MOC tips

Tryptic digests of {alpha}-casein, fetuin, and phosvitin (2.5 µg/tip) were purified using lactic acid-modified titania MOC tips. Recovery was calculated based on peak areas in the extracted ion chromatograms of the phosphopeptide-enriched sample and sample without enrichment. Peak intensities were from samples without enrichment. Bold and underlined letters represent identified and ambiguous phosphorylated sites, respectively.

 
The values of relative standard deviation of triplicate analysis for 13 peptides (Table II) ranged from 9.1 to 81% (average, 24%). Linearity was also investigated in the range from 0.125 to 25 µg of total loaded amount of digested peptides per titania MOC tip, with six data points, as shown in Supplemental Fig. S1. Five phosphopeptides with the smallest peak areas in Table II were selected to avoid saturation of the MS detector. The correlation coefficients of these five phosphopeptides ranged from 0.927 to 0.999, indicating that the method is satisfactory for quantitation.

Enrichment of Phosphopeptides in HeLa Cytoplasmic Extracts—
There have been only a few reports of the application of phosphopeptide enrichment methods to real complex samples such as crude cell extracts including very recent large scale studies (40, 41). In those studies, ~10 mg of starting materials were used for strong cation exchange chromatography prior to phosphopeptide enrichment. To our knowledge there has been no report describing the successful use of a single MOC-based method for phosphopeptide analysis of cell lysates using less than 1 mg as the starting material. It is an extremely challenging task to enrich phosphopeptides from real complex mixtures with a high dynamic range. Therefore we examined the applicability of aliphatic hydroxy acid-modified MOC tips for phosphopeptide enrichment from the cytoplasmic fraction of HeLa cells with less than a 1-mg amount. Fig. 2 shows the number of identified phosphopeptides and non-phosphopeptides following enrichment with three titania MOC tips, i.e. a lactic acid-modified tip, a DHB-modified tip, and a tip without modifier. The ratio of the number of identified phosphopeptides to that of all identified peptides was very low (ratio = 0.008) when no modifier was used. The use of DHB slightly improved the enrichment selectivity as well as the number of phosphopeptides (ratio = 0.111). The lactic acid-modified titania tip gave the highest selectivity (ratio = 0.607) as well as the largest number of identified phosphopeptides. It is noteworthy that ~350 phosphopeptides were obtained in a single 2-h nano-LC-MS/MS gradient run without repetition. Similar results were also obtained for an HPA-modified zirconia tip in comparison with a DHB-modified zirconia tip and a zirconia tip without any modifier. Table III compares the results obtained after enrichment with lactic acid-modified titania MOC and HPA-modified zirconia MOC tips. In total, 1100 and 1181 phosphopeptides were identified by four replicated experiments using titania and zirconia MOC tips, respectively (Table III and Supplemental Table S2). Of 1645 distinct identified phosphopeptides, 636 were found by both methods, and the other peptides were uniquely identified by a single method. Of 1629 phosphorylated sites, 1467 phosphorylation sites from 1502 peptides were unambiguously determined. On the other hand, the exact phosphorylated sites were unknown for the other 143 phosphorylated peptides because of the lack of sufficient fragmentation ions, although these peptides were unambiguously phosphorylated. Thus, complementary use of titania and zirconia MOC tips may allow a relatively comprehensive analysis of phosphopeptides. Note that zirconia did not show any tendency to enrich singly phosphorylated peptides in our final list, contrary to the finding of Kweon and Hakansson (19).


Figure 2
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  		FIG. 2. Enrichment of phosphopeptides in the cytoplasmic fraction of HeLa cells. The cell extract was digested with trypsin and enriched using titania MOC tips without modifier, with DHB, and with lactic acid. The enriched samples were analyzed by nano-LC-MS/MS with the LTQ-Orbitrap. The numbers of phospho- and non-phosphopeptides were obtained by averaging duplicate experimental results.

 

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  		TABLE III Identification of phosphoproteins, phosphopeptides, and their phosphorylated sites in the cytoplasmic fraction of HeLa cells

Nano-LC-MS/MS was performed using the LTQ-Orbitrap. These data were obtained from four replicated experimental results with four nano-LC-MS/MS runs. The number of phosphorylated peptides was based on unique sequences containing missed cleavage products, oxidization of methionine, and phosphorylation of different sites. The number of phosphorylated sites was calculated from phosphopeptides with modification sites determined unambiguously. Note that 66 peptides of a total 1645 phosphopeptides were ambiguous for either phosphorylation or sulfation.

 
Conclusion—
We developed highly selective enrichment methods for phosphopeptides using lactic acid-modified titania and HPA-modified zirconia MOC tips. Both methods were applied to the cytoplasmic fraction of HeLa cells, and over a thousand phosphopeptides were identified. The combination of these methods is expected be useful for proteome-wide experiments to study cell signaling networks, which require the enrichment of many phosphopeptides from real complex mixtures with a wide dynamic range.


   ACKNOWLEDGMENTS
 
We thank Sumiko Ohnuma and Yasuyuki Igarashi for technical assistance and Motomu Matsui for developing tools for Mascot data analysis. We also thank Dr. Jesper V. Olsen (Max Planck Institute) for kind support for the LTQ-Orbitrap.


  FOOTNOTES
 
Received, November 9, 2006, and in revised form, February 21, 2007.

Published, MCP Papers in Press, February 23, 2007, DOI 10.1074/mcp.T600060-MCP200

1 The abbreviations used are: MOC, metal oxide chromatography; HPA, ß-hydroxypropanoic acid; DHB, 2,5-dihydroxybenzoic acid. Back

* This work was supported by a research fund from Yamagata Prefecture and Tsuruoka City. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

S The on-line version of this article (available at http://www.mcponline.org) contains supplemental material. Back

|| To whom correspondence should be addressed. Tel.: 81-235-29-0571; Fax: 81-235-29-0536; E-mail: y-ishi{at}ttck.keio.ac.jp


   REFERENCES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 

  1. Hunter, T. (2000) Signaling—2000 and beyond. Cell 100, 113 –127[CrossRef][Medline]

  2. Aebersold, R., and Mann, M. (2003) Mass spectrometry-based proteomics. Nature 422, 198 –207[CrossRef][Medline]

  3. de Godoy, L. M., Olsen, J. V., de Souza, G. A., Li, G., Mortensen, P., and Mann, M. (2006) Status of complete proteome analysis by mass spectrometry: SILAC labeled yeast as a model system. Genome Biol. 7, R50[CrossRef][Medline]

  4. Steen, H., Kuster, B., Fernandez, M., Pandey, A., and Mann, M. (2002) Tyrosine phosphorylation mapping of the epidermal growth factor receptor signaling pathway. J. Biol. Chem. 277, 1031 –1039[Abstract/Free Full Text]

  5. Rush, J., Moritz, A., Lee, K. A., Guo, A., Goss, V. L., Spek, E. J., Zhang, H., Zha, X. M., Polakiewicz, R. D., and Comb, M. J. (2005) Immunoaffinity profiling of tyrosine phosphorylation in cancer cells. Nat. Biotechnol. 23, 94 –101[CrossRef][Medline]

  6. Gronborg, M., Kristiansen, T. Z., Stensballe, A., Andersen, J. S., Ohara, O., Mann, M., Jensen, O. N., and Pandey, A. (2002) A mass spectrometry-based proteomic approach for identification of serine/threonine-phosphorylated proteins by enrichment with phospho-specific antibodies: identification of a novel protein, Frigg, as a protein kinase A substrate. Mol. Cell. Proteomics 1, 517 –527[Abstract/Free Full Text]

  7. Stensballe, A., Andersen, S., and Jensen, O. N. (2001) Characterization of phosphoproteins from electrophoretic gels by nanoscale Fe(III) affinity chromatography with off-line mass spectrometry analysis. Proteomics 1, 207 –222[CrossRef][Medline]

  8. Kokubu, M., Ishihama, Y., Sato, T., Nagasu, T., and Oda, Y. (2005) Specificity of immobilized metal affinity-based IMAC/C18 tip enrichment of phosphopeptides for protein phosphorylation analysis. Anal. Chem. 77, 5144 –5154[Medline]

  9. Haydon, C. E., Eyers, P. A., Aveline-Wolf, L. D., Resing, K. A., Maller, J. L., and Ahn, N. G. (2003) Identification of novel phosphorylation sites on Xenopus laevis Aurora A and analysis of phosphopeptide enrichment by immobilized metal-affinity chromatography. Mol. Cell. Proteomics 2, 1055 –1067[Abstract/Free Full Text]

  10. Ficarro, S. B., McCleland, M. L., Stukenberg, P. T., Burke, D. J., Ross, M. M., Shabanowitz, J., Hunt, D. F., and White, F. M. (2002) Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae. Nat. Biotechnol. 20, 301 –305[CrossRef][Medline]

  11. Ndassa, Y. M., Orsi, C., Marto, J. A., Chen, S., and Ross, M. M. (2006) Improved immobilized metal affinity chromatography for large-scale phosphoproteomics applications. J. Proteome Res. 5, 2789 –2799[CrossRef][Medline]

  12. Ballif, B. A., Villen, J., Beausoleil, S. A., Schwartz, D., and Gygi, S. P. (2004) Phosphoproteomic analysis of the developing mouse brain. Mol. Cell. Proteomics 3, 1093 –1101[Abstract/Free Full Text]

  13. Beausoleil, S. A., Jedrychowski, M., Schwartz, D., Elias, J. E., Villen, J., Li, J., Cohn, M. A., Cantley, L. C., and Gygi, S. P. (2004) Large-scale characterization of HeLa cell nuclear phosphoproteins. Proc. Natl. Acad. Sci. U. S. A. 101, 12130 –12135[Abstract/Free Full Text]

  14. Ikeguchi, Y., and Nakamura, H. (1997) Determination of organic phosphates by column-switching high performance anion-exchange chromatography using on-line preconcentration on titania. Anal. Sci. 13, 479 –483

  15. Sano, A., and Nakamura, H. (2004) Chemo-affinity of titania for the column-switching HPLC analysis of phosphopeptides. Anal. Sci. 20, 565 –566[CrossRef][Medline]

  16. Ishihama, Y., and Mann, M. (2003) Development of nanoLC-MS/MS systems for proteomics. Chromatography 24, Suppl. 1,12 –13

  17. Pinkse, M. W., Uitto, P. M., Hilhorst, M. J., Ooms, B., and Heck, A. J. (2004) Selective isolation at the femtomole level of phosphopeptides from proteolytic digests using 2D-NanoLC-ESI-MS/MS and titanium oxide precolumns. Anal. Chem. 76, 3935 –3943[Medline]

  18. Larsen, M. R., Thingholm, T. E., Jensen, O. N., Roepstorff, P., and Jorgensen, T. J. (2005) Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns. Mol. Cell. Proteomics 4, 873 –886[Abstract/Free Full Text]

  19. Kweon, H. K., and Hakansson, K. (2006) Selective zirconium dioxide-based enrichment of phosphorylated peptides for mass spectrometric analysis. Anal. Chem. 78, 1743 –1749[Medline]

  20. Wolschin, F., Wienkoop, S., and Weckwerth, W. (2005) Enrichment of phosphorylated proteins and peptides from complex mixtures using metal oxide/hydroxide affinity chromatography (MOAC). Proteomics 5, 4389 –4397[CrossRef][Medline]

  21. Annan, R. S., Huddleston, M. J., Verma, R., Deshaies, R. J., and Carr, S. A. (2001) A multidimensional electrospray MS-based approach to phosphopeptide mapping. Anal. Chem. 73, 393 –404[Medline]

  22. Gruhler, A., Olsen, J. V., Mohammed, S., Mortensen, P., Faergeman, N. J., Mann, M., and Jensen, O. N. (2005) Quantitative phosphoproteomics applied to the yeast pheromone signaling pathway. Mol. Cell. Proteomics 4, 310 –327[Abstract/Free Full Text]

  23. Beausoleil, S. A., Villen, J., Gerber, S. A., Rush, J., and Gygi, S. P. (2006) A probability-based approach for high-throughput protein phosphorylation analysis and site localization. Nat. Biotechnol. 24, 1285 –1292[CrossRef][Medline]

  24. Schroeder, M. J., Shabanowitz, J., Schwartz, J. C., Hunt, D. F., and Coon, J. J. (2004) A neutral loss activation method for improved phosphopeptide sequence analysis by quadrupole ion trap mass spectrometry. Anal. Chem. 76, 3590 –3598[Medline]

  25. Syka, J. E., Coon, J. J., Schroeder, M. J., Shabanowitz, J., and Hunt, D. F. (2004) Peptide and protein sequence analysis by electron transfer dissociation mass spectrometry. Proc. Natl. Acad. Sci. U. S. A. 101, 9528 –9533[Abstract/Free Full Text]

  26. Nuhse, T. S., Stensballe, A., Jensen, O. N., and Peck, S. C. (2003) Large-scale analysis of in vivo phosphorylated membrane proteins by immobilized metal ion affinity chromatography and mass spectrometry. Mol. Cell. Proteomics 2, 1234 –1243[Abstract/Free Full Text]

  27. Nawrocki, J., Dunlap, C., McCormick, A., and Carr, P. W. (2004) Part I. Chromatography using ultra-stable metal oxide-based stationary phases for HPLC. J. Chromatogr. A 1028, 1 –30[CrossRef][Medline]

  28. Saito, H., Oda, Y., Sato, T., Kuromitsu, J., and Ishihama, Y. (2006) Multiplexed two-dimensional liquid chromatography for MALDI and nanoelectrospray ionization mass spectrometry in proteomics. J. Proteome Res. 5, 1803 –1807[CrossRef][Medline]

  29. Rappsilber, J., Ishihama, Y., and Mann, M. (2003) Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. Anal. Chem. 75, 663 –670[Medline]

  30. Ishihama, Y., Rappsilber, J., and Mann, M. (2006) Modular stop and go extraction tips with stacked disks for parallel and multidimensional peptide fractionation in proteomics. J. Proteome Res. 5, 988 –994[CrossRef][Medline]

  31. Ishihama, Y., Rappsilber, J., Andersen, J. S., and Mann, M. (2002) Microcolumns with self-assembled particle frits for proteomics. J. Chromatogr. A 979, 233 –239[CrossRef][Medline]

  32. Olsen, J. V., de Godoy, L. M., Li, G., Macek, B., Mortensen, P., Pesch, R., Makarov, A., Lange, O., Horning, S., and Mann, M. (2005) Parts per million mass accuracy on an Orbitrap mass spectrometer via lock mass injection into a C-trap. Mol. Cell. Proteomics 4, 2010 –2021[Abstract/Free Full Text]

  33. Olsen, J. V., Ong, S. E., and Mann, M. (2004) Trypsin cleaves exclusively C-terminal to arginine and lysine residues. Mol. Cell. Proteomics 3, 608 –614[Abstract/Free Full Text]

  34. Mann, M., and Wilm, M. (1994) Error-tolerant identification of peptides in sequence databases by peptide sequence tags. Anal. Chem. 66, 4390 –4399[Medline]

  35. Tani, K., and Ozawa, M. (1999) Investigation of chromatographic properties of titania. I. On retention behavior of hydroxyl and other substituent aliphatic carboxylic acids: comparison with zirconia. J. Liq. Chromatogr. Relat. Technol. 22, 843 –856[CrossRef]

  36. Tunesi, S., and Anderson, M. (1991) Influence of chemisorption on the photodecomposition of salicylic acid and related compounds using suspended titania ceramic membranes. J. Phys. Chem. 95, 3399 –3405[CrossRef]

  37. Connor, P. A., and McQuillan, A. J. (1999) Phosphate adsorption onto TiO2 from aqueous solutions: an in situ internal reflection infrared spectroscopic study. Langmuir 15, 2916 –2921[CrossRef]

  38. Tani, K., and Miyamoto, E. (1999) Investigation of chromatographic properties of titania. II. Influence of calcination temperature. J. Liq. Chromatogr. Relat. Technol. 22, 857 –871[CrossRef]

  39. Tani, K., Kitada, M., Tachibana, M., Koizumi, H., and Kiba, T. (2003) Retention behavior of monosaccharides and disaccharides on titania. Chromatographia 57, 409 –412[CrossRef]

  40. Olsen, J. V., Blagoev, B., Gnad, F., Macek, B., Kumar, C., Mortensen, P., and Mann, M. (2006) Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell 127, 635 –648[CrossRef][Medline]

  41. Villen, J., Beausoleil, S. A., Gerber, S. A., and Gygi, S. P. (2007) Large-scale phosphorylation analysis of mouse liver. Proc. Natl. Acad. Sci. U. S. A. 104, 1488 –1493[Abstract/Free Full Text]


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X. Zhang, J. Ye, O. N. Jensen, and P. Roepstorff
Highly Efficient Phosphopeptide Enrichment by Calcium Phosphate Precipitation Combined with Subsequent IMAC Enrichment
Mol. Cell. Proteomics, November 1, 2007; 6(11): 2032 - 2042.
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