Highly Efficient Phosphopeptide Enrichment by Calcium Phosphate Precipitation Combined with Subsequent IMAC Enrichment*

  • Xumin Zhang
    Footnotes
    Affiliations
    Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense, Denmark
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  • Juanying Ye
    Footnotes
    Affiliations
    Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense, Denmark
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  • Ole N. Jensen
    Footnotes
    Affiliations
    Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense, Denmark
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  • Peter Roepstorff
    Correspondence
    To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark. Tel.: 45-6550-2404; Fax: 45-6593-2661;
    Affiliations
    Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense, Denmark
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  • Author Footnotes
    * This work was supported in part by a Ph.D. fellowship from The Danish Research Agency (to X. Z.), a Young Investigator Award (to O. N. J.), and the Danish Biotechnology Instrument Center (for instrumentation). 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.
    The on-line version of this article (available at http://www.mcponline.org) contains supplemental material.
    ‡ Both authors contributed equally to this work.
    § A Lundbeck Foundation Professor.
      A new method for enrichment of phosphopeptides in complex mixtures derived by proteolytic digestion of biological samples has been developed. The method is based on calcium phosphate precipitation of the phosphopeptides prior to further enrichment with established affinity enrichment methods. Calcium phosphate precipitation combined with phosphopeptide enrichment using Fe(III) IMAC provided highly selective enrichment of phosphopeptides. Application of the method to a complex peptide sample derived from rice embryo resulted in more than 90% phosphopeptides in the enriched sample as determined by mass spectrometry. Introduction of a two-step IMAC enrichment procedure after calcium phosphate precipitation resulted in observation of an increased number of phosphopeptides.
      Protein phosphorylation plays a crucial role in almost all cellular and developmental processes including signal transduction, translocation, and protein-protein interaction (
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      Signaling—2000 and beyond.
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      ). However, because phosphopeptides are often of low abundance and their ionization efficiencies are poor, selective enrichment is required. To date, numerous methods have been introduced for enrichment of phosphoproteins or phosphopeptides. The methods used for selective enrichment of phosphopeptides fall in two main categories: chemical derivatization (
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      ) and affinity chromatography-based methods. Although the chemical derivatization methods are highly selective, they are not widely applied in the phosphoproteome studies most likely due to sample loss caused by the multiple reaction steps and increased sample complexity by unavoidable side reactions (
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      ), and metal oxide chromatography (
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      Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns.
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      ). In several studies the combination of different enrichment methods has been found to be advantageous for selective phosphopeptide enrichment (
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      ). In our laboratory affinity chromatography using Fe(III) IMAC and titanium dioxide (TiO2)
      The abbreviations used are: TiO2, titanium dioxide; DHB, 2,5-dihydroxybenzoic acid.
      1The abbreviations used are: TiO2, titanium dioxide; DHB, 2,5-dihydroxybenzoic acid.
      in combination with MALDI MS and MS/MS has been widely used for phosphopeptide analysis (
      • 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.
      ,
      • Stensballe A.
      • Andersen S.
      • Jensen O.N.
      Characterization of phosphoproteins from electrophoretic gels by nanoscale Fe(III) affinity chromatography with off-line mass spectrometry analysis.
      ,
      • Stensballe A.
      • Jensen O.N.
      Phosphoric acid enhances the performance of Fe(III) affinity chromatography and matrix-assisted laser desorption/ionization tandem mass spectrometry for recovery, detection and sequencing of phosphopeptides.
      ). Ion exchange chromatography and IMAC have been proven to be very efficient for phosphoproteomics analysis of complex samples (
      • Nuhse T.S.
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      • Jensen O.N.
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      Large-scale analysis of in vivo phosphorylated membrane proteins by immobilized metal ion affinity chromatography and mass spectrometry.
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      ). However, although a variety of methods are available for phosphopeptide enrichment, the complete mapping of the phosphoproteome is still a challenging task (
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      ), and development of new enrichment methods is needed.
      In our effort to develop a simple, sensitive, and LC-ESI-MS-compatible method for the enrichment of phosphopeptides, we investigated phosphopeptide precipitation efficiency using a variety of metal ions. We describe a novel phosphopeptide precipitation method based on precipitation of phosphopeptides with an excess of calcium phosphate. Phosphopeptides were selectively separated from non-phosphopeptides by this method. In addition, calcium phosphate precipitation and IMAC were found to be complementary enrichment methods. Application of the new method of calcium phosphate precipitation and IMAC to studies of the rice embryo phosphoproteome demonstrated that a highly selective enrichment of phosphopeptides was achieved in very complex peptide samples.

      EXPERIMENTAL PROCEDURES

       Materials—

      Pure water was obtained from a Milli-Q system (Millipore, Bedford, MA). CaCl2, Na2HPO4, and NH3·H2O were from Sigma-Aldrich. 2,5-Dihydroxybenzoic acid (DHB) was from Fluka (St. Louis, MO). FeCl3 was purchased from Merck KGaA. Modified trypsin was from Promega (Madison, WI). Poros Oligo R3 reversed-phase material was from PerSeptive Biosystems (Framingham, MA). GELoader tips were from Eppendorf (Hamburg, Germany). 3M Empore™ C8 disks were from 3M Bioanalytical Technologies (St. Paul, MN). TiO2 beads were obtained from a disassembled titanium dioxide cartridge purchased from GL Sciences Inc. (Tokyo, Japan). Nickel-nitrilotriacetic acid-silica resin was purchased from Qiagen (Hilden, Germany). All other reagents and solvents were of the highest commercial quality and were used without further purification.

       Digestion of Standard Phosphoproteins—

      α-Casein (bovine), β-casein (bovine), serum albumin (bovine), β-lactoglobulin (bovine), and carbonic anhydrase (bovine) (all from Sigma-Aldrich) were individually dissolved in 25 mm ammonium bicarbonate, reduced with DTT, and alkylated with iodoacetamide followed by digestion with modified trypsin at 37 °C overnight.

       Calcium Phosphate Precipitation—

      The total volume of the peptide solution was adjusted to 50 μl. 2 μl of 0.5 m Na2HPO4 and 2 μl of 2 m NH3·H2O were added and mixed followed by the addition of 2 μl of 2 m CaCl2. It should be noted that the pH value of the buffer prior to adding CaCl2 should be around 10. The solution was vortexed and centrifuged at 20,000 × g for 10 min at room temperature. Subsequently the supernatant was removed, and 60 μl of 80 mm CaCl2 was applied to suspend and wash the pellet. After centrifugation as described above, the washing solution was removed, and the resulting pellet was dissolved in 20 μl of 5% formic acid.

       Desalting—

      The resulting peptide solution was loaded onto a home-made Poros R3 microcolumn (
      • Gobom J.
      • Nordhoff E.
      • Mirgorodskaya E.
      • Ekman R.
      • Roepstorff P.
      Sample purification and preparation technique based on nano-scale reversed-phase columns for the sensitive analysis of complex peptide mixtures by matrix-assisted laser desorption/ionization mass spectrometry.
      ). To minimize sample loss, the length of the column varied depending on the sample amount. After washing with 20 μl of 5% formic acid, the bound peptides were eluted with 20 μl 80% ACN and 0.6% acetic acid and dried in vacuum. For subsequent analysis on TiO2, the peptides were directly eluted onto the TiO2 column with 5% TFA and 80% ACN.

       Fe(III) Immobilized Metal Ion Affinity Chromatography—

      A slurry of Fe(III)-loaded nitrilotriacetic acid-silica resin was prepared as described previously (
      • Boeri Erba E.
      • Bergatto E.
      • Cabodi S.
      • Silengo L.
      • Tarone G.
      • Defilippi P.
      • Jensen O.N.
      Systematic analysis of the epidermal growth factor receptor by mass spectrometry reveals stimulation-dependent multisite phosphorylation.
      ). An aliquot of the IMAC material (about 0.5-μl bed volume, if not stated otherwise) was added into the tube containing dried peptides, and the pH was adjusted to ∼3.0 with 0.1 m acetic acid to a total volume of 30 μl. The peptide solution and IMAC material were incubated for 1 h with constant slow end-over-end rotation at room temperature. After incubation, the slurry was packed into a GELoader tip (
      • Gobom J.
      • Nordhoff E.
      • Mirgorodskaya E.
      • Ekman R.
      • Roepstorff P.
      Sample purification and preparation technique based on nano-scale reversed-phase columns for the sensitive analysis of complex peptide mixtures by matrix-assisted laser desorption/ionization mass spectrometry.
      ).
      The beads were washed with 10 μl of washing solution containing 0.1 m acetic acid and 30% ACN. Subsequently the bound peptides were eluted with 20 μl of NH3·H2O (pH 10.5). The eluate was immediately acidified with 1 μl of 100% formic acid and desalted using Poros R3 microcolumns prior to analysis by MS.
      For the complex rice embryo samples a larger amount of IMAC material was used, and the flow-through from packing the IMAC columns was incubated a second time with new IMAC material. This procedure allowed recovery of more singly phosphorylated peptides.

       TiO2 Microcolumn—

      TiO2 microcolumns were packed in GELoader tips according to Thingholm et al. (
      • Thingholm T.E.
      • Jorgensen T.J.
      • Jensen O.N.
      • Larsen M.R.
      Highly selective enrichment of phosphorylated peptides using titanium dioxide.
      ). A small plug of C8 material was punched out of a 3M Empore C8 extraction disk using an HPLC syringe needle and placed at the narrow end of the GELoader tip. The TiO2 beads were suspended in 100% ACN, and an aliquot of this suspension was loaded onto the GELoader tip. Following the loading of peptides in loading buffer (5% TFA and 80% ACN), the column was washed with 20 μl of loading buffer. The bound peptides were eluted with 20 μl of NH3·H2O (pH 10.5). After acidification with 1 μl of 100% formic acid, the eluate was desalted on a Poros R3 microcolumn prior to analysis by MS.

       Extraction and Digestion of Rice Embryo Proteins—

      The seed of rice (Oryza sativa L. ssp. indica) cultivars, 9311, was used in this work. The bran coats on rice seed were completely removed, and the rice embryo was carefully separated with a scalpel. A portion (44 mg) of the rice embryo preparation was prepared by TCA-acetone precipitation (
      • Xie Z.
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      • Cao M.
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      • Zhao K.
      • Shao J.
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      • Xu N.
      • Liu S.
      Pedigree analysis of an elite rice hybrid using proteomic approach.
      ). The resulting pellet was dried in vacuum and subsequently resuspended in 750 μl of lysis buffer containing 7 m urea, 2 m thiourea, 1:100 protein phosphatase inhibitor cocktails 1 and 2 (Sigma), and 5 mm DTT. The solution was further sonicated for 3 min (2-s sonication time with 5-s intervals) followed by centrifugation at 20,000 × g for 20 min. The protein concentration was determined by the Bradford assay. The protein solution was reduced at 37 °C for 45 min, alkylated by 15 mm iodoacetamide at room temperature for 45 min, and subsequently digested with Lys-C at room temperature for 4 h. After 6-fold dilution with H2O, trypsin was added, and the solution was kept at 37 °C for overnight digestion. Note that the pH value should be around 8.0 and was checked before each digestion step. NH3·H2O was used to adjust the pH instead of ammonium bicarbonate to avoid precipitation of calcium carbonate in later steps of the procedure.

       MALDI-TOF MS and MS/MS Analysis—

      Analyses by MALDI-TOF MS were used for fast screening of the sample quality and composition. Prior to MALDI analysis, the peptide samples were desalted on Poros R3 microcolumns and directly eluted onto the sample supports with matrix solution. In this work, 20 mg/ml DHB in 50% ACN and 1% phosphoric acid was used as the matrix solution because it has been demonstrated previously to be efficient for analysis of phosphopeptides (
      • Stensballe A.
      • Jensen O.N.
      Phosphoric acid enhances the performance of Fe(III) affinity chromatography and matrix-assisted laser desorption/ionization tandem mass spectrometry for recovery, detection and sequencing of phosphopeptides.
      ,
      • Kjellstrom S.
      • Jensen O.N.
      Phosphoric acid as a matrix additive for MALDI MS analysis of phosphopeptides and phosphoproteins.
      ). MALDI MS and MS/MS were performed using a Bruker Ultraflex Tof/Tof mass spectrometer (Bruker, Bremen, Germany). All spectra were obtained in positive reflector mode. Mass spectrometric data analysis was performed using the Bruker Daltonics flexAnalysis Software version 2.4. Sequence analysis and peptide assignment were accomplished using the General Protein/Mass Analysis for Windows (GPMAW) software (Lighthouse data).

       Nanoflow LC-ESI-MS/MS—

      LC-ESI-MS/MS analysis was performed using a Q-TOF Micro mass spectrometer (Waters/Micromass UK Ltd., Manchester, UK) using automated data-dependent acquisition. A nanoflow high pressure LC system (Ultimate, Switchos2, Famos, LC Packings, Amsterdam, The Netherlands) was used to deliver a flow rate of 2 μl/min (loading) and 100 nl/min (elution). Samples were loaded onto a home-made 2-cm fused silica precolumn (75-μm inner diameter/375-μm outer diameter, Resprosil C18-AQ, 3 μm (Dr. Maisch, Ammerbuch-Entringen, Germany)) using an autosampler. The mobile phases consisted of Solution A (0.5% acetic acid) and Solution B (80% ACN and 0.5% acetic acid). Sequential elution of peptides was accomplished using a three-step linear gradient of 0–10% B in 5 min, 10–50% B in 85 min, 50–100% B in 5 min, and 100% B in 5 min over the precolumn in line with a home-made 10–15-cm analytical column (375-μm outer diameter/50-μm inner diameter, Resprosil C18-AQ, 3 μm (Dr. Maisch)). The analytic column was connected via a fused silica transfer line (20-μm inner diameter) to a distally coated fused silica emitter (New Objective, Cambridge, MA) (360-μm outer diameter/20-μm inner diameter/10-μm tip inner diameter) biased to 1.8 kV.
      The mass spectrometer was operated in positive ion mode with a source temperature of 80 °C and a countercurrent gas flow rate of 150 liters h−1. Data-dependent analysis was used (the four most abundant ions present in the survey spectrum were automatically mass-selected and fragmented by collision-induced dissociation in each cycle): 1-s MS m/z 350–1500 and maximum 4-s MS/MS m/z 50–2000 (continuum mode), 30-s dynamic exclusion.

       Database Search—

      Raw data were processed using MassLynx 3.5 ProteinLynx (smooth 3/2 Savitzky Golay and center four channels/80% centroid), and the MS/MS datasets were exported in the Micromass pkl format. The resulting pkl file was searched against the National Center for Biotechnology Information non-redundant (NCBInr) protein sequence database (February 10, 2007; 4,565,699 sequences) with O. sativa (rice) (78,302 sequences) as taxonomy using an in-house Mascot server (Version 2.1.04, Matrix Sciences, London, UK). All datasets were searched twice, first with relatively large peptide mass tolerances (0.8 Da) followed by internal mass recalibration by an in-house software algorithm using theoretical masses from unambiguously identified peptides (Mascot score ≥35) obtained from the first search. The recalibrated MS and MS/MS datasets were searched against the protein sequence database using the following parameters: only tryptic peptides with up to one missed cleavage site were allowed; 50 ppm mass tolerances for MS and 0.3 Da for MS/MS fragment ions; carbamidomethylcysteine as fixed modification; and protein N-acetylation, oxidized methionine, and phosphorylation (serine, threonine, and tyrosine) as variable modifications. Phosphopeptides identified were considered to be potential candidates if the Mascot score was ≥25 and the peptides ranked number 1. To evaluate the parameters used for threshold, the decoy database search in Mascot was performed, and it revealed the false positive rate of ∼2%. This was found sufficient because the purpose of the study was to evaluate the performance of the different enrichment methods and not to perform high confidence protein and phosphorylation site identification. When peptides matched to multiple members of a protein family, only the protein hit with the most matching peptides was accepted as correct result. Further manual verification was carried out by the observation of consecutive y or b ions. First, the result was accepted if at least four consecutive y or b ions were present; second, for the results with only three consecutive y or b ions, it was accepted only if other information was present, e.g. another series of three consecutive y or b ions or ions presenting a 98-Da neutral loss. Furthermore the phosphorylated sites were determined by the presence of a 69-Da distance between fragment ions for phosphoserine and an 83-Da distance for phosphothreonine. When several potential phosphorylation sites were present in a peptide, the site could be determined because only the phosphorylated site showed complete neutral loss of the phosphoric acid, whereas neural loss of water from unmodified serine and threonine residues was only partial.

      RESULTS AND DISCUSSION

       Calcium Phosphate Precipitation—

      Our hypothesis was that phosphorylated peptides could be precipitated just like calcium phosphate by the addition of calcium ions (Fig. 1A). We decided to use α- and β-casein as test phosphoproteins because these proteins have been extensively used as models in the development of methods for phosphoproteomics (
      • 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.
      ,
      • Stensballe A.
      • Andersen S.
      • Jensen O.N.
      Characterization of phosphoproteins from electrophoretic gels by nanoscale Fe(III) affinity chromatography with off-line mass spectrometry analysis.
      ,
      • Imanishi S.Y.
      • Kochin V.
      • Eriksson J.E.
      Optimization of phosphopeptide elution conditions in immobilized Fe(III) affinity chromatography.
      ) due to their multiple phosphorylated sites (Table I). In the present study, a tryptic digest of an α,β-casein mixture (1:1) was used as starting material. Stock solutions containing 1 μg/μl α-casein and β-casein in 25 mm ammonium bicarbonate (pH 8.0) were prepared and digested with trypsin followed by mixing 1:1. The test solution was made by dilution of the stock solution with H2O to contain 0.5 μg of α-casein and β-casein/50 μl (400 fmol/μl each). Precipitation was performed on 50 μl of solution containing a total of 20 pmol each of α- and β-casein. In a first attempt the procedure shown in Fig. 1B was followed with omission of the addition of Na2HPO4. No visible precipitate was observed, and no precipitate could be obtained after high speed centrifugation (20,000 × g for 20 min). Therefore co-precipitation with calcium phosphate was attempted by addition of Na2HPO4 to the buffer prior to the addition of CaCl2 based on the hypothesis that a mixed calcium phosphate and calcium phosphopeptide precipitate might be formed allowing precipitation of the phosphopeptides. The obtained pellet was dissolved in 20 μl of 5% formic acid and desalted as described under “Experimental Procedures.” Although our purpose is to develop a new LC-MS-compatible method, MALDI-TOF MS was used for fast checks of the efficiency of the method. The MALDI mass spectra obtained from 5% of the total sample volume from the different stages of the procedure are shown in Fig. 2. In the spectrum resulting from the original sample, five phosphopeptide signals were observed (Fig. 2A, labeled with asterisks). The supernatants after precipitation and the subsequent washing procedures (Fig. 2, B and C) were dominated by non-phosphopeptides. Only one phosphopeptide signal, m/z 1660, could be detected with low intensity.
      Figure thumbnail gr1
      Fig. 1Overview of the reactions and procedures used for the precipitation methods.A, calcium phosphate precipitation and co-precipitation of phosphopeptides with calcium phosphate. B, schematic workflow in the phosphopeptide precipitation
      Table IOverview of the phosphopeptides obtained by tryptic digestion of α -casein S1 (α -S1) and S2 (α -S2) and β -casein (β -C) observed in different enrichment experiments
      Peptide sequence
      a The peptides sequences are cited from Larsen. et al.(
      • 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.
      ) with minor modification.
      No. of phosphoryl groups[M + H]+α,β-Casein CPP1:1 dilution CPP1:50 dilution
      CPPIMACTiO2CPP-IMACCPP-desalting-IMAC
      EQLpSTpSEENSK, α-S2, 141–15121411.50+
      TVDMEpSTEVFTK, α-S2, 153–16411466.61++++++
      TVDMoEpSTEVFTK, α-S2, 153–164
      b Mo, oxidation on methionine.
      11482.61
      VPQLEIVPNpSAEER, α-S1, 121–13411660.79++++++
      DIGpSEpSTEDQAMEDIK, α-S1, 58–7321927.69+++++++
      DIGpSEpSTEDQAMoEDIK, α-S1, 58–73
      b Mo, oxidation on methionine.
      21943.69
      FQpSEEQQQTEDELQDK, β-C, 33–4812061.83+++++++
      NVPGEIVEpSLpSpSpSEESITR, β-C, 7–2542352.85++++++
      NTMEHVpSpSpSEEpSIISQETYK, α-S2, 17–3642619.04++++++
      VNELpSKDIGpSEpSTEDQAMEDIK α-S1, 52–7332678.01+
      Q*MEAEpSIpSpSpSEEIVPNpSVEAQK, α-S1, 74–94
      c Q*, pyroglutamylation on the N-terminal Gln.
      52703.89++++++
      QMEAEpSIpSpSpSEEIVPNpSVEAQK, α-S1, 74–9452720.91++++++
      NTMEHVpSpSpSEEpSIISQETYKQ, α-S2, 17–3742747.10++++
      ELEELNVPGEIVEpSLpSpSpSEESITR, β-C, 17–4042966.16++++++
      NANEEEYSIGpSpSpSEESAEVATEEVK, α-S2, 61–8543008.01++++++
      RELEELNVPGEIVEpSLpSpSpSEESITR, β-C, 16–4043122.27++++++
      a The peptides sequences are cited from Larsen. et al.(
      • 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.
      ) with minor modification.
      b Mo, oxidation on methionine.
      c Q*, pyroglutamylation on the N-terminal Gln.
      Figure thumbnail gr2
      Fig. 2MALDI mass spectra of 5% of the various fractions (1 pmol of sample) from the calcium phosphate precipitation of α,β-casein digest. A, sample prior to precipitation; B, supernatant; C, wash; and D, pellet. The phosphopeptide signals are labeled with asterisks, and doubly charged phosphopeptide signals are labeled with double asterisks.
      The mass spectra of the pellet (Fig. 2D) demonstrated that the pellet mainly contained phosphopeptides. In addition to the phosphopeptide signals observed in the spectrum of the original sample (Fig. 2A), 10 additional phosphopeptide signals were observed. Signals for six new phosphopeptides representing different phosphorylation sites were present. Signals at m/z 1717 and 2118 correspond to the overalkylated form of the m/z 1660.8 and 2061.8, and signals at m/z 830 and 1031 correspond to the doubly charged form of the m/z 1660.8 and 2061.8. That these two ions were doubly charged was confirmed by the distance between the isotopic ions as well as a neutral loss of 49 Da upon MS/MS. Compared with the original sample, the number of phosphopeptide to total peptide signals increased from 5:46 to 15:18 and demonstrated high sensitivity and specificity of the precipitation method toward phosphopeptides. In addition, the relative intensities of the phosphopeptide signals increased significantly. With these promising preliminary results it was decided to apply the precipitation method to more complex mixtures to simulate complex real life samples.
      The tryptic digests from the α-casein and β-casein stock solution were mixed with the tryptic digests of a stock solution containing three non-phosphoproteins (BSA, β-lactoglobulin, and carbonic anhydrase, all at 1 μg/μl). The results obtained by applying the precipitation method using 50 μl of solution containing the five proteins in a 1:1:1:1:1 ratio (400 fmol/μl each) are shown in Fig. 3. 5% of the sample volume in each step was applied on the MALDI target. Only one phosphopeptide signal (m/z 1660) could be observed in the MALDI MS spectrum of the mixture sample prior to precipitation (Fig. 3A). No phosphopeptide signal was observed in the spectrum of the supernatant solution (Fig. 3B), and only a low abundance signal at m/z 1660 in the spectrum of the washing solution (Fig. 3C) was observed. A total of 13 singly charged phosphopeptide signals were present in the spectrum obtained from the pellet (Fig. 3D) plus a few more signals from non-phosphopeptides. Thus, increasing sample complexity does not significantly affect the specificity of the phosphopeptide precipitation method (compare Fig. 2D and Fig. 3D).
      Figure thumbnail gr3
      Fig. 3MALDI mass spectra of 5% of the different fractions (1 pmol of sample) from the calcium phosphate precipitation of digests from α,β-casein mixed 1:1 with a mixture of three non-phosphorylated proteins. A, sample prior to precipitation; B, supernatant after precipitation; C, wash; and D, pellet. The phosphopeptide signals are labeled with asterisks.
      To further assess the enrichment efficiency of the precipitation method α-casein and β-casein were mixed with BSA, β-lactoglobulin, and carbonic anhydrase in the ratio 1:1:50:50:50. Samples corresponding to 1 pmol each of α- and β-casein were prepared with the precipitation method and analyzed by MALDI MS (Fig. 4). The top spectrum (Fig. 4A) shows the MALDI mass spectrum from the original peptide sample with 50-fold substoichiometric levels of the casein (phospho)peptides. Only the signal at m/z 1927 might represent a phosphopeptide. The spectra of the supernatant and washing fractions (Fig. 4, B and C) were almost identical to that of the original sample. The spectrum of the pellet fraction (Fig. 4D) showed some enrichment, although only four weak phosphopeptide signals were detected. Based on these results we concluded that the precipitation method might have some shortcomings for the analysis of highly diluted phosphopeptides present at substoichiometric levels.
      Figure thumbnail gr4
      Fig. 4MALDI spectra of different fractions obtained from the same sample as in but mixed with phosphoproteins/non-phosphoproteins 1:50.A, digested sample prior to enrichment; B, supernatant after precipitation; C, wash; and D, pellet fraction from calcium phosphate precipitation. The insets in A–D show the MS/MS spectra of the peak at m/z 1927. E, IMAC enriched digest; F, TiO2 enriched digest. The phosphopeptide signals are labeled with asterisks, and the three non-phosphopeptide signals used for comparison of enrichment efficiency are labeled with triangles.
      The above mentioned peak at m/z 1927 was present in all four fractions, whereas it was only observed in the original sample and the pellet in the spectra obtained with the casein standard (Fig. 2). Analysis of the digests of the three non-phosphorylated proteins in the mixture revealed that a tryptic peptide, CCAADDKEACFAVEGPK, from BSA was present at almost the same m/z. To investigate whether the m/z 1927 signal was derived from the BSA or α-casein, MS/MS was performed after selection of m/z 1927 in all the different fractions to check whether a neutral loss of phosphoric acid (loss of 97.97 Da) was observed (Fig. 4, insets). This demonstrated that the phosphopeptide was only present as a low abundance component in the original sample, whereas it was present at high abundance in the pellet. This confirms that the precipitation method is highly efficient as a phosphopeptide enrichment method and that the recovery of some phosphopeptides is high.
      Precipitation with other salts, e.g. calcium carbonate, ferric phosphate, and barium sulfate, was also tried. However, none of them were found to be as efficient as calcium phosphate presumably due to low yields (calcium carbonate), excessive oxidation (ferric phosphate), or too low solubility of the precipitate (barium sulfate).

       Coupling Precipitation with IMAC and TiO2

      The spectra obtained with the diluted (phospho)peptide mixture showed that the enrichment obtained with the precipitation method was not sufficient for enriching 50-fold substoichiometric levels of phosphopeptides in such complex mixtures for subsequent unseparated analysis by MALDI. Direct enrichment using IMAC and TiO2 was tested with the highly diluted sample (α-casein:β-casein:BSA:β-lactoglobulin:carbonic anhydrase = 1:1:50:50:50). Sample volumes corresponding to the presence of 1 pmol of α- and β-casein were used. In both cases a better phosphopeptide enrichment efficiency was obtained (Fig. 4, E and F). However, both methods also resulted in signals derived from non-phosphopeptides. IMAC showed a preferential enrichment of multiple phosphopeptides (Fig. 4E), whereas the monophosphopeptides dominated after enrichment on TiO2 (Fig. 4F). It must be noted that the TiO2 enrichment was made under suboptimal conditions because the addition of DHB was omitted in the loading buffer to make the procedure compatible with subsequent analysis by LC-ESI-MS (
      • Thingholm T.E.
      • Jorgensen T.J.
      • Jensen O.N.
      • Larsen M.R.
      Highly selective enrichment of phosphorylated peptides using titanium dioxide.
      ,
      • 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.
      ). Comparing the spectra resulting from the different methods using the three most intense non-phosphopeptide signals in the spectra of the pellet fraction at m/z 1245, m/z 1446, and m/z 1749 (Fig. 4D), it is seen that these three peaks are present in the spectrum obtained after enrichment by TiO2 (Fig. 4F), whereas they are absent in the spectrum obtained after enrichment by IMAC (Fig. 4E). This indicates that precipitation and IMAC might be complementary enrichment methods and that further IMAC enrichment of precipitated fraction might increase the specificity toward phosphopeptides by reducing the amount of non-phosphopeptides.
      To test this, the pellet obtained by calcium phosphate precipitation of the diluted, substoichiometric phosphopeptide sample was dissolved in 5% formic acid, completely dried down in vacuum, redissolved in 100 mm acetic acid, and incubated with IMAC resin. After incubation, the IMAC material was packed in a microcolumn and washed, and the peptides were eluted and analyzed by MALDI MS as described under “Experimental Procedures.” The resulting spectrum (Fig. 5A) was dominated by phosphopeptide signals. However, the signals for several of the monophosphopeptides, e.g. m/z 1466 and m/z 1660, were missing. This might be due to the presence of phosphate in the loading buffer. Therefore desalting before the IMAC step was attempted. The resulting spectrum (Fig. 5B) showed that the combination of precipitation, desalting, and IMAC resulted in efficient enrichment of the phosphopeptides. Signals for all the expected phosphopeptides were present (Table I) plus a few additional signals for doubly charged phosphopeptide ions. An unexpected additional signal at m/z 1539 could be assigned to a phosphopeptide based on a neutral loss of phosphoric acid. Its origin could not be determined because MS/MS mainly resulted in the neutral loss and did not give enough sequence information for identification. Similar experiments combining precipitation with TiO2 enrichment confirmed that these two methods were not complementary, although a certain improvement was observed (data not shown). The supernatant and wash fractions were applied to IMAC. No phosphopeptide signals could be observed in the resulting spectra indicating that the loss of phosphopeptides is minimal during the precipitation. In summary, the combination of calcium phosphate precipitation and IMAC enable efficient recovery of substoichiometric levels (50-fold dilution) of phosphopeptides in a complex peptide mixture.
      Figure thumbnail gr5
      Fig. 5The performance obtained by combining calcium phosphate precipitation and IMAC. After precipitation the peptides in the pellet fraction were further enriched with IMAC without (A) or with (B) a prior desalting procedure. The phosphopeptide signals are labeled with asterisks, and doubly charged phosphopeptide signals are labeled with double asterisks.

       Application of the Combined Precipitation and IMAC Enrichment to Investigate the Rice Phosphoproteome—

      To investigate whether the new combined enrichment method was applicable to a very complex biological sample, it was tested in one of our ongoing projects, the rice embryo proteome. 300 μg of rice embryo tryptic digest was submitted to calcium phosphate precipitation followed by Fe(III) IMAC enrichment. By comparing the MALDI MS spectrum of the original digestion mixture (Fig. 6A) with those obtained after phosphopeptide enrichment by IMAC only (Fig. 6B), precipitation only (Fig. 6C), and the combined method of calcium phosphate precipitation and IMAC (Fig. 6D), it is clear that an enrichment is obtained by precipitation, and that this is further enhanced by combining precipitation with IMAC. To identify phosphopeptide signals the 10 most intense peptide peaks in the spectra obtained with IMAC or precipitation were checked for neutral loss of phosphoric acid by MALDI-TOF/TOF MS. None of these signals could be assigned to phosphopeptides. Analysis of the same two fractions by LC-ESI-MS/MS resulted in identification of 3 and 11 phosphopeptides in the fractions obtained after IMAC only and precipitation only, respectively, corresponding to 1.1 and 6.2%, respectively, of the total number of the detected peptides in these fractions (data not shown). For the sample obtained by combining enrichment of precipitation and IMAC (Fig. 6D) all abundant signals were submitted to MALDI MS/MS, and all of them showed neutral loss of phosphoric acid (examples are shown in Fig. 6E). This observation was confirmed by treatment with alkaline phosphatase (results not shown).
      Figure thumbnail gr6
      Fig. 6Enrichment of phosphopeptides from rice embryo using different enrichment methods and assessed by MALDI MS and MS/MS.A, the original sample prior to any enrichment. B, enrichment by IMAC. C, enrichment by precipitation. D, enrichment combining precipitation, desalting, and IMAC. E, examples of MS/MS spectra obtained from three peaks in D. The phosphopeptide signals are labeled with asterisks.
      Because the loading capacity of the reversed-phase column in nanospray LC-ESI-MS is limited, we used a limited amount (1.5-μl bed volume) of IMAC resin to enrich phosphopeptides. This might result in loss of some of the phosphopeptides due to saturation of the IMAC resin. Therefore the flow-through from preparing the IMAC microcolumn was applied to the second IMAC enrichment, and the 98-Da loss test on MALDI MS/MS and 80-Da loss by alkaline phosphatase treatment revealed some phosphorylated peptides in the resulting eluate.
      Next the phosphopeptide samples prepared by calcium phosphate precipitation and IMAC were analyzed by nanoliter flow LC-ESI-MS/MS on a Q-TOF tandem mass spectrometer. The LC-MS/MS datasets were searched using iterative data analysis and stringent criteria for identification and assignment of phosphorylation sites (see “Experimental Procedures”).
      The analysis of the peptide samples from the first IMAC enrichment made it possible to identify 181 peptides derived from 102 different proteins. Among these 171 phosphopeptides (derived from 95 phosphoproteins) and 10 regular peptides were assigned. Thus, 94% of all the identified peptide sequences originated from phosphopeptides. In the second IMAC fraction, 100 phosphopeptides were assigned representing 67 different proteins, corresponding to 86 and 85% of all identified peptides and proteins, respectively. In total, 242 phosphopeptides (92%) representing 125 phosphoproteins (92%) were identified.
      The results also clearly show that the two subsequent IMAC steps were needed because the overlap between the phosphopeptides observed after the first and the second IMAC enrichments is rather small (Fig. 7A). In total, 227 non-redundant phosphorylated sites (213 on serines and 14 on threonines) were determined (see Supplemental Fig. S1 and Table S1). No peptides containing phosphorylated tyrosines were observed. We believe that this is mainly due to the fact that the frequency ratio of phosphoserine:phosphothreonine:phosphotyrosine in general is 1800:200:1 (
      • Hunter T.
      • Sefton B.M.
      Transforming gene product of Rous sarcoma virus phosphorylates tyrosine.
      ). Thus the chance of finding a peptide with phosphotyrosine among the 242 phosphopeptides observed is minimal. In our experience specific enrichment of the phosphotyrosine-containing peptides by immune precipitation is needed to observe these peptides. In principle the precipitation should be efficient also for phosphotyrosine-containing peptides. The distribution of number of phosphoryl groups per peptide is shown in Fig. 7B. In the first IMAC enrichment, the monophosphopeptides constitute only 30% of all observed phosphopeptides, whereas they accounted for about 75% in the second enrichment. Surprisingly very highly phosphorylated peptides (up to seven phosphoryl groups) were identified in the second IMAC enrichment. This might be due to less suppression in the second enrichment because fewer peptide signals were present in each spectrum. To our knowledge, such highly phosphorylated peptides have rarely been reported in previous studies using mass spectrometry. The observation of the different phosphopeptides and different preference for number of phosphoryl groups obtained with the two consecutive IMAC enrichment steps suggests that prefractionation with limited amounts of IMAC resin might be used to reduce sample complexity.
      Figure thumbnail gr7
      Fig. 7Results obtained by LC-ESI-MS/MS of the first IMAC enrichment and the second IMAC enrichment of the flow-through from the first.A, number of phosphopeptides identified in each of the serial IMAC steps and the overlap between these datasets. B, distribution of the number of phosphoryl groups per phosphopeptide in different enrichment steps.
      Finally an examination of the amino acid composition on the total number of identified phosphopeptides and non-phosphopeptides using the GPMAW software showed a high frequency of acidic amino acids (Asp and Glu) in the sequences of non-phosphopeptides and a high frequency of serine in the sequences of phosphopeptides. This means that the calcium phosphate precipitation, although reducing complexity and increasing phosphopeptide enrichment, like IMAC enriches very acidic peptides. The complementarity to IMAC thus might be a result of reduced complexity of the sample prior to IMAC enrichment. The sequence information of non-phosphopeptides is shown in Supplemental Table S2.

       Conclusion—

      A method for phosphopeptide enrichment based on calcium phosphate precipitation has been developed. In combination with IMAC, it has been demonstrated to be highly selective. Even with very complex biological samples such as the total enzymatic digest of rice embryo proteins high enrichment of the phosphopeptides can be achieved with minimal contamination with non-phosphopeptides. In addition, it might be possible to reduce the complexity of the samples by successive IMAC enrichments using a limited amount of IMAC material in each step. Altogether we have demonstrated that serial phosphopeptide enrichment initiated by a precipitation step improves the selectivity of phosphopeptide enrichment and allows identification of more phosphopeptides. Further analyses to examine the rice phosphoproteome in detail are now in course.

      Acknowledgments

      Dr. Pia Hønnerup Jensen, Lene Jakobsen, and Kate Rafn are acknowledged for help with LC-MS/MS measurements, and Dr. Siqi Liu is acknowledged for supply of the rice seed samples. Dr. Martin R. Larsen and Tine Thingholm are acknowledged for valuable discussions.

      Supplementary Material

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