Effect of Peptide-to-TiO2 Beads Ratio on Phosphopeptide Enrichment

Sep 17, 2009 - recently, including online/offline mode and optimization of sample loading/elution buffer; however, beads usage has rarely been explore...
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Effect of Peptide-to-TiO2 Beads Ratio on Phosphopeptide Enrichment Selectivity Qing-run Li,† Zhi-bin Ning,† Jia-shu Tang, Song Nie, and Rong Zeng* Key Laboratory of Systems Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200031, China Received July 24, 2009

Abstract: Titanium dioxide (TiO2) has been proven to be a highly efficient strategy and widely used for phosphopeptide enrichment. Many advances have been made recently, including online/offline mode and optimization of sample loading/elution buffer; however, beads usage has rarely been explored. In the current study, we found that the peptide-to-TiO2 beads ratio was a significant factor for phosphopeptide enrichment, and insufficient or excessive beads could decrease the selectivity. Specifically, for HeLa total cell lysate, the optimum peptide-tobeads ratio is about 1:2-1:8 (mass/mass) to obtain the highest enrichment selectivity and the maximum phosphopeptides identification with single incubation. Preexperiments are recommended to decide an optimum peptide-to-TiO2 beads ratio when it comes to different samples. Interestingly, deficient beads can help identify much more multiphosphorylated peptides than the optimum peptide-to-beads ratio by consecutive incubations. Therefore, if multiphosphorylated peptides identification is desired, deficient beads amount is preferred. In addition, consecutive incubation using deficient beads could be used as a fractionation of phosphopeptides besides as an enrichment method. Keywords: peptide-to-TiO2 beads ratio • phosphorylation • enrichment • selectivity

Introduction Due to the relative low abundance of phosphoproteins in the global phosphoproteome analysis, it is still a formidable but critical task to study phosphorylation in a large-scale manner. The evolution of mass spectrometry-based proteomics helped to develop powerful analytical strategies for posttranslational modification (PTM) analysis such as phosphorylation.1-4 On the other hand, for sensitive and highly selective phosphorylation analysis, various techniques have been introduced to enrich phosphopeptides prior to mass spectrometric analyses in recent years, such as the antibody-based method,5,6 strong anion/cation exchange chromatography (SCX/SAX),7-9 immobilized metal affinity chromatography (IMAC),10,11 and the hydrophilic interaction chromatography (HILIC) as well.12,13 * To whom correspondence should be addressed. Prof. Rong Zeng, e-mail: [email protected]. † These authors contributed equally to this work. 10.1021/pr900659n CCC: $40.75

 2009 American Chemical Society

Titanium dioxide (TiO2) chromatography has been recently proven to be a highly efficient strategy for enrichment of phosphopeptides before MS analysis.14-16 With this approach, an online TiO2 precolumn was used in a two-dimensional system for successful enrichment of phosphopeptides from complex mixtures. Recently, offline mode, such as TiO2 tip, was developed for phosphopeptide purification.17,18 Moreover, to improve the binding selectivity of phosphopeptides on TiO2, a novel modification by introducing 2,5-dihydroxybenzoic acid (DHB) as a competitor to prevent adsorption of nonphosphopeptides was developed.17 The optimized strategy presents substantial enhancement for phosphopeptide enriched on a TiO2 column. However, given that the loading capacity is the limit for a TiO2 microcolumn, a more straightforward protocol (“batch” mode) was employed to add TiO2 beads to protein digest directly and several incubations were performed.19 This workflow provided a simple but robust way to perform phosphopeptide enrichment and therefore is widely used for phosphoproteome exploration.20-23 To improve the phosphopeptide binding specificity for TiO2 column/beads, subsequent development focused on the optimization of sample loading buffer, such as DHB, ammonium glutamate,24 and glutamic acid25 and other acidic reagents were evaluated as modifiers.18,26-28 More recently, Bodenmiller et al. has mentioned the influence of the peptide-to-resin ratio on the selectivity and percentage of singly phosphorylated peptides in the case of IMAC and TiO2.27 However, the presented data is limited and therefore did not draw much attention in this field. To date, there are no further investigations on TiO2 beads dosage except the above study to our knowledge. In this work, we performed intensive experiments to investigate the phosphopeptides binding behavior to TiO2 beads. We found that the peptide-to-TiO2 beads ratio was a substantial factor for the selectivity of isolation to phosphorylation peptides, and different peptide-to-beads ratios gave distinct selectivity profiles with consecutive incubations. In detail, for HeLa lysate, the optimum ratio is about from 1:2 to 1:8 (mass/ mass). With the optimum beads amount, we obtained the highest enrichment selectivity (higher than 80%). Besides, more multiphosphorylated peptides could be identified by deficient TiO2 beads. On the basis of the results in the current study, we recommend performing pre-experiment to determine the peptide-to-beads ratio to get higher selectivity. On the other hand, as an alternative experimental strategy, the deficient beads usage can also be employed to get multiphosphorylated peptides. Journal of Proteome Research 2009, 8, 5375–5381 5375 Published on Web 09/17/2009

technical notes Materials and Methods Chemicals. Acetonitrile (ACN) and trifluoroacetic acid (TFA) were obtained from Merck (Darmstadt, Germany). Formic acid (FA) was purchased from Aldrich (Milwaukee, WI). Urea, dithiothreitol (DTT), ammonium bicarbonate (ABC), CHAPS, and iodoacetamide (IAA) were all purchased from Bio-Rad (Hercules, CA). Sodium orthovanadate (Na3VO4), sodium fluoride (NaF) and glutamic aid were obtained from Sigma (St. Louis, MO). Trypsin was purchased from Promega (Madison, WI). All of the chemicals were of analytical purity grade except ACN and FA, which were of HPLC grade. All of the water used in the experiment was prepared using a Milli-Q system (Millipore, Bedford, MA). Sample Preparation. The HeLa cells were cultured in 10cm plates supplemented with 30 mL of Dulbecco’s modified Eagle’s medium and 10% FBS. When reached 90% confluence, the cells was washed twice with 10 mL of ice-cold PBS and frozen in liquid nitrogen. Then, the cells were lysed in lysis buffer (8 M urea, 4% CHAPS, 40 mM Tris-base, 65 mM DTT) with a mixture of phosphatase inhibitors (1 mM Na3VO4 and 10 mM NaF, Roche Diagnostics protease inhibitor mixture) on ice. The lysate was sonicated for 10s triplicate following centrifugation at 15 000 g for 45 min (4 °C). The supernatants were collected and the protein concentration was determined with Bradford assay (Bio-Rad). The tryptic digestion was processed according to the method as described elsewhere,29 and the protein digests were lyophilized completely and stored at -80 °C for further use. Phosphopeptide Enrichment. The phosphopeptide enrichment procedure was described elsewhere30,31 with some modification. In detail, the TiO2 beads (GL Sciences, Tokyo, Japan) were preincubated first in 200 µL loading buffer (65% ACN/ 2% TFA/saturated by glutamic acid) for acidifying. The peptide samples were resolved in 200 µL loading buffer, and then incubated with different amount of the TiO2 beads. For consecutive incubations, the peptide-beads slurry was incubated and centrifugated, then the supernatant was incubated with another aliquot of freshly prepared TiO2 beads for the next enrichment. The incubated beads were then washed with 800 µL wash buffer I (65% ACN/0.5% TFA) and wash buffer II (65% ACN/0.1% TFA). The bound peptides were eluted once with the 200 µL elution buffer I (300 mM NH4OH/50% ACN) and twice with 200 µL elution buffer II (500 mM NH4OH/60% ACN). All the incubation, washing as well as elution procedures were rotated end-overend for 20 min at room temperature. The eluates were dried down and reconstituted in 0.1% FA/H2O for MS analysis. MS Acquisition. All the samples were analyzed on nanoscale HLPC-MS system. A Surveyor liquid chromatography system coupled online to LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose, CA) equipped with a nanoelectrospray interface operated in positive ion mode. Separation was performed on a 75 µm I.D., 15 cm in length C18 reversed phase column (Column Technology Inc., CA). Pump flow was split to achieve a flow rate at 4 µL/min for sample loading and 300 nL/min for MS analysis. Peptides were eluted using a linear gradient of increasing mobile phase B (0.1% FA in ACN) from 2 to 35% in 80 min. The spray voltage was set to 2.1 kV and the temperature of heated capillary was 200 °C. The instrument method consisted of one full MS scan from 400 to 1800 m/z followed by data-dependent MS/MS scan of the seven most intense ions, a dynamic exclusion repeat count of 2, and a 5376

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Li et al. repeat duration of 90s. The full mass was scanned in orbitrap analyzer with R ) 100 000 (defined at m/z 400), and the subsequent MS/MS analysis were performed in LTQ analyzer. To improve the mass accuracy, all the measurements in the orbitrap mass analyzer were performed with on-the-fly internal recalibration (“Lock Mass”) described elsewhere32 at m/z 445.120025. The charge state rejection function was enabled, and for charge states “unassigned” and single charge state were rejected. All data were recorded with Xcalibur software (Thermo Fisher Scientific, San Jose, CA). Data Analysis. All 64 raw files were analyzed with software MaxQuant (Version 1.0.12.31).33 The generated .par and .msm files were searched using Mascot search engine (version 2.2.0, Matrix Science)34 against a database concatenating forward and reversed human International Protein Index protein sequence database (IPI human, version 3.52), including 262 commonly observed contaminants (http://www.maxquant.org/). For Mascot search, the following parameters were used: cysteine carbamidomethylation was selected as a fixed modification, the methionine oxidation, protein N-terminal acetylation, and phosphorylation on serine, threonine and tyrosine were selected as variable modifications. Up to two missing cleavage points were allowed. The precursor ion mass tolerances were 7 ppm, and fragment ion mass tolerance was 0.5 Da for MS/ MS spectra. The database search results (.dat files) were further processed with MaxQuant. The false discovery rate (FDR) of 1% and a minimum length of six amino acids were used for peptide identification.

Results and Discussion Effect of Peptide-to-Beads Ratio on Enrichment Selectivity. The selectivity of TiO2 to phosphopeptides suffered from nonspecifically binding of nonphosphorylated peptides. Although considerable efforts have been focused on minimization of the nonspecific interactions, the usage of a peptide-to-beads ratio has rarely been described. Recently, Bodenmiller et al. mentioned the effect of the peptide-to-resin ratio on the selectivity and percentage of singly phosphorylated peptides in the case of TiO2 and IMAC.35 However, there was no further intensive information. From experiment of the phosphopeptide enrichment using TiO2 beads, we found that the reproducibility of enrichment selectivity (measured by the ratio of the phosphopeptides to all peptides identified) varied along with the amount of sample and beads to start-up. For a systematic study on the influence of beads usage on phosphopeptide enrichment, we designed an experiment using different peptide-tobeads ratios to test if it really has something to do with the enrichment selectivity. HeLa cell digests (500 µg) were mixed with 125, 250, 500, 1000, 2000, 4000, 10 000, and 20 000 µg of TiO2 beads, respectively, and performed enrichment procedures in parallel. All enrichment incubations were performed exactly the same except for beads usage. All enrichment experiments were repeated to decrease variation. The numbers of phosphorylated peptides identified in each MS run including replicate experiment are shown in Figure 1. A remarkable changing tendency could be observed. The number of phosphopeptides increased with larger amount of TiO2 beads used from peptide-to-beads ratio of 1:0.25 (125 µg TiO2 beads), however, it fell down in the last two TiO2 beads amount (10 000 and 20 000 µg TiO2 beads). Obviously, the peptides number variation cannot simply be attributed to the different amount of TiO2 beads usage, because the nonphosphopeptide number did not follow the same profile as phos-

Effect of Peptide-to-TiO2 Beads Ratio

technical notes

Figure 1. Profiles of phosphopeptides identified when different peptide-to-beads ratio applied. The bar and curve indicate the number of phosphopeptides identified and the phosphopeptide ratio (the number of phosphopeptides divided by the total peptides identified in each incubation) in each bin. The x-axis shows the experiments for different peptide-to-beads ratio. A1 to A8 represent 125, 250, 500, 1000, 2000, 4000, 10 000, and 20 000 µg TiO2 beads respectively, and “-1” and “-2” correspond to duplicates of each experiment. For each incubation, 500 µg HeLa total cell lysate digest was used.

phopeptides (Figure S1, Supporting Information). We used the phosphopeptide ratio (defined as the number of phosphopeptides divided by the total peptides identified in each MS run) as the evaluation indicator, which could be interpreted directly as the enrichment selectivity or binding specificity. The phosphopeptide ratio varied dramatically when a different peptideto-beads ratio was applied. For example, only 317 phosphopeptides (1197 total peptides) were detected when 125 µg beads were applied (peptide-to-beads ratio ) 1:0.25, Figure 1, A1-1), and the phosphopeptide ratio is 26%. On the contrary, 1563 phosphopeptides (1903 total peptides) were identified using 2000 µg beads (peptide-to-beads ratio ) 1:4, Figure 1, A5-1), and the phosphopeptide ratio is about 82%. However, the phosphopeptides ratio decreased to less than 40% when the beads were increased to 20 000 µg (peptide-to-beads ratio ) 1:40). These results showed that the peptide-to-beads ratio did affect the enrichment selectivity to a large extent and the effective enrichment of phosphopeptides by TiO2 beads could be achieved by a specific peptide-to-beads ratio. Here, for nonstimulated HeLa cell lysate, the optimum peptide-to-beads ratio is about 1:2 to 1:8 by quantity. The optimized phosphopeptide ratios were all above 80%, and the number of identified phosphopeptides climbed to summit at a peptide-to-beads ratio of 1:4. Less or more TiO2 beads usage would not obtain more phosphopeptides or higher enrichment selectivity. Different Enrichment Selectivity Profiles of Consecutive Incubations. From Figure 1 we can see that the peptide-tobeads ratio did have a great effect on the enrichment selectivity of phosphopeptides by TiO2. One phenomenon should be noted, especially when beads dosage is deficient: the phosphopeptide number is much less than the optimum peptideto-beads ratio. If the inadequate beads cannot pull-down all of the phosphopeptides once, the remaining phosphopeptides should be recovered by subsequent incubation of supernatant after the first run. In addition, for the optimum peptide-tobeads ratio incubation, whether all the phosphopeptides were recovered by single incubation is still needed to be checked. Therefore, we did eight consecutive incubations to explore the above questions.

Figure 2A and B shows the phosphopeptides identified and the enrichment selectivity in each consecutive incubation for peptide-to-beads ratio of 1:0.25 and 1:4, respectively. The first incubation in Figure 2A is of good reproducibility with the corresponding result in Figure 1 (A1-1 and A1-2), from which we can deduce the overall repeatability of the experiment. It is remarkable that the subsequent incubations of peptide-tobeads ratio 1:0.25 contribute a great deal to phosphopeptide identification (Figure 2A). Particularly, the fourth incubation identified the maximum phosphopeptides and highest enrichment selectivity (higher than 80%). It was interesting that the summit of phosphopeptide number and enrichment selectivity did not appear in the first incubation. And still, there was also an “optimum” enrichment even with deficient beads to startup. In addition, we analyzed the data published recently in which eight consecutive incubations were also performed.23 Although different biological samples and protocols were employed, a similar distribution pattern could be drawn (Figure S2, Supporting Information). Theoretically, if TiO2 beads preferentially bind to phosphopeptides, when beads were deficient, the number of phosphopeptides and enrichment selectivity should remain constant at a high level in several early incubations and then drop down in later incubations when phosphopeptides were depleted. However, the hypothesis disagreed with the facts. The above results suggested that multiple rounds of consecutive incubation were needed to recover as many phosphopeptides as possible when insufficient TiO2 beads were used. The number of incubation rounds needed may depend on characteristic of specific sample, as well as the peptide-tobeads ratio employed. For experiment here, there were still considerable phosphopeptides identified in the eighth incubation (767 and 742 phosphopeptides respectively, 140 and 98 distinct phosphopeptides sequence found only in this fraction). The 125 µg beads here may be too inadequate for 500 µg HeLa digest to perform the TiO2 enrichment. On the other hand, consecutive incubation using deficient TiO2 beads usage itself can also be used as a pluripotent strategy of fractionation and enrichment. As shown in Figure S3A (Supporting Information), each subsequent incubation contributes a lot to the phosphoJournal of Proteome Research • Vol. 8, No. 11, 2009 5377

technical notes

Figure 2. Eight incubations of different amount of TiO2 beads to HeLa lysate (500 µg). (A) and (B) 125 µg and 2000 µg TiO2 beads applied (peptide-to-beads ratio of 1:0.25 and 1:4, respectively), and saturated glutamic acid was added (Glu+) for sample loading. (C) 2000 µg TiO2 beads without glutamic acid (Glu-) in loading buffer.

peptides identification, and eventually there are 3159 unique phosphopeptide identifications, compared with 2143 from the optimum consecutive incubation (Figure S3B, Supporting Information) and 1227 from single optimum incubation (Figures 1, A5-1). The results shown here may suggest that the insufficient TiO2 beads could be a new alternative of phosphopeptide enrichment approach, which is robust but easy to perform. From the multiple incubations for the optimum peptide-tobeads ratio (Figure 2B), a quite different profiling could be observed. Compared with deficient beads, less phosphopeptides identified although consecutive incubation were applied. In detail, the phosphopeptide ratio declined dramatically from 86 and 88% (1756 and 1760 phosphopeptides) in the first incubation to less than 30% (420 and 485) in the second incubation and even fell to less than 4% (67 and 68) in the third incubation. Combining the last six rounds of incubation together, only 71 unique phosphopeptides were identified. The phosphopeptides number profile suggested that there was no need to perform multiple incubations for optimum peptideto-beads ratio enrichment. The word “optimum” here not only 5378

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Li et al. means that it has the highest enrichment selectivity among different peptide-to-beads ratio, but it also infers that the least incubation rounds are needed for enrichment. Effect of Saturated Glutamic Acid on Nonphosphopeptide Binding. Recently, glutamic acid was introduced into loading buffer to reduce nonspecific bindings on TiO2.25 In that work, the binding of nonphosphopeptides was significantly inhibited and the binding specificity was greatly improved. In the current study, we therefore employed the strategy that the saturated glutamic acid was added in the loading buffer. However, in consecutive incubations of the two peptide-to-beads ratios (1: 0.25 and 1:4, Figure 2A and B), a large number of nonphosphorylated peptides were identified, especially in later incubations of a peptide-to-beads ratio of 1:4, as well as early and later incubations of a peptide-to-beads ratio of 1:0.25. The same situation also occurred in the data of Zanivan et al., although 2,5-dihydroxy benzoic acid (DHB) was added as the nonphosphopeptides inhibitor in loading buffer (Figure S2, Supporting Information).23 Given that glutamic acid performs a real robust competitor with the nonphosphopeptides as assumed, there should be less nonphosphopeptides binding to TiO2 beads with saturated glutamic acid in later incubations of the ratio 1:4 (Figure 2B), as well as two side incubations of ratio 1:0.25. We then did a parallel experiment as a negative control to the experiment as in Figure 2B using peptide-to-beads ratio 1:4, in which there was no saturated glutamic acid in loading buffer. The identification result is shown in Figure 2C. Intriguingly, a quite similar elution profile was obtained and the phosphopeptides were mainly isolated in the first and second incubations. However, the absolute identification number is a little less than the experiment with saturated glutamic acid: in the first elution, the phosphopeptides number was 1756 and 1760 vs 1432 and 1434, whereas the phosphopeptide ratio was 86 and 88% vs 72 and 73%, respectively. The result indicated that the glutamic acid exhibited moderate competitive ability to inhibit the nonphosphopeptides binding. However, glutamic acid is still preferred in our experiment, because it is a quite economic and easy procedure which did increase the enrichment selectivity and phosphopeptide identification. DHB was also reported as a nonspecific binding inhibitor, therefore the situation without DHB should be more or less the same situation as without glutamic acid. Effect of Deficient Beads on the Identification of Multiphosphorylated Peptides. It is reported that TiO2 tends to bind monophosphorylated peptides.27 We then checked the distributions of peptides with mono- and multiphosphorylation sites (double, triple, and more) in this experiment. To our surprise, abundant multiphosphorylated peptides were identified (Figure S4, Supporting Information). Moreover, we found that less beads helped to identify more peptides with multiple phosphorylation sites. Notably, in the incubation with deficient TiO2 beads used (125 µg), nearly 90% of the phosphopeptides identified were with multiple phosphorylation sites as shown in Figure 3A (Incubation A1). With the increase of beads dosage, the monophosphorylated peptides became dominant, even up to 95% (Figure 3A, incubations A7 and A8), while the number of multiphosphorylation peptides dropped down gradually. From Figure 3A we can see that the number of phosphopeptides with three and four phosphorylation sites have a constant dropping trend, and doubly phosphorylated peptides get its summit a bit later (the second incubation), while monophosphorylated peptides have a constant increasing trend. A quite similar profile for eight consecutive incubation with deficient

Effect of Peptide-to-TiO2 Beads Ratio

technical notes why monophosphopeptide dominated in all incubations when the peptide-to-beads ratio is 1:4 as shown in Figure 3C.

Figure 3. Distribution of phosphosite(s) per peptide. The ratio shown is an average of the replicates. (A) Phosphosites distribution based on different beads used. The x-axis is peptide-to-beads ratio as in figure 1. (B) Multiple incubation by peptide-to-beads ratio of 1:0.25. (C) Multiple incubation by peptide-to-beads ratio of 1:4. The phosphorylation site(s) per peptide was marked as “1P” (one phosphosite) to “4P” (four phosphosites), respectively.

beads usage (peptide-to-beads ratio ) 1:0.25) is shown in Figure 3B. These profiles with different number of phosphorylation site(s) could possibly be attributed to the affinity of phosphopeptides to TiO2 beads surface. Theoretically, the more phosphate groups in a peptide, the higher affinity (smaller dissociation constant) it would show to TiO2, which therefore have a relatively priority to be bound on the TiO2 beads. Eventually peptides with multiphosphorylation sites were favored in early fractions, while monophosphorylation peptides appeared in later fractions. If beads usage is sufficient, and the binding sites on TiO2 are sufficient for all the phosphorylation sites in sample, monophosphopeptides would have a depression effect on the MS identification of multiple phosphorylation peptides due to higher abundance. That might be the reason

Recently, a two-step elution approach was developed to purify monophosphorylated peptides from multiply phosphorylated peptides from IMAC, in which pH from low to high was used.36 The same year, Simon and co-workers presented the same strategy for phosphopeptide enrichment on TiO2.21 Upon the basis of the results in this experiment, we found deficient beads usage could serve to multiphosphorylated peptide enrichment, and the bias that TiO2 tend to bind monophosphopeptides only happened when TiO2 beads were sufficient. When insufficient beads used, although the total amount of phosphopeptides was reduced, the multiply phosphorylated peptides became prevalent. Therefore, deficient TiO2 beads were preferred if multiphosphorylated peptides were desired. Hypothetical Model for TiO2 Enrichment Selectivity. On the basis of the above results, it is reasonable that multiphosphorylated peptides appeared earlier than monophosphorylated ones from TiO2. However, we still wondered the exact mechanism for this “up and down” profile of phosphopeptide ratio in consecutive incubations by deficient beads (Figure 2A), and incubations with different beads amount (Figure 1). In detail, why there were so many nonphosphorylated peptides in the early fractions and why the phosphopeptide identifications drop down with large amount of TiO2 beads. We examined some popular characteristics (such as pI and amino acid composition) of phosphopeptides and nonphosphopeptides from each incubation (Figure S5, Supporting Information), and found little attribute reasonably related. We then proposed a hypothetical model (Figure S6, Supporting Information) from our experiment results trying to explain the above questions. In this model, deficient amount of TiO2 beads will bind those nonphosphopeptides with special affinity preferentially, as well as some multiphosphorylated peptides. On the contrary, the optimized beads amount happened to cover most of phosphopeptides, and the enrichment efficiency is therefore very high. However, when the TiO2 beads are excessive, nonphosphopeptides with less affinity to TiO2 beads are also incorporated. It is well-known that the presence of nonphosphopeptides could inhibit the identification of phosphopeptides. Similarly, with excessive TiO2 beads for enrichment, the presence of nonphosphopeptides may greatly suppress the identification of phosphopeptides. Hereby, the “up and down” profile is reasonable. It is imprudent to attribute all of the nonphosphopeptides to nonspecific binding, and it is still surprising that there are a group of nonphosphopeptides with special high affinity to TiO2. Therefore, further studies still needed to be performed. Perhaps more efficient loading buffer would be expected to decrease the level of nonphosphorylated peptide in the future. Quick Pre-Experiment Recommendation. We recommend a quick pre-experiment for the optimum enrichment with specific samples to maximize the identifications efficiently. For nonprocessed (e.g., without biochemical stimulation) HeLa cell lysate in this experiment, we found that optimum peptide-tobeads ratio is about 1:2 to 1:8. However, it should be noted that this ratio could be changed for different samples, considering specific biological samples have distinct phosphorylation level and complexity. An obvious indicator is the phosphopeptide ratio of the second incubation. If it is higher than or equal to the first enrichment, it is probable that the beads amount is insufficient for phosphopeptides contained in the sample. On the other hand, if the ratio drops dramatically, a good starting Journal of Proteome Research • Vol. 8, No. 11, 2009 5379

technical notes point might have been chosen. However, further incubation is still needed to check the overall profile. Therefore, when it comes to an unknown sample, four consecutive enrichments of the sample are recommended for three peptide-to-beads ratios based on our experience. The optimum peptide-to-beads ratio performs a quite high selectivity in the first incubation (up to 90% or higher), while there is a dramatic decrease in the second ratio (Figure 2B). However, the “optimum” ratio did not identify as many multiphosphorylation peptides as the deficient beads amount. In addition, consecutive incubation using deficient beads could also be used as a fractionation strategy. In this sense, deficient beads amount is also an “optimum” starting point for the multiphosphorylation identification. Apparently, the optimum peptide-to-beads ratio as well as the appropriate incubation rounds is not universal either.

Li et al.

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Conclusions From the results in the current study, we conclude that the peptide-to-beads ratio plays an important role in phosphopeptide enrichment selectivity by TiO2 and that there is an optimum peptide-to-beads ratio for a given sample. Excess or deficient beads could not obtain the best enrichment by single incubation. We recommend performing a multiple elution experiment using different peptide-to-beads ratios first to decide the optimum ratio if a high enrichment efficiency is favored. In addition, multiphosphorylated peptides emerged when TiO2 beads were deficient, and consecutive elution of the same sample with deficient beads, could be used as a fractionation approach besides enrichment. The usually used inhibitor for nonspecific bindings such as glutamic acid do improve the specificity to some extent, but it seems not to be the key factor for the enrichment selectivity based on the result of this work. So far we still do not know the exact mechanism for why deficient beads have such a selectivity profile of consecutive incubations, especially why there are more nonphosphopeptides bound in early fractions. The number of negative solution charges in a peptide may play an important role in the binding process. Further study is still needed to explore the TiO2 beads’ binding behavior for phosphopeptides.

Acknowledgment. This work was supported by the National Natural Science Foundation (30425021, 30521005), Basic Research Foundation (2006CB910700), CAS Project (KSCX2-YW-R-106, KSCX1-YW-02), and High-technology Project (2007AA02Z334). Supporting Information Available: Additional information as noted in text is provided as supplemental figures, and all peptides identified with detail information are in evidence_list.txt. This material is available free of charge via the Internet at http://pubs.acs.org.

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technical notes

Effect of Peptide-to-TiO2 Beads Ratio

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