Figure 1. Specific enrichment of phosphopep- tides by metal oxide chromatography. 500 fmol of a mixture of 10 phosphorylated and 2 unphosphorylated peptides were separated by nano-RP-HPLC coupled to an LTQ Orbitrap mass spectrometer (a) without prior TiO2 purification and (b) after being subjected to a TiO2 purification step using the modified proto- col. In (b), following the use of TiO 2 tips for phosphopeptide enrichment and clean-up; the signals of the unphosphorylated peptides disappeared or were greatly reduced, whereas the recovery of phosphorylated peptides turned out to be very efficient. A new loading solvent composition and a new wash procedure for phosphopeptide enrichment by metal oxide chromatography was tested to improve our previously published method (Mazanek et al., 2006). In the loading solvent the concentration of OSA and DHB was increased and HFBA was added as a new component. The wash procedure was extended by an extra wash step using the loading solvent and the ACN concentration in the second wash step was increased. Additionally, we compared the performance of TiO 2 and ZrO 2 columns. Applied to a peptide test mix, the new procedure showed improved results, both in terms of higher phosphopeptide binding efficiency (on TiO 2 ) and better exclusion of unphosphorylated peptides (on both TiO 2 and ZrO 2 ). Applied to two immunopurifed human complexes, the new enrichment protocol led to the identification of a significantly higher number of phosphopeptides when compared to the old procedure or the analysis of the untreated samples. Both, TiO 2 and ZrO 2 performed similarly well and led to the identification of an partially overlapping set of phosphopeptides. A New Acid Mix Enhances Phosphopeptide Enrichment on Titanium- and Zirconium Dioxide for Mapping of Phosphorylation Sites on Protein Complexes Michael Mazanek, Goran Mitulovic, Otto Hudecz, Björn Hegemann, James Hutchins, Jan-Michael Peters, Elisabeth Roitinger, Karl Mechtler. I.M.P - Research Institute of Molecular Pathology (Protein Chemistry Facility and Mass Spectrometry). 1030 Vienna, Dr. Bohrgasse 7; Austria Homepage: http://www.imp.univie.ac.at/protein Figure 5. Phospho- peptides identified in the APC and the Condensin complex with different analytical methods. A significantly higher number of unique peptides, especially for the condensin complex, were identified when applying the new load and wash Acknowledgements: Research in the lab of Karl Mechtler is supported by the 6th Framework Programme of the European Union via the Integrated Project MitoCheck and the Austrian Proteomics Platform (APP) within the Austrian Genome Program (GEN-AU), Vienna, Austria. A big THANK YOU goes to the protein group: Ines Steinmacher, Christoph Stingl, Matthias Madalinksi, Gabi Krssakova. Old enrichment method: Load: 20% acetic acid, containing 1-Octanesulfonic acid sodium salt and Dihydroxybenzoic acid. Wash: 2 x 25 μl of 30% ACN Elute: 3 x 13.3 μl) of elution buffer (125 mM ammonium bicarbonate, phosphoric acid to produce 50 mM ammonium phosphate, adjusted with ammonia to pH 10.5. New enrichment method: Load: 20% acetic acid, containing 1-Octanesulfonic acid sodium salt, Dihydroxybenzoic acid and Heptafluorobutyric acid. Wash: Loading solvent followed by 80% ACN, 0.1% TFA. Elute: 50 mM ammonium dihydrogenphosphate, pH 10.5 (adjusted with ammonia solution). Phospho- and non-phosphopeptides used in this study: Name Sequence PP1 TASDTDSSpY AIPTAGMSPSR PP2 SVENLPEAGIpTHEQR PP3 NpSVEQGRRL PP4 APPDNLPSPGGpSR PP5 LIEDNEpYTAR NP6 APPDNLPSPGGSR PP7 RpSDGGHTVLHR PP8 ENIMRpSENSESQLTSK PP9 QLGEPEKpSQDSSPVLpSELK PP10 QLGEPEKpSQDpSSPVLpSELK PP11 THILLFLPKpSVSDYEGK GluFib EGVNDNEEGFFSAR Chromatographic system: UltiMate + Nano-LC system (LC-Packings – A Dionex company) Mass spectrometry: Thermo Finnigan – Orbi Trap operated in positive nano-ESI mode. IS (spray voltage) = 1500 V The selective enrichment of phosphorylated peptides prior to reversed phase separation and mass spectrometric detection signifi- cantly improves the analytical results in terms of higher number of detected phospho- rylation sites and spectra of higher quality. We have described a protocol for titanium dioxide based enrichment of phosphopep- tides which is directly compatible with subse- quent analysis by online nano-reversed phase HPLC-MS/MS (Mazanek et al., 2006). In the present work we have tested a new combination of loading and washing condi- tions and two different column materials (TiO 2 vs ZrO 2 ) in order to improve the perfor- mance of the method used in our laboratory. To confirm the improvement of the resulting optimized method in terms of the number of identified phosphopeptides, tryptic digests of two immunopurified human protein complexes were either analyzed by nano- reversed phase HPLC-MS/MS without prior enrichment or they were loaded on TiO 2 according to the previous enrichment proto- col (Mazanek et al., 2006) or on TiO 2 and ZrO 2 according to the optimized method. Follow- ing TiO 2 treatment according to the new enrichment method 128 phosphopeptides were identified in both protein complexes compared to 78 phosphopeptides after using the previous protocol and 67 phosphopep- tides in the untreated samples. Using ZrO 2 110 phosphopeptides were identified. Summing up the results obtained with both TiO 2 and ZrO 2 chromatography, 159 different phosphopeptides were identified in both complexes indicating that there is only a partial overlap of the peptides enriched on each material and that the application of both materials to the same sample in parallel could lead to a more comprehensive result. In summary, our improved method is highly effective for the enrichment of phosphopep- tides from biological samples prior to mass spectrometry, and is suitable for high-throughput phosphoproteomic projects that aim to uncover phosphorylation- dependent signaling pathways. Figure 6. MS/MS spectra showing the condensin peptide YQPLASTASDNDFVTPEPR in three different phosphorylation states which were enriched via TiO 2 . The fragmentation pattern for the single, double and triple phosphorylated peptides (all doubly charged) look very similar. All three spectra are reliable because of their strong peaks and almost complete b and y ion series. Selectivity and Recovery (continued) IV. Biological samples V. Abstract Selectivity and Recovery III. MS/MS Spectra VII. Experimentals (overview) II. Summary VIII. Untreated Ti_old Ti_new Zr_new phosphorylation sites 35 37 60 46 phosphopeptides 43 38 73 49 APC Complex Untreated Ti_old Ti_new Zr_new phosphorylation sites 20 26 36 45 phosphopeptides 24 40 55 61 Condensin Complex 0% 20% 40% 60% 80% 100% PP1 PP2 PP3 PP4 PP5 NP6 PP7 PP8 PP9 PP10 PP11 GluFib Percentage recovery of peptides Ti_old Ti_new Zr_new be superior to TiO2 as both unphosphorylated peptides were almost completely excluded from binding to ZrO2. Figure 2. Effect of the new procedure regarding selectivity and recovery rate. 500fmol of the 12 peptide mix were either directly analysed by nano-RP-HPLC- MS/MS or were subjected to TiO 2 enrich- ment prior to the analysis applying either the old or the newly modified protocol. Additionally, the new protocol was applied to phosphopeptide enrichment using ZrO 2 tips. The experiment was repeated 10 times and the mean value of the recovery rate of each peptide was taken. The new protocol applied on TiO 2 led to a slightly higher recovery rate for phosphopeptides and to a clear signal reduction of the two unphosphorylated acidic peptides when compared to the old protocol. For unspe- cifically binding peptides ZrO 2 showed to Biological samples (continued) VI. Figure 4 Two Protein complexes were immunopuri- fied from HeLa cell extracts. The Anaphase Promoting Complex (APC) was immu- nopurified from nocodazole arrested HeLa cells using APC3 antibodies and the Condensin-I (Condensin) complex was immunopurified from a nocodazole arrested HeLa-‘Kyoto’ cell line express- ing Kleisin-γ-GFP-FLAG using FLAG-antibodies. 10% of each sample was subjected to SDS-PAGE followed by silver staining. 90% of each sample was digested with trypsin and split into 4 equal parts, which were subjected to the different MOC methods as described below. Table 1. Phosphopeptides and phosphorylation sites identified in the APC and Condensin complex following phosphopeptide enrichment according to three analytical approaches. method to TiO 2 and ZrO 2 chromatography in comparison to the old enrichment method and to the direct approach without enrichment. APC APC Condensin I. Condensin (b) Figure 3. Inclusion of a wash step using the loading buffer affects the retention of acidic unphosphorylated peptides on TiO 2 . A mix containing 500 fmol trypsinised BSA plus 500fmol of the 12 peptide mix were enriched (TiO 2 tips), using the new protocol either excluding (a), or including (b) an additional wash step using the loading buffer. Shown here are the selected ion traces of different BSA-derived and synthetic peptides which eluted from TiO 2 after application of the two different wash conditions. The reduction in peak heights for unphospho- rylated peptides in (b) compared to (a) indicates that the addition of loading buffer significantly increased the efficiency of the wash step. All 10 phosphopeptides showed similarly high signal intensities after the additional wash step. (a)