Detection of Phosphorylated Peptides in Proteomic Analyses Using

a gift from Ulf Hellman, Ludwig Institute for Cancer Research,. Uppsala, Sweden. A phosphopeptide test sample (PRG03) consist- ing of 5 pmol tryptic d...
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Anal. Chem. 2004, 76, 5864-5871

Detection of Phosphorylated Peptides in Proteomic Analyses Using Microfluidic Compact Disk Technology Daniel Hirschberg,† Theres Ja 1 gerbrink,† Jenny Samskog,† Magnus Gustafsson,‡ Marie Sta˚hlberg,† † Gunvor Alvelius, Bolette Husman,§ Mats Carlquist,§ Hans Jo 1 rnvall,† and Tomas Bergman*,†

Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden, Gyros AB, Uppsala Science Park, SE-751 83 Uppsala, Sweden, and Karo Bio AB, Novum, SE-141 57 Huddinge, Sweden

A compact disk (CD)-based microfluidic method for selective detection of phosphopeptides by mass spectrometry is described. It combines immobilized metal affinity chromatography (IMAC) and enzymatic dephosphorylation. Phosphoproteins are digested with trypsin and processed on the CD using nanoliter scale IMAC with and without subsequent in situ alkaline phosphatase treatment. This is followed by on-CD matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Dephosphorylation of the IMAC-enriched peptides allows selective phosphopeptide detection based on the differential mass maps generated (mass shifts of 80 Da or multiples of 80 Da). The CD contains 96 microstructures, each with a 16 nL IMAC microfluidic column. Movement of liquid is controlled by differential spinning of the disk. Up to 48 samples are distributed onto the CD in two equal sets. One set is for phosphopeptide enrichment only, the other for identical phosphopeptide enrichment but combined with in situ dephosphorylation. Peptides are eluted from the columns directly into MALDI target areas, still on the CD, using a solvent containing the MALDI matrix. After crystallization, the CD is inserted into a MALDI mass spectrometer for analysis down to the femtomole level. The average success rate in phosphopeptide detection is over 90%. Applied to noncharacterized samples, the method identified two novel phosphorylation sites, Thr 735 and Ser 737, in the ligand-binding domain of the human mineralocorticoid receptor. Phosphorylation is an important functional modification of eukaryotic proteins, affecting their activity, interactions, and localization.1 Protein phosphorylation is also the major molecular mechanism for signal transduction by which extracellular signals produce their cellular responses.2 Although there is a wealth of genomic data for organisms of many types, characterization of the posttranslational phosphorylations of the expressed protein * Corresponding author. Phone: +46-8-524 8 7780. Fax: +46-8-337 462. E-mail: [email protected]. † Karolinska Institutet. ‡ Gyros AB. § Karo Bio AB. (1) Hunter, T. Cell 2000, 100, 113-127. (2) Cohen, P. Nat. Rev. Drug Discovery 2002, 1, 309-315.

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products requires proteome studies and efficient analytical tools for direct protein analysis, including selective detection and mass spectrometric identification.3,4 Characterization of phosphoproteins involves detection of phosphate-containing peptides and localization of their phosphorylation site(s). One strategy for selective recovery is to use the negative charge of the phosphate group in metal-binding affinity chromatography, as now demonstrated. For mapping of proteolytic fragments and identification of phosphorylation sites, matrixassisted laser desorption/ionization (MALDI) mass spectrometry complemented with electrospray ionization (ESI) tandem mass spectrometry (MS/MS) are useful techniques.5-7 Because of the impact of enzyme-catalyzed phosphorylation and dephosphorylation on the function of key metabolic proteins,2 there is a constant demand to improve the methods for reliable analysis of phosphopeptides. A difficulty in this respect is that phosphoproteins often exist in low abundance in cells, and are often mixed with the corresponding nonphosphorylated parent form, which decreases the phosphopeptide amount. Another difficulty is that the phosphopeptides are easily suppressed in both MALDI and ESI mass spectrometry. The low stoichiometry of phosphorylation in proteins makes it important to increase the selectivity in the analysis.8,9 A solution is to enrich the phosphopeptides from proteolytic mixtures by immobilized metal affinity chromatography (IMAC)5,6,10 before mass spectrometric analysis. Sample handling prior to analysis is critical, and miniaturization has become a key issue. To meet these requirements, microfluidic devices have been developed, initially for handling sample solutions by pressure-driven flow (nano-LC/capillary LC), by electroosmotic pumping (capillary electrophoresis), or by a combi(3) Quadroni, M.; James, P. Proteomics in Functional Genomics; Jolle`s, P., Jo ¨rnvall, H., Eds.; Birkha¨user Verlag: Basel, Switzerland, 2000; pp 199213. (4) Aebersold, R.; Mann, M. Nature 2003, 422, 198-207. (5) Larsen, M. R.; Roepstorff, P. Fresenius’ J. Anal. Chem. 2000, 366, 677690. (6) Mann, M.; Ong, S.-E.; Grønborg, M.; Steen, H.; Jensen, O. N.; Pandey, A. Trends Biotechnol. 2002, 20, 261-268. (7) Hirschberg, D.; Rådmark, O.; Jo ¨rnvall, H.; Bergman, T. J. Protein Chem. 2003, 22, 177-181. (8) Ficarro, S. B.; McCleland, M. L.; Stukenberg, P. T.; Burke, D. J.; Ross, M. M.; Shabanowitz, J.; Hunt, D. F.; White, F. M. Nat. Biotechnol. 2002, 20, 301-305. (9) McLachlin, D. T.; Chait, B. T. Anal. Chem. 2003, 75, 6826-6836. (10) Andersson, L.; Porath, J. Anal. Biochem. 1986, 154, 250-254. 10.1021/ac040044g CCC: $27.50

© 2004 American Chemical Society Published on Web 09/03/2004

nation of those two principles.11 A recent technique, based on a different concept, is microfluidic electrocapture of peptides and proteins in a flow stream for desalting and concentration into nanoliter volumes followed by MALDI-MS.12 Another novel approach, based on compact disk (CD) technology, was recently reported,13 where the liquid is driven by centrifugal force through capillary microstructures within the CD. Portions of the microstructures are packed with reversed-phase resins for preparation of peptide samples for on-CD MALDI-MS. The CD technology offers better recovery than off-line methods since it integrates sample preparation and MS analysis in a single platform format. The improved sample handling is combined with high throughput, an important aspect in proteomics, by parallel processing of up to 96 samples per CD. In the present study, IMAC resin was used within the CD format to enrich phosphopeptides generated by tryptic digestion of phosphoproteins (from chromatographies or SDS/PAGE) for on-CD MALDI-MS analysis. For efficient detection of phosphorylated peptides, differential mass maps were employed using alkaline phosphatase.14 The method was applied to the ligandbinding domain (LBD) of the human mineralocorticoid receptor (MR)15-18 in order to identify novel phosphorylation sites. EXPERIMENTAL PROCEDURES Materials. Bovine R- and β-casein, bovine serum albumin (BSA), bovine brain myelin basic protein (MBP), ovalbumin, phosphorylase b, enolase (yeast), and bovine alkaline phosphatase were purchased from Sigma. A synthetic peptide with a single phosphorylation site at a tyrosine residue (pYQQVDEEFLR) was a gift from Ulf Hellman, Ludwig Institute for Cancer Research, Uppsala, Sweden. A phosphopeptide test sample (PRG03) consisting of 5 pmol tryptic digest of bovine protein disulfide isomerase (PDI), spiked with 1 pmol each of two synthetic phosphopeptides derived from the PDI sequence (residues 266-273 phosphorylated at Ser 268 and 257-273 phosphorylated at Ser 266) was obtained from the Association of Biomolecular Resource Facilities (ABRF) Proteomics Research Group (PRG). BSA and β-casein tryptic peptides were prepared by in-solution digestion.13 MBP was phosphorylated at threonine residues 94 and 97.7 Recombinant human MR-LBD was produced in a baculovirus expression system.18 A protein stock solution at 5.0 µM each of MBP, R- and β-casein, ovalbumin, phosphorylase b, enolase, and MR-LBD was prepared in water. It should be noted that phosphorylase b and enolase do not contain phosphorylated residues; these proteins were included as negative controls and to increase the complexity (11) Lemmo, A. V.; Jorgenson, J. W. Anal. Chem. 1993, 65, 1576-1581. (12) Astorga-Wells, J.; Jo ¨rnvall, H.; Bergman, T. Anal. Chem. 2003, 75, 52135219. (13) Gustafsson, M.; Hirschberg, D.; Palmberg, C.; Jo ¨rnvall, H.; Bergman, T. Anal. Chem. 2004, 76, 345-350. (14) Zhang, X.; Herring, C. J.; Romano, P. R.; Szczepanowska, J.; Brzeska, H.; Hinnebusch, A. G.; Qin, J. Anal. Chem. 1998, 70, 2050-2059. (15) Arriza, J. L.; Weinberger, C.; Cerelli, G.; Glaser, T. M.; Handelin, B. L.; Housman, D. E.; Evans, R. M. Science 1987, 237, 268-275. (16) Alnemri, E. S.; Maksymowych, A. B.; Robertson, N. M.; Litwack, G. J. Biol. Chem. 1991, 266, 18072-18081. (17) Sheppard, K. E. Am. J. Physiol. Gastrointest. Liver Physiol. 2002, 282, G742746. (18) Kauppi, B.; Jakob, C.; Fa¨rnegårdh, M.; Yang, J.; Ahola, H.; Alarcon, M.; Calles, K.; Engstro ¨m, O.; Harlan, J.; Muchmore, S.; Ramqvist, A.-K.; Thorell, S.; O ¨ hman, L.; Greer, J.; Gustafsson, J-Å.; Carlstedt-Duke, J.; Carlquist, M. J. Biol. Chem. 2003, 278, 22748-22754.

of in-solution digested samples. Water was from a MilliQ purification unit (Millipore), and all chemicals were of analytical grade. SDS/PAGE. The protein stock solution was diluted to 0.5, 0.1, 0.05, 0.03, 0.01, and 0.005 µM. Protein samples (40 µL) were mixed with 20 µL of sample buffer (NuPAGE LDS sample buffer 4×, Invitrogen) and 20 µL of 0.2 M dithiothreitol (DTT). The samples were heated (70 °C, 10 min) before loading onto Trisbuffered (pH 6.4) 12% polyacrylamide gels. The running buffer (2.5 mM Tris base, 19 mM glycine, and 0.1% SDS) was supplemented with 1.25 mM DTT to prevent reoxidation of reduced proteins. Electrophoresis was carried out in a Mini-Protean II system (BioRad) at room temperature and 120 V for 1-1.5 h (until the dye marker bromophenol blue had reached the edge of the gel). After electrophoresis, proteins were stained with Coomassie R-250, 0.1% (w/v) in 40% methanol containing 10% acetic acid, for 4 h at room temperature. Destaining was carried out overnight in the methanol/acetic acid solution until the protein bands were clearly visible against the background. In-Gel Digestion. Protein bands were excised manually from the Coomassie-stained gels. Gel pieces were processed and digested with trypsin using a robotic protein handling system (MassPREP, Waters) employing a protocol based on in-gel digestion methods.19 Gel pieces were destained twice in 100 µL of 50 mM ammonium bicarbonate (Ambic)/50% (v/v) acetonitrile at 40 °C for 10 min. Proteins were reduced by treatment in 10 mM DTT/100 mM Ambic for 30 min, gel pieces were shrunk in acetonitrile, and alkylation was carried out in 55 mM iodoacetamide/100 mM Ambic for 20 min. Trypsin (25 µL of a 12 ng/µL solution in 50 mM Ambic) was added, and digestion was allowed to proceed for 4.5 h at 40 °C. Peptides were extracted with 30 µL of 1% acetic acid/50% 2-propanol, followed by 24 µL of 1% acetic acid/50% 2-propanol. Alternatively, the peptides were extracted with 30 µL of 5% formic acid/2% acetonitrile followed by 24 µL of 2.5% formic acid/50% acetonitrile. The first approach was used for the CD-based IMAC phosphopeptide enrichment, the latter for ESI MS/MS peptide sequence analysis. In-Solution Digestion. To 80 µL of the protein stock solution diluted to 0.5 µM in water, 10 µL of 0.5 M Ambic and 3 µL of 20 mM DTT were added and the solution was incubated for 15 min at room temperature. Then, 5 µL of 55 mM iodoacetamide was added followed by incubation for 15 min at 37 °C. Trypsin (2 µL of a 1 µg/µL solution in 0.5 M Ambic) was added, and digestion was allowed to proceed for 4.5 h at 37 °C. After digestion, aliquots of the tryptic digest were diluted to 250, 50, 25, 15, 5, and 2.5 nM prior to IMAC enrichment. CD-Based Sample Concentration, Cleanup, and Crystallization. For sample preparation with the Gyrolab MALDI SP1 CD in a Gyrolab Workstation (Gyros AB, Uppsala, Sweden), 1 µL of peptide solution was applied to each sample inlet used on the CD. The CD contains 96 microcolumns for parallel concentration/desalting by reversed-phase chromatography (RPC).13 Each column contains a hydrophobic stationary phase with a volume of 10 nL. The columns were conditioned using 50% acetonitrile in water. The samples were loaded onto the reversed-phase columns (after removal of acetonitrile under a stream of nitrogen), and solvents were passed through the microstructures by spinning (19) Oppermann, M.; Cols, N.; Nyman, T.; Helin, J.; Saarinen, J.; Byman, I.; Toran, N.; Alaiya, A. A.; Bergman, T.; Kalkkinen, N.; Gonzalez-Duarte, R.; Jo¨rnvall, H. Eur. J. Biochem. 2000, 267, 4713-4719.

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Figure 1. MALDI mass spectra showing the phosphopeptide detection principle using on-CD IMAC and enzymatic dephosphorylation. Two aliquots of each sample are transferred by robotics from the 96-well microtiter plate, containing the digests, to the microfluidic CD. (A) One aliquot is applied to a section for IMAC enrichment of phosphopeptides followed by on-CD MALDI-MS, (B) the other aliquot to a section for identical IMAC enrichment followed by alkaline phosphatase treatment and MALDI-MS. A characteristic mass shift of 80 Da (or multiples of 80 Da) is detected for phosphate-containing peptides.

the disks. The wash solution (200 nL of 0.1% trifluoroacetic acid (TFA)) along with salts and other polar components was directed to a waste exit. Peptides were eluted from the columns using 200 nL of 50% acetonitrile containing 1 mg/mL R-cyano-4-hydroxycinnamic acid (CHCA) MALDI matrix and 0.1% TFA. The eluates were captured in open MALDI target areas to allow for solvent evaporation and peptide/matrix cocrystallization. CD-Based IMAC: Phosphopeptide Enrichment, Cleanup, and Crystallization. For phosphopeptide enrichment with the Gyrolab MALDI IMAC1 CD in a Gyrolab Workstation (Gyros AB, Uppsala, Sweden), 1 µL of peptide solution was applied to each microstructure of the CD. The CD has a construction similar to the Gyrolab MALDI SP1 CD,13 containing 96 microcolumns and open MALDI target areas of 200 µm × 400 µm. Each column contains 16 nL of IMAC resin (Poros 20 MC) activated with Ga(III). Half of the CD (48 microcolumns) was used for phosphopeptide enrichment, the other half for combined phosphopeptide enrichment and dephosphorylation by alkaline phosphatase (Figure 1). The columns were conditioned with 50% acetonitrile/0.6% acetic acid in water, samples were loaded onto the columns, and solvents passed, all by spinning the disks. Each sample was distributed in equal volumes to one microstructure for phosphopeptide enrichment and another microstructure for enrichment and dephosphorylation (Figure 1). Columns were washed with 50% acetonitrile/ 5% acetic acid, followed by 50% 2-propanol/5% acetic acid. Nonphosphorylated peptides, salts, and other polar components were directed to a waste exit. Dephosphorylation was carried out by addition of 200 nL of 0.1 U/µL alkaline phosphatase in 50% acetonitrile/25 mM Ambic. Peptides were eluted from the columns using 200 nL of 50% acetonitrile/1% H3PO4 containing 10 mg/mL dihydroxybenzoic acid (DHBA) MALDI matrix. The eluates were captured in the open MALDI target areas to allow for solvent evaporation and peptide/matrix cocrystallization. Mass Spectrometry. Peptides were analyzed by MALDI-MS in a Voyager DE-Pro instrument (Applied Biosystems). Tryptic 5866

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fragments of the MR-LBD were also analyzed by ESI quadrupole time-of-flight tandem MS in a Q-TOF instrument (Waters). Before on-CD MALDI analysis, the CD was cut (precision cutter, Gyros AB, Uppsala, Sweden) to fit into the target compartment of the MALDI instrument. For peptides with m/z less than 4000, the MALDI instrument was operated in reflectron mode, while larger peptides were analyzed in linear mode. Mascot was used for database searches of peptide mass fingerprint data (www. matrixscience.com). The Q-TOF tandem MS instrument was equipped with an orthogonal sampling ES interface (Z-spray, Waters). Samples were introduced via gold-coated nano-ES needles (Proxeon). A capillary voltage of 1200-1500 V was applied together with a cone voltage of 40-45 V. The sample aerosol was desolvated in a stream of nitrogen. During the collision-induced dissociation (CID), the collision energy was 15-30 eV, and argon was used as collision gas. For ESI MS/MS, digests were desalted employing C18 ZipTips (Millipore). Before desalting, acetonitrile was evaporated from samples under a stream of nitrogen. The ZipTips were activated and equilibrated using 10 µL of 70% acetonitrile/0.1% TFA twice, 10 µL of 50% acetonitrile/0.1% TFA twice, and 10 µL of 0.1% TFA twice. The samples were loaded onto ZipTips by pipetting 20 times and washed using 10 µL of 0.1% TFA twice. The tryptic fragments were eluted with 60% acetonitrile/1% acetic acid. RESULTS Establishment of CD-Based Selective Detection of Phosphopeptides. Model peptides (β-casein tryptic phosphopeptides at 0.1 µM, a phosphotyrosine peptide at 0.025 µM, and BSA tryptic peptides at 0.4 µM) were used to test the feasibility of CD-based IMAC to enrich phosphorylated peptides for MALDI-MS analysis (Figure 2). The phosphopeptides from β-casein correspond to residues 48-63 and 16-40, containing one phosphoserine residue at position 51, and four at positions 30, 32, 33, and 34, respectively,

Figure 2. MALDI mass spectra from on-CD analysis of a mixture of β-casein phosphorylated fragments (m/z 2061 and 3122, 100 fmol, from β-casein tryptic digest), a synthetic phosphotyrosine peptide (m/z 1407, 25 fmol), and BSA tryptic fragments (nonphosphorylated, 400 fmol) after (A) on-CD concentration/desalting of the mixture by RPC, (B) on-CD IMAC enrichment, and (C) on-CD dephosphorylation by alkaline phosphatase. The phosphopeptide peaks correspond to (1) the phosphotyrosine peptide at m/z 1407, (2) β-casein tryptic fragment at m/z 2061, and (3) β-casein tryptic fragment at m/z 3122. The prime signs denote the corresponding dephosphorylated species in each case.

while the phosphotyrosine peptide is singly phosphorylated at the N-terminus. The peptide mixture was first concentrated/desalted using a Gyrolab RPC CD and analyzed by MALDI-MS, generating a peptide mass map (Figure 2A). The mixture was then analyzed using Gyrolab MALDI IMAC1 CD sample preparation before MALDI-MS analysis (Figure 2B) and finally treated by a combination of IMAC enrichment and alkaline phosphatase treatment before MALDI-MS analysis using the same IMAC CD (Figure 2C). The results clearly demonstrate the importance of IMAC sample preparation combined with on-column dephosphorylation for efficient identification of phosphopeptides in mixture with nonphosphorylated tryptic fragments. The loss of 80 Da (corresponding to a phosphate group) and multiples of 80 Da can readily be detected in the spectra (Figure 2B and C). It should be noted that the phosphopeptide samples were contaminated with BSA tryptic fragments at concentrations that were 4- and 16-fold greater than the concentration of the β-casein peptides and the phosphotyrosine peptide, respectively. Nevertheless, the spectra after IMAC treatment do not show significant signals from BSA tryptic fragments. However, at m/z 927, 1283, 1749, and 3026, minor signals are detected (Figure 2B and C), corresponding to peptides with 10-20% Asp- and/or Glu-residues. In a series of analyses of the phosphotyrosine peptide, the singly phosphorylated β-casein peptide and the quadruply phosphorylated β-casein peptide (about 100 analyses of each peptide, mixed with BSA tryptic peptides), it was demonstrated that both the phosphorylated and the dephosphorylated species were reproducibly detected at high signal-to-noise ratios (Table 1). Successful detection after IMAC enrichment was achieved on the

Table 1. Success Rates in Phosphopeptide Analysis by MALDI-MS after On-CD IMAC Enrichment and Alkaline Phosphatase Treatmenta

phosphopeptide

successful phosphopeptide detectionb (%)

successful detection of the dephosphorylated peptidec (%)

1P-Tyr 1P-Ser 4P-Ser

94d 96e 97e

97d 96e 79e

a The peptides tested were a synthetic phosphotyrosine peptide (1PTyr, 1407 Da), a singly phosphorylated β-casein peptide (1P-Ser, 2061 Da), and a quadruply phosphorylated β-casein peptide (4P-Ser, 3122 Da). Samples were mixed with BSA tryptic peptides to yield complex mixtures for CD separation. b At a signal-to-noise ratio of minimally 3:1. c At a signal-to-noise ratio of minimally 10:1. d N ) 72 analyses.e N ) 120 analyses.

average in 96% of the analyses at MALDI-MS signal-to-noise ratios of at least 3:1. Detection of the dephosphorylated peptide after alkaline phosphatase treatment was achieved on the average in 91% of the analyses at signal-to-noise ratios that were minimally 10:1 (Table 1). A tryptic digest of bovine protein disulfide isomerase (PRG03, 5 pmol) spiked with two synthetic phosphopeptides derived from the PDI structure (1 pmol each) was dissolved in 10 µL of 10% ethanol/0.1 M acetic acid. Peptide mass fingerprinting after onCD concentration/desalting (Gyrolab MALDI SP1) was tested (Figure 3A). The peptide mass list was searched using Mascot at 120 ppm mass accuracy which resulted in correct identification Analytical Chemistry, Vol. 76, No. 19, October 1, 2004

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Figure 3. MALDI mass spectra from on-CD analysis of the phosphopeptide-containing PRG03 sample. (A) Peptide mass fingerprint after RPC concentration/desalting. A database search showed that the sample contained bovine protein disulfide isomerase. (B) Phosphopeptide enrichment by IMAC. (C) Phosphopeptide enrichment followed by enzymatic on-column dephosphorylation using alkaline phosphatase. Two phosphopeptides at m/z 964 and 2027 were recognized from the mass shifts of 80 Da resulting from dephosphorylation.

(PDI) and 48% sequence coverage corresponding to 23 peptides identified at a MOWSE score of 267. After successful protein identification, the PRG03 sample was diluted with 10 µL of acetonitrile to a final concentration of 250 fmol digest/µL, containing 50 fmol/µL of the phosphopeptides. Two aliquots of 1 µL each were transferred by the Gyrolab workstation to the CD, where one was used for IMAC enrichment of phosphate-containing peptides only (Figure 3B), the other for IMAC enrichment followed by enzymatic dephosphorylation using alkaline phosphatase (Figure 3C). Both phosphopeptides were efficiently enriched and dephosphorylated using on-CD IMAC (Figure 3B and C). Peptide sequences were shown to be SVSDYEGK and THILLFLPKSVSDYEGK by a molecular weight search against the PDI amino acid sequence (residues 266-273 and 257-273, with one phosphorylation each). Analysis of Phosphoproteins from Gel Separations. Protein samples containing MBP, R- and β-casein, ovalbumin, phosphorylase b, enolase, and MR-LBD were separated by SDS/PAGE followed by Coomassie-staining, trypsin in-gel digestion, and onCD IMAC processing with subsequent MALDI-MS analysis. The MALDI spectra derived from the β-casein band in different concentrations revealed that it was possible to detect the phosphopeptide with m/z 2061 down to a level of 1 pmol applied to gel electrophoresis (Figure 4A). The large, quadruply phosphorylated β-casein fragment at m/z 3122 was not abundant in the mass spectra, probably due to a low degree of extraction from the gel.20 The MALDI spectra for MBP tryptic peptides after IMAC enrichment demonstrated that the doubly phosphorylated peptide was clearly seen when 5 pmol of protein was loaded onto the gel (Figure 4B). (20) Jonsson, A. P.; Aissouni, Y.; Palmberg, C.; Percipalle, P.; Nordling, E.; Daneholt, B.; Jo ¨rnvall, H.; Bergman, T. Anal. Chem. 2001, 73, 5370-5377.

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For comparison, the same proteins in mixture were digested in solution with trypsin and subjected to IMAC processing and MALDI-MS. On-CD IMAC treatment removed most of the irrelevant peptides from the digest, and signals in the MALDI spectra were enhanced, for some of the phosphopeptides by large factors, greatly improving detection limits (Figure 5). Despite the fact that the proteins were not separated, but digested and analyzed in mixture, the detection limit for the phosphopeptides was generally lower than that for gel-separated proteins, about 15 fmol (Figure 5). Identification of Phosphorylation Sites in MR-LBD. To identify possible phosphorylation sites in MR-LBD, the recombinant protein, purified by affinity and ion exchange chromatography,18 was digested with trypsin and analyzed by on-CD IMAC and MALDI-MS before and after alkaline phosphatase treatment. Diagnostic mass shifts of 80 and 160 Da were detected for two of the tryptic fragments (Figure 6A). From a comparison of the peptide mass data with the known amino acid sequence of MR-LBD,18 it was clear that the two phosphopeptides correspond to residues 733-771 of MR-LBD with one and two phosphate groups attached, respectively. Since there are more than two possible phosphorylation sites in this segment of the protein, MS/ MS analysis was applied to assign the phosphorylated residues. The tandem MS data confirmed that the tryptic fragment exists in two phosphorylation states, one fraction phosphorylated at Ser 737 only, and the other at both Thr 735 and Ser 737 (Figure 6B). To our knowledge, phosphorylation at these sites has not previously been reported for recombinant human MR-LBD. DISCUSSION The microfluidic CD-based IMAC method now presented is designed for selective and sensitive screening of phosphopeptides. The CD format allows processing of multiple samples at high throughput down to the femtomole level. We have shown that nanoliter affinity columns in combination with in situ enzymatic dephosphorylation is efficient. The method has been extensively tested with model peptides and gel-separated proteins and applied to a recombinant protein to identify novel phosphorylation sites. Mixtures of phosphorylated and nonphosphorylated peptides were tested to establish the efficiency and success rate of the method. In particular, the technique was applied to a phosphopeptide sample (PRG03) difficult to characterize and submitted to a worldwide investigation in 2003 (www.abrf.org/ResearchGroups/Proteomics/EPosters/ABRF_PRG03.pdf). At the time we analyzed this sample, the phosphorylation sites were known to us. Nevertheless, the results clearly show the benefit of using a combination of on-CD IMAC and IMAC plus dephosphorylation, before MALDI-MS analysis (Figure 3). From the peptide masses and mass shifts detected, it was straightforward to assign both the number of phosphate groups involved and the position of each fragment in the PDI sequence. In repetitive analysis of singly and multiply phosphorylated peptides, the success rate for detection in MALDI-MS was high, both directly after IMAC enrichment and after combined IMAC enrichment/dephosphorylation (Table 1). The results thus reveal a high analytical consistency and reproducibility from microstructure to microstructure, and from CD to CD. Notably, the phosphopeptides were mixed with BSA tryptic fragments to increase the analytical complexity and the risk of signal suppres-

Figure 4. MALDI mass spectra after on-CD IMAC enrichment of in-gel digested samples. (A) β-casein singly Ser-phosphorylated tryptic peptide at m/z 2061 (asterisk, 1 pmol protein applied to gel electrophoresis). (B) MBP doubly Thr-phosphorylated tryptic peptide at m/z 1651 (asterisk, 5 pmol protein applied to gel electrophoresis). The component at m/z 2005 is a nonphosphorylated MBP peptide (residues 74-90).

Figure 5. MALDI mass spectra (A) before and (B) after on-CD IMAC enrichment of an in-solution tryptic digest of a mixture of ovalbumin, enolase, R- and β-casein, phosphorylase b, MBP, and MR-LBD. The detection limit for the phosphorylated tryptic peptides in (B) is about 15 fmol. The tryptic phosphopeptide peaks correspond to (1) MBP fragment at m/z 1651, (2) R-casein fragment at m/z 1660, (3) R-casein fragment at m/z 1927, (4) β-casein fragment at m/z 2061, (5) β-casein fragment at m/z 3122. Phosphopeptides from ovalbumin and MR-LBD were not detected at this concentration (1 µL of a 15 nM digest was applied to the CD) while enolase and phosphorylase b did not contain phosphorylated residues.

sion. It is particularly encouraging to detect the signals of a multiply phosphorylated peptide with four phosphoserine residues, both directly after IMAC enrichment and after dephosphorylation, where the intensities are comparable to those of singly phosphorylated peptides at high signal-to-noise ratios, of the order 20:1 or more (Figure 2B and C, Figure 5B). Furthermore, it has been reported that multiply phosphorylated peptides frequently are difficult to detect after IMAC sample preparation, which has been attributed to strong binding of these peptides to the IMAC resin.21,22 In contrast, using the present protocol, the success rate (21) Posewitz, M. C.; Tempst, P. Anal. Chem. 1999, 71, 2883-2892.

in detection of the quadruply Ser-phosphorylated peptide after IMAC processing is well over 90% which is also found for a singly Ser-phosphorylated peptide (both results are based on 120 analyses, Table 1). In addition, the CD-based IMAC method appears not to be influenced by differences between Tyr-phosphorylated and Ser-phosphorylated peptides, since they are detected at similar success rates (Table 1). Elution of the phosphopeptides from the IMAC resin is most often carried out at alkaline pH by the use of a high-concentration phosphate (22) Hart, S. R.; Waterfield, M. D.; Burlingame, A., L.; Cramer, R. J. Am. Soc. Mass Spectrom. 2002, 13, 1042-1051.

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Figure 6. MALDI and tandem mass spectra of a phosphorylated peptide recovered after tryptic digestion of MR-LBD showing that the peptide exists both singly and doubly phosphorylated and additionally revealing the location of the phosphorylation sites. (A) MALDI spectra from on-CD analysis of the phosphorylated peptide before and after dephosphorylation with alkaline phosphatase: (1) doubly phosphorylated peptide, (2) singly phosphorylated peptide, and (3) peptide without phosphate groups. The relative signal intensities were shifted 80 Da (for 2) and 160 Da (for 1) toward lower mass after dephosphorylation. (B) Section of CID spectrum for the triply charged parent ion at m/z 1469.84, providing evidence for the presence and location of two phosphate groups in the 39-residue tryptic MR-LBD fragment. The peptide corresponds to residues 733-771 of MR-LBD with phosphate groups attached to Thr 735 and Ser 737 (denoted by p). The complete sequence of the peptide could be assigned from the tandem MS data and is AL(pT)P(pS)PVMVLENIEPEIVYAGYDSSKPDTAENLLSTLNR, in full agreement with the MR-LBD structure.18 Losses of one and two phosphoric acid molecules were also detected in the CID spectrum. Asterisk indicates y373+.

buffer.21 In the present method, the elution is at low pH using 1% phosphoric acid in combination with DHBA (instead of CHCA as MALDI matrix), which we have found to be a powerful eluent that does not require desalting before MALDI-MS analysis. 5870 Analytical Chemistry, Vol. 76, No. 19, October 1, 2004

To assess the performance with samples originating from gel separations, protein bands were excised from SDS/polyacrylamide gels, digested with trypsin, and applied to the IMAC CD technology. For comparison, a mixture of the same proteins was

in-solution treated with trypsin and directly applied to on-CD IMAC sample preparation. From these results it can be concluded that the extraction of tryptic peptides from gels, in particular of large fragments, e.g., β-casein at m/z 3122 now detected as a strong signal after in-solution digestion (Figure 5B), may be difficult in accordance with a previous report.20 In addition, R-casein phosphopeptides at m/z 1660 and 1927, the β-casein phosphopeptide at m/z 2061, and the MBP fragment at m/z 1651 were detected in the MALDI-MS analysis of the in-solution digest processed by on-CD IMAC (Figure 5). However, not all potential phosphopeptides were detected at this concentration (1 µL of 15 nM digest) in the positive ion mode MALDI-MS analysis which is most certainly related to structural properties including a high content of acidic groups. The comparison between in-gel and in-solution digestion also revealed a better sensitivity for the latter, partly due to low yield in the extraction of tryptic peptides from the gels, but also because of the multistep nature that includes application of sample to the gel, electrophoresis, staining, excision of gel piece with protein band/spot, in-gel digestion, and finally extraction of tryptic peptides. For detection in MALDI-MS analysis after onCD IMAC processing, gel applications in the range 1-5 pmol are suitable (Figure 4), while for in-solution samples, low femtomole of protein is sufficient (Figure 5). The yield in on-CD dephosphorylation of phosphothreonine was relatively low for MBP (Figure 5B) and MR-LBD (Figure 6A). Notably, however, the branched β-carbon in the threonine side chain, may cause steric hindrance, and regarding MBP the two phosphorylated threonine residues are flanking a positively charged arginine residue which might lower the accessibility to alkaline phosphatase. Novel phosphorylation sites in the ligand-binding domain of the human mineralocorticoid receptor were identified by a combination of CD-based IMAC/MALDI-MS and ESI tandem MS. MR is a member of the steroid/thyroid/retinoid receptor family.15-18 The human renal MR has been reported to be phosphorylated upon baculovirus-mediated expression in Spodoptera frugiperda insect cells.16 The biological function of MR phosphorylation is not known but transformation to a DNA-binding form has been shown for native rat kidney MR upon treatment with phosphatases.23 The human MR-LBD studied in this work was also expressed in a baculovirus system,18 and the purified recombinant protein was in-solution digested with trypsin followed by on-CD

IMAC enrichment and dephosphorylation (Figure 6A). The MALDI mass spectra before and after dephosphorylation clearly indicate the presence of three peptide species differing by 80 or 160 Da. From the lowest mass detected, a search of the sequence for MR-LBD18 revealed a clear match with a segment constituting the N-terminal 39 residues. It was then clear that the two higher masses correspond to this fragment with one and two phosphate groups attached. Since this segment contains more than two potential phosphorylation sites, tandem MS was applied to localize the phosphorylated residues. Despite the large size (39 residues, corresponding to 4406 Da with two phosphate groups), CID of the triply charged parent ion at m/z 1469.84 resulted in fragment ions covering the complete sequence and clearly positioned the phosphorylation sites to Thr 735 and Ser 737 (Figure 6B). Although phosphorylation of the human MR-LBD has previously been shown,16 these two sites have to our knowledge not been reported. The MR-LBD data illustrate an efficient strategy in phosphoprotein characterization: (i) selective screening for phosphopeptides using MALDI-MS, after on-CD IMAC/dephosphorylation, and if required (ii) tandem MS to assign the phosphorylation sites. In conclusion, the CD-based IMAC enrichment of phosphopeptides from proteolytic digests combined with dephosphorylation and on-CD MALDI-MS for generation of differential mass maps is highly efficient for selective and sensitive phosphopeptide detection. The CD technology enables routine analyses at the femtomole level, combined with high throughput, and good reproducibility, all important factors in phosphoproteome characterization.

(23) Galigniana, M. D. Biochem. J. 1998, 333, 555-563.

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ACKNOWLEDGMENT We are grateful to the ABRF Proteomics Research Group (Chair: Kaye D. Speicher) for supplying the PRG03 phosphopeptide sample, to Ulf Hellman, Ludwig Institute for Cancer Research, Uppsala, Sweden, for supplying the phosphotyrosine peptide, and to Allan Stensballe, University of Southern Denmark, Odense, Denmark, for fruitful discussions. This work was supported by Grants from the Swedish Research Council, the Swedish Cancer Society, and Karolinska Institutet. Received for review March 10, 2004. Accepted July 1, 2004.

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