Analysis of Histidine Phosphorylation Using Tandem MS and Ion

Sep 7, 2007 - with the typically low abundance of signaling proteins. Detection .... (CID), electron-based tandem MS methods are very useful to locali...
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Anal. Chem. 2007, 79, 7450-7456

Analysis of Histidine Phosphorylation Using Tandem MS and Ion-Electron Reactions Anne J. Kleinnijenhuis,† Frank Kjeldsen,† Birgitte Kallipolitis,† Kim F. Haselmann,‡ and Ole N. Jensen*,†

Department of Biochemistry and Molecular Biology and Department of Physics and Chemistry, University of Southern Denmark, DK-5230 Odense M, Denmark

Phosphorylation of proteins is essential in intracellular signal transduction pathways in eukaryotic and prokaryotic cells. Histidine phosphorylation plays an important role in two-component signal transduction in bacteria. In this study, we describe the characterization of a synthetic histidine-phosphorylated peptide with four different mass spectrometric (MS) fragmentation techniques: Collisioninduced dissociation (CID), electron capture dissociation, electron-transfer dissociation, and electron detachment dissociation. Furthermore, LC-MS methods were developed to detect histidine-phosphorylated peptides, which are acid-labile, in more complex samples. From these results, we concluded that nonacidic solvent systems or fast LC methods provide the best conditions for separation of histidine-phosphorylated peptides prior to electrospray ionization mass spectrometry analysis. Electron-based fragmentation methods should be used for determination of histidine phosphorylation sites, since CID results in very facile phosphate-related neutral losses. The developed LC-MS/MS methods were successfully applied to a tryptic digest of the cytoplasmic part of the histidine kinase EnvZ, which was in vitro autophosphorylated. Finally, a new method is described for nonretentive solidphase extraction of histidine-phosphorylated peptides using polymeric Strata-X microcolumns. Many signal transduction pathways in eukaryotic and prokaryotic cells are regulated by protein phosphorylation events.1 It has been estimated that about one-third of mammalian proteins contain covalently bound phosphate at some point.2 Most studies on protein phosphorylation consider O-phosphorylated hydroxyamino acid residues: serine, threonine, and tyrosine. In a typical proteomics experiment, complex protein mixtures or purified proteins are subjected to proteolytic cleavage, followed by liquid chromatography (LC) and mass spectrometric (MS) analysis of the peptides. Phosphopeptides are generally difficult to detect, because of the often low phosphorylation stoichiometry combined with the typically low abundance of signaling proteins. Detection of phosphopeptides with MS is further hampered by suppression effects in the presence of non-phosphopeptides during the ionization event. To circumvent these problems, a number of enrichment * To whom correspondence should be addressed. E-mail: [email protected]. Phone: +45-6550-2368. Fax: +45-6593-2661. † Department of Biochemistry and Molecular Biology. ‡ Department of Physics and Chemistry. (1) Cohen, P. Nature 1982, 296, 613-620. (2) Zolnierowicz, S.; Bollen, M. EMBO J. 2000, 19, 483-488.

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methods for phosphopeptides have been developed, such as TiO2 chromatography3,4 and immobilized metal affinity chromatography (IMAC).5-8 In both these methods, acidic conditions are used, because the binding selectivity of TiO2 and IMAC material for phosphopeptides is significantly reduced or absent at pH g7. For LC separation and ionization of the peptides, with matrix-assisted laser desorption/ionization (MALDI) or electrospray ionization (ESI), acidic conditions are used routinely as well. These are the main reasons why acid-labile phosphorylations, such as phosphorylation of histidine (half-life time ∼30 min at pH 39), have not been studied on a large scale. Histidine phosphorylation plays an important role in two-component signal transduction. Twocomponent systems are found in eubacteria, archaea, plants, and eukaryotic organisms10-12 and are dedicated to sense and interpret signals, such as pH, osmolarity, temperature, and the presence of toxins. Histidine phosphorylation may even account for 6% of total protein phosphorylation in eukaryotes,13 which makes it ∼100-fold more abundant than phosphotyrosine, but less abundant than phosphoserine. Two-component systems transmit information via histidine phosphorylation on a histidine kinase and subsequent phosphate transfer to an aspartate residue on their response regulator protein partner, which is usually a separate protein.10 An example of a histidine kinase is EnvZ of Escherichia coli that responds to osmolarity change in the extracellular medium by regulating the phosphorylation state of its response regulator partner OmpR.14,15 EnvZ is located in the inner membrane with two transmembrane domains and extends into the cytoplasm and into the periplasm.16 At high osmolarity, EnvZ dimers transautophosphorylate at the His-243 residue. The phosphate group (3) Pinkse, M. W. H.; Uitto, P. M.; Hilhorst, M. J.; Ooms, B.; Heck, A. J. R. Anal. Chem. 2004, 76, 3935-3943. (4) Larsen, M. R.; Thingholm, T. E.; Jensen, O. N.; Roepstorff, P.; Jorgensen, T. J. D. Mol. Cell Proteomics 2005, 4, 873-886. (5) Neville, D. C. A.; Rozanas, C. R.; Price, E. M.; Gruis, D. B.; Verkman, A. S.; Townsend, R. R. Protein Sci. 1997, 6, 2436-2445. (6) Posewitz, M. C.; Tempst, P. Anal. Chem. 1999, 71, 2883-2892. (7) Stensballe, A.; Andersen, S.; Jensen, O. N. Proteomics 2001, 1, 207-222. (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) Sickman, A.; Meyer, H. E. Proteomics 2001, 1, 200-206. (10) Appleby, J. L.; Parkinson, J. S.; Bourrett, R. B. Cell 1996, 86, 845-848. (11) Loomis, W. F.; Kuspa, A.; Shaulsky, G. Curr. Opin. Microbiol. 1998, 1, 643648. (12) Perraud, A.-L.; Weiss, V.; Gross, R. Trends Microbiol. 1999, 7, 115-120. (13) Matthews, H. R. Pharmacol. Ther. 1995, 67, 323-350. (14) van Alphen, W.; Lugtenberg, B. J. Bacteriol. 1977, 131, 623-630. (15) Hall, M.; Silhavy, T. J. J. Mol. Biol. 1981, 151, 1-15. (16) Forst, S.; Corneau, D.; Norioka, S.; Inouye, M. J. Biol. Chem. 1987, 262, 16433-16438. 10.1021/ac0707838 CCC: $37.00

© 2007 American Chemical Society Published on Web 09/07/2007

is then transferred to Asp-55 on OmpR. Phosphorylated OmpR represses OmpF and activates OmpC, resulting in a lower influx of solutes. OmpC and OmpF are two outer membrane porin proteins, with OmpC having a smaller pore size and allowing a slower flow rate than OmpF.17 Currently, more than 300 histidine kinases are known in prokaryotes.18 Also, histidine kinases have been detected in mammals. In rats, for instance, a nuclear protein kinase phosphorylates histone H4 on histidine.19 Motojima and Goto20 and Hedge and Das21 have detected histidine phosphorylation on the proteins p36 and p38 from rat, respectively. Both proteins are phosphorylated in response to the presence of ras protein and guanine nucleotides. To identify the histidine-phosphorylated proteins, they used 32P labeling in combination with SDS-PAGE and performed pH stability studies. Noiman and Shaul22 developed a protocol for large-scale and rapid detection of histidine phosphoproteins in crude cellular extracts also using radioactive 32P labeling and SDS-PAGE. In this report, we describe the analysis of a synthesized histidine-phosphorylated peptide and a histidine-phosphorylated protein using LC and mass spectrometry without the use of radioactive 32P labeling. The synthesized histidine-phosphorylated peptide was used to develop and optimize mass spectrometric and chromatographic methods to detect the histidine-phosphorylated tryptic peptide from the histidine kinase EnvZ. Also a method is described for nonretentive solid-phase extraction of histidine-phosphorylated peptides with polymeric Strata-X microcolumns. Tandem MS was used to localize phosphohistidine sites. In contrast to collision-induced dissociation (CID), electron-based tandem MS methods are very useful to localize labile peptide modifications, because backbone bond fragmentation preferentially takes place even in the presence of labile groups. Electron capture dissociation (ECD)23 has been used to localize phosphorylation sites in peptides24-26 and other labile modified amino acid residues such as oxidized methionine.27 In ECD, polycations are irradiated with low-energy electrons, resulting in very fast dissociation.23 With electron detachment dissociation (EDD)28 it is possible to fragment polyanions by irradiating them with >10 eV electrons. ECD and EDD are primarily used in combination with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR). Electron-transfer dissociation (ETD)29 is similar to ECD, although in ETD electrons are transferred to polycations by reacting them with (radical) anions, which can be achieved in an ion trap. ETD is a promising tandem MS technique for phosphoproteomics. First reports on the use of ETD in largescale phosphoproteomic studies suggest that ETD and CID generate complementary information.30,31 (17) Nikaido, H.; Rosenberg, E. Y. J. Bacteriol. 1983, 153, 241-252. (18) Grebe, T. W.; Stock, J. B. Adv. Microbiol. Physiol. 1999, 41, 139-227. (19) Chen, C. C.; Bruegger, B. B.; Kern, C. W.; Lin, Y. C.; Halpern, R. M.; Smith, R. A. Biochemistry 1977, 16, 4852-4855. (20) Motojima, K.; Goto, S. FEBS Lett. 1993, 319, 75-79. (21) Hedge, A. N.; Das, M. R. FEBS Lett. 1987, 217, 74-80. (22) Noiman, S.; Shaul, Y. FEBS Lett. 1995, 364, 63-66. (23) Zubarev, R. A.; Kelleher, N. L.; McLafferty, F. W. J. Am. Chem. Soc. 1998, 120, 3265-3266. (24) Stensballe, A.; Jensen, O. N.; Olsen, J. V.; Haselmann, K. F.; Zubarev, R. A. Rapid Commun. Mass Spectrom. 2000, 14, 1793-1800. (25) Shi, S. D.; Hemling, M. E.; Carr, S. A.; Horn, D. M.; Lindh, I.; McLafferty, F. W. Anal. Chem. 2001, 73, 19-22. (26) Chalmers, M. J.; Hakansson, K.; Johnson, R.; Smith, R.; Shen, J.; Emmett, M. R.; Marshall, A. G. Proteomics 2004, 4, 970-981. (27) Guan, Z.; Yates, N. A.; Bakhtiar, R. J. Am. Soc. Mass Spectrom. 2003, 14, 605-613. (28) Budnik, B. A.; Haselmann, K. F.; Zubarev, R. A. Chem. Phys. Lett. 2001, 342, 299-302. (29) Syka, J. E. P.; Coon, J. J.; Schroeder, M. J.; Shabanowitz, J.; Hunt, D. F. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 9528-9533.

METHODS Histidine phosphorylation was analyzed using a synthesized peptide and the cytoplasmic part of EnvZ (EnvZc), a histidine kinase from Escherichia coli. Peptide Synthesis and Tandem MS Analysis. The peptide ALLMAGVAHDLR was synthesized using an EPS221 automated peptide synthesizer (Intavis, AG). This peptide is an analogue of the tryptic peptide containing the histidine phosphorylation site from the bacterial histidine kinase EnvZ: TLLMAGVSHDLR. The histidine residue of this peptide was phosphorylated with the method of Wei and Matthews32 using phosphoryl chloride. The phosphopeptide products of this reaction were characterized with an LC/MSD Trap XCT Ultra (Agilent Technologies) and an LTQFT linear ion trap-Fourier transform mass spectrometer (ThermoFisher, Bremen, Germany), using CID, ETD, ECD, and EDD as fragmentation techniques. ESI was used as ionization method. The spray voltage used for positive ESI was ∼2800 V and for negative ESI -1600 V. Electron irradiation times for ECD and EDD and reaction time for ETD were 70 and 150 ms, respectively. Reagent accumulation time for ETD was 30 ms. EnvZc. DNA encoding for the cytoplasmic part of EnvZ (Arg180-Gly450) with an N-terminal His6 tag was cloned into the pUHE24-2 vector and transformed into E. coli TOP10 harboring pMS421.33 The bacteria were grown in Luria-Bertani medium to an OD600 of 0.5, induced with isopropyl β-D-1-thiogalactopyranoside, and grown for an additional 1 h. Then the bacteria were harvested and sonicated in lysis buffer (50 mM sodium phosphate, 300 mM NaCl, pH 7 and protease inhibitor cocktail (Roche)). Histagged EnvZc was purified with Talon metal affinity resin (Clontech) using the batch/gravity-flow column purification protocol. SDS-PAGE was performed to determine which of the eluted fractions contained the protein. EnvZc was in vitro autophosphorylated by incubating the protein at room temperature with ATP in phosphorylation buffer (50 mM Tris‚HCl pH 8.0, 0.8 mM dithiothreitol, 0.1 mM EDTA, 4% glycerol, 10 mM MgCl2), following the method of Inouye and co-workers.34 After the autophosphorylation reaction, EnvZc was digested with trypsin in 50 mM NH4HCO3 for 16 h at 37 °C. LC-MS. LC-MS analysis was performed on either an 1100 nanoflow system connected to an LC/MSD Trap XCT Ultra (Agilent Technologies, Santa Clara, CA), an HPLC-Chip/MS system (Agilent Technologies), or an Ultimate 3000 LC system (Dionex, Sunnyvale, CA) coupled to a QTOF Micro (Waters/ Micromass) tandem mass spectrometer. With the HPLC-Chip/ MS system, a Protein ID chip #1 (Agilent Technologies, Santa Clara, CA), containing a 40-nL enrichment column and a 43 × 75 µm analytical column, packed with Zorbax 300SB C18, was used. Otherwise, trap and analytical columns were packed with Reprosil C18 (Dr Maisch) material. Different LC solvents were tested to optimize conditions for histidine-phosphorylated peptides, namely, acidic (0.5% acetic acid, pH 2.9 or 0.1% formic acid, pH 2.7), “neutral” (10 mM ammonium acetate, pH 6.7), and basic solvents (30) Chi, A.; Huttenhower, C.; Geer, L. Y.; Coon, J. J.; Syka, J. E. P.; Bai, D. L.; Shabanowitz, J.; Burke, D. J.; Troyanskaya, O. G.; Hunt, D. F. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 2193-2198. (31) Molina, H.; Horn, D. M.; Tang, N.; Mathivanan, S.; Pandey, A. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 2199-2204. (32) Wei, Y. F.; Matthews, H. R. Methods Enzymol. 1991, 200, 388-414. (33) Jørgensen, C. I.; Kallipolitis, B. H.; Valentin-Hansen, P. Mol. Microbiol. 1998, 27, 41-50. (34) Forst, S.; Delgado, J.; Inouye, M. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 6052-6056.

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(25 mM triethyl ammonium bicarbonate (TEAB), pH 8.4). Peptides were eluted from the analytical column using an acetonitrile gradient. The histidine-phosphorylated peptide ALLMoxAGVApHDLR was spiked into 400 fmol of tryptic digest of 12 standard proteins: transferrin (human), serum albumin (bovine), β-lactoglobulin (bovine), carbonic anhydrase (bovine), β-casein (bovine), R-casein (bovine), ovalbumin (chicken), ribonuclease B (bovine pancreas), alcohol dehydrogenase (baker yeast), myoglobin (whale skeletal muscle), lysozyme (chicken), and R-amylase (bacillus species), that were all obtained from Sigma (St. Louis, MO). Phosphohistidine Peptide Enrichment. Constricted GELoader tips were packed35 with Strata-X (Phenomenex, Torrance, CA) material, to obtain microcolumns with a length of ∼1 cm. Strata-X is highly cross-linked polystyrene divinylbenzene material that retains peptides by hydrophobic interactions; hydrophilic peptides are not retained.36 The column was washed with 20 µL of acetonitrile and equilibrated with 20 µL of deionized water. Samples containing histidine-phosphorylated peptides were loaded on a Strata-X column in 25 mM TEAB buffer (pH 8.4). The flow-through was loaded directly on a Poros Oligo R3 (PerSeptive Biosystems, Framingham, MA) microcolumn. The R3 column was washed with 30 µL of deionized water, and the peptides were eluted with 70% acetonitrile for LC-MS analysis or with 1 µL of 20 mg/mL 2,5-dihydroxybenzoic acid in 70% acetonitrile and 1% phosphoric acid directly onto a MALDI plate.37 MALDI-TOF MS analysis was performed using a Bruker/Daltonics Ultraflex MALDI ToF/ToF mass spectrometer. RESULTS AND DISCUSSION Peptide synthesis resulted in the nearly pure end product ALLMAGVAHDLR. A phosphate group was added to the peptide after the reaction with phosphoryl chloride.32 The yield for peptide phosphorylation was >50%. In addition, extensive methionine oxidation (Mox) was observed. The synthesized phosphopeptide was stable at pH g7 but very unstable at acidic pH (see also Figure 2), typical for phosphorylation on histidine residues.9 To confirm the location of the phosphorylation site in ALLMoxVApHDLR, [M + 2H]2+ ions of this peptide were subjected to CID (Figure 1a). Collisional activation resulted in extensive loss of H3PO4. Minor HPO3 loss was observed, which would be expected for a histidinephosphorylated peptide.38 The apparent H3PO4 loss was thus most probably a combined loss of HPO3 and water. When CID MS/ MS was performed on the ions that had lost 98 Da from the precursor ions, the origin of the water loss turned out to be the Asp10 residue next to the His9 residue. Because of the very abundant neutral loss of 98 Da, it was difficult to extract sequence information with CID MS/MS. The CID MS3 spectra of [M + 2H - 98]2+ ions were more informative as backbone bond cleavages were much more abundant (results not shown). Unfortunately, with CID, almost all information was lost about the location of the phosphorylation site. The only observed phosphorylated fragment ions were b10 and b11. Therefore, we investigated (35) Gobom, J.; Nordhoff, E.; Mirgorodskaya, E.; Ekman, R.; Roepstorff, P. J. Mass Spectrom. 1999, 34, 105-116. (36) Kapkova, P.; Lattova, E.; Perreault, H. Anal. Chem. 2006, 78, 7027-7033. (37) Stensballe, A.; Jensen, O. N. Rapid Commun. Mass Spectrom. 2004, 18, 1721-1730. (38) Medzihradszky, K. F.; Phillipps, N. J.; Senderowicz, L.; Wang, P.; Turck, C. W. Protein Sci. 1997, 6, 1405-1411.

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electron-based techniques to fragment phosphorylated ALLMoxAGVAHDLR (Figure 1b-d). In contrast to CID, ECD of [M + 2H]2+ ions resulted in abundant backbone bond cleavages and the phosphorylation site could be assigned to the histidine residue (Figure 1b). However, the z4•-z8• and c9-c11 fragment ions that all contain the phosphohistidine residue were observed in both the phosphorylated and the nonphosphorylated state. The phosphorylated fragment ions were less abundant than the corresponding nonphosphorylated fragment ions. c-type ions retained the phosphate group better than z-type ions. Furthermore, extensive H3PO4 loss (probably again a combined HPO3 and H2O loss) was observed from the [M + 2H]+• product ions. This is typically not observed when O-phosphorylated peptides are subjected to ECD.24 Phosphoramidate bonds do not seem to be selectively cleaved with ECD, because in this case, the 80 Da neutral loss signal would be much more prominent. ETD MS/MS of doubly protonated ALLMoxAGVApHDLR ions (Figure 1c) gave results similar to ECD. Only minor differences in the fragmentation pattern were observed. In general, phosphorylated fragment ions were slightly higher in intensity with ETD than with ECD. The z-type ions retained the phosphate group better than c-type ions. Because ETD was performed in an ion trap instead of an FT-ICR MS, the resolution of the ETD spectra was lower as compared to the ECD spectra (Figure 1). Interestingly, many radical c•-type ions and nonradical z-type ions were observed. These ions can also be formed as products in ECD of peptides (47% occurrence),39,40 but appear to be much more prominent in ETD spectra. Previously, Savitski et al.40 concluded that the degree of hydrogen atom transfer between c’and z• fragments is influenced by the lifetime of a short-lived product complex [c’ + z•]complex f [c• + z’]complex f c• + z’. In quadrupole ion traps, ions are subjected to much higher damping pressure than in Penning traps, which we believe leads to enhanced stabilization of this product complex prior to the dissociation. As such, this could explain why hydrogen atom transfer appears to be more frequent in ETD than ECD. Finally, doubly deprotonated ALLMoxAGVApHDLR anions were subjected to EDD in the negative ion mode, producing predominantly a- and x-type ions.41 Despite more abundant loss of phosphoric acid in EDD from the oxidized species [M - 2H]-• than observed with ECD or ETD (reduced species [M + 2H]+•), the phosphate group on histidine was retained more efficiently on the fragment ions than with either ECD or ETD (Figure 1d). The a-type ions exhibited no phosphate loss at all, whereas the x-type ions showed only minor phosphate-related losses. EDD fragmentation efficiency was low as compared to ECD and ETD (approximately 4 and 5 times lower, respectively). The fragmentation behavior of the synthetic histidine-phosphorylated peptide highlights the extreme lability of phosphohistidine groups. Even with electron-based dissociation techniques, phosphate-related losses are observed. Phosphohistidine was more stable during the EDD process than during ETD, ECD, and CID. (39) Zubarev, R. A.; Horn, D. M.; Fridriksson, E. K.; Kelleher, N. L.; Kruger, N. A.; Lewis, M. A.; Carpenter, B. K.; McLafferty, F. W. Anal. Chem. 2000, 72, 563-573. (40) Savitski, M. M.; Kjeldsen, F.; Nielsen, M. L.; Zubarev, R. A. J. Am. Soc. Mass Spectrom. 2007, 18, 113-120. (41) Kjeldsen, F.; Silivra, O. A.; Ivonin, I. A.; Haselmann, K. F.; Gorshkov, M.; Zubarev, R. A. Chemistry 2005, 11, 1803-1812.

Figure 1. (a) CID, (b) ECD, and (c) ETD spectrum of ALLMoxAGVApHDLR [M + 2H]2+ ions, (d) EDD spectrum of ALLMoxAGVApHDLR [M - 2H]2- ions. Phosphorylated fragment ions are indicated by the suffix +p. ∼ is a harmonic. The fragmentation pattern is shown in each spectrum. Small arrows represent fragment ions that were only observed nonphosphorylated. Big arrows represent fragment ions that were observed only phosphorylated (a-type ions from EDD) or both phosphorylated and nonphosphorylated. In each spectrum, a zoom of the m/z 1050-1100 region is shown to illustrate the obtained resolution.

The exact location of the phosphorylation site could be deduced with ECD and EDD; with ETD, it could not be determined whether the phosphorylation site was His9 or Asp10. Only ECD gave complete amino acid sequence coverage of the peptide, while CID, ETD, and EDD gave nearly complete sequence coverage (10 out 11 interresidue bonds were cleaved). To establish an LC-MS method to analyze histidine-phosphorylated peptides, different chromatographic solvent systems were tested: 0.5% acetic acid/acetonitrile (pH 2.9), 25 mM TEAB/ acetonitrile (pH 8.4), and 10 mM ammonium acetate/acetonitrile (pH 6.7). The synthesized peptide ALLMoxVApHDLR was spiked into a 400-fmol tryptic digest of 12 standard proteins to mimic more complex biological samples. Due to its acid lability, the histidine-phosphorylated peptide was poorly detected using acidic conditions (0.5% acetic acid/acetonitrile, Figure 2a). Using 25 mM TEAB/acetonitrile (pH 8.4) as solvent, the phosphorylated peptide was observed, but the sensitivity and chromatographic resolution were very low (Figure 2b). Finally, 10 mM ammonium acetate/ acetonitrile provided the most optimal conditions for detecting the histidine-phosphorylated peptide. The peptide was clearly detected by LC-MS when only 100 fmol of the phosphopeptide

was spiked into 400 fmol of the tryptic digest of 12 standard proteins (Figure 2c). Theoretically, analysis of histidine-phosphorylated peptides can also be performed with standard acidic solvents (pH close to 3) using a very fast LC method. The half-life time of phosphohistidine is ∼30 min at pH 3,9 while the exposure time to acidic conditions can be limited to several minutes using a neutral loading buffer and an HPLC-Chip for separation. With a steep gradient, going from 3 to 80% solvent B (0.1% FA, 90% ACN) in 7 min, the peptide ALLMoxAGVApHDLR eluted after ∼5 min (Figure 2d) and very little phosphate loss was observed. Doubly protonated peptide ions were automatically selected and subjected to CID and ETD to localize the labile phosphoistidine site. The peptide was detectable with this method when only 40 fmol of sample was injected. Next, we used the neutral solvent system, 10 mM ammonium acetate/acetonitrile, and the fast chip LC method to try to detect the histidine-phosphorylated tryptic peptide from the protein EnvZc. First, purified EnvZc was analyzed with SDS-PAGE and detected with Coomassie stain (Figure 3). The observed mass of the protein on SDS-PAGE corresponded to the theoretical mass of 31 kDa. After in vitro autophosphorylation of EnvZc, digestion Analytical Chemistry, Vol. 79, No. 19, October 1, 2007

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Figure 2. LC-MS analysis under acidic (0.5% acetic acid, pH 2.9), basic (25 mM TEAB, pH 8.4), and neutral (10 mM ammonium acetate, pH 6.7) conditions using a long gradient (1 h) and LC-MS analysis under acidic conditions (0.1% formic acid, pH 2.7) using a short gradient (7 min). Shown are the extracted ion chromatograms of the 2+ ions of phosphorylated ALLMoxAGVApHDLR (a-d) and nonphosphorylated ALLMoxAGVAHDLR (e-h).

Figure 3. Coomassie-stained SDS-PAGE of the first three fractions after Talon purification. The observed mass of His-tagged EnvZc (31 kDa) corresponds to the theoretical mass.

with trypsin, and desalting, an amino acid sequence coverage of 66% was obtained using LC-MS and 10 mM ammonium acetate/ acetonitrile as the solvent system, but the signal intensity for phosphorylated TLLMAGVSHDLR (m/z 696.8) was poor. However, the [M + 2H]2+ ions of phosphorylated TLLMAGVSHDLR were automatically selected for CID MS/MS several times. CID induced intense 98 Da loss and a few backbone fragment ions (Figure 4). The spectra were very similar to those obtained for the synthesized peptide analogue. Similar results were obtained using the HPLC-Chip and CID/ETD fragmentation. The peptide was still very low in intensity, and therefore, the ETD spectrum did not contain any fragment ions. To reduce the complexity of the sample and increase the signal for TLLMAGVSpHDLR, we tested several methods to make a sample enriched for histidinephosphorylated peptides. TiO2 chromatography and IMAC, commonly used for enrichment of O-phosphorylated peptides, are not very suitable to enrich for histidine-phosphorylated peptides because they employ acidic conditions. The selectivity of TiO2 toward phosphopeptides is highest at low pH.4 Only few histidine-phosphorylated peptides will survive these conditions, even though samples can be loaded on the TiO2 column within minutes. With IMAC, higher pH values can be used in the loading buffer (pH 3), but typically it requires a loading time of 1 h or more; thus, IMAC also is not optimal for 7454

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the enrichment of histidine-phosphorylated peptides. An enrichment method for histidine-phosphorylated peptides operating at basic or neutral pH would be highly desirable. Recently, the nonretentive extraction of O-phosphorylated peptides from R- and β-casein digests using Strata-X polymeric material and basic conditions was reported.36 Due to hydrophobic interactions, Strata-X material retains non-phosphopeptides better than the intrinsically hydrophilic phosphopeptides. Therefore, the flowthrough from a Strata-X column will contain a higher fraction of phosphopeptides than the original sample. To test this, the synthesized peptide ALLMAGVA(p)HDLR, in both the phosphorylated and nonphosphorylated states, was loaded on a Strata-X microcolumn in 25 mM TEAB and the column flow-through was collected. The retained peptides were eluted with 20% acetonitrile. The MALDI spectrum from the flow-through fraction showed a much higher peak for the phosphorylated peptide at m/z 1362.7 than the MALDI spectra from the eluate and the untreated sample (Figure 5), showing that the nonphosphorylated peptide is retained by the column material. Due to the acidity of the matrix, quite an abundant signal from the nonphosphorylated peptide is present in the MALDI spectrum of the hydrophilic flow-through fraction. Nonetheless, with a Strata-X nonretentive extraction step, it should be possible to analyze more complex samples containing histidine-phosphorylated peptides. Next, we performed nonretentive extraction with Strata-X of the tryptic digest of EnvZc, followed by LC-MS/MS analysis. An ETD spectrum of TLLMAGVSpHDLR was recorded, which contained several informative fragment ions (Figure 6a). In contrast, LC-MS spectra of TLLMAGVSpHDLR from the untreated tryptic digest of EnvZc produced only weak ion signals and no ETD fragment ions were observed. We attribute the better quality of the mass spectra after Strata-X fractionation to the reduced complexity of the sample. When the amount of peptides in a sample is reduced, fewer peptides will coelute during LC analysis. This increases the probability for low abundant peptides to be selected for MS/MS and decreases ionization bias. This is illustrated by the extracted ion chromatograms for phosphorylated and nonphosphorylated TLLMAGVSHDLR with and without Strata-X extraction (Figure 6b, c, e, f): the signal for nonphosphorylated TLLMAGVSHDLR is greatly reduced after Strata-X

Figure 4. CID spectrum of phosphorylated TLLMAGVSpHDLR. Phosphorylated fragment ions are indicated by the suffix +p. The fragmentation pattern is shown in the spectrum. Small arrows represent fragment ions that were only observed nonphosphorylated. Big arrows represent fragment ions that were observed both phosphorylated and nonphosphorylated.

Figure 5. MALDI-ToF spectra of ALLMAGVAHDLR: (a) untreated sample, (b) flow-through Strata-X column, and (c) elution with 20% acetonitrile. Peaks in the spectra are from (A) the unmodified, (B) the methionine-oxidized, (C) the phosphorylated, and (D) the methionine-oxidized and phosphorylated peptide.

treatment while the signal for TLLMAGVSpHDLR is in the same order of magnitude. The ETD fragmentation pattern of TLLMAGVSpHDLR (Figure 6a) points to phosphorylation on Ser8 rather than His9. However, similar to ALLMoxAGVApHDLR, the abundant collision induced H3PO4 loss (Figure 4) and the acid lability observed for TLLMAGVSpHDLR strongly indicate phosphorylation on the histidine residue. When LC-MS analysis was performed on the EnvZc digest using a long gradient and acidic conditions, the TLL-

MAGVSpHDLR phosphopeptide was not detected (Figure 6d, g). Therefore, we attribute the absence of phosphorylated z4 ions to electron-transfer induced phosphate loss from z4 ions, which was also observed for ALLMoxAGVApHDLR. CONCLUSION Histidine phosphorylation is difficult to analyze using standard MS and LC-MS methods. The results presented in this paper show that the application of fast LC-MS methods or neutral/ Analytical Chemistry, Vol. 79, No. 19, October 1, 2007

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Figure 6. (a) ETD spectrum of phosphorylated TLLMAGVSpHDLR. Phosphorylated fragment ions are indicated by the suffix +p. The fragmentation pattern is shown in the spectrum. Small arrows represent fragment ions that were only observed non-phosphorylated. Big arrows represent fragment ions that were observed both phosphorylated and nonphosphorylated. (b-g) LC-MS analysis under acidic conditions (0.1% formic acid, pH 2.7) using a short gradient (7 min), with and without Strata-X treatment, and using a long gradient (1 h). Shown are the extracted ion chromatograms of the 2+ ions of phosphorylated TLLMAGVSpHDLR (b-d) and nonphosphorylated TLLMAGVSHDLR (e-g).

basic LC solvents in combination with nonretentive solid-phase extraction with Strata-X and electron-based fragmentation methods are extremely useful tools for the analysis of histidine-phosphorylated peptides and proteins. The pH stability characteristics of synthesized ALLMoxAGVApHDLR combined with the ECD, ETD, and EDD fragmentation pattern unambiguously demonstrated the presence of a phosphorylated histidine residue. Several LC-MS methods were tested to optimize conditions for the detection of histidinephosphorylated peptides. From these results, we concluded that either nonacidic solvent systems or fast LC methods provide the best conditions for histidine-phosphorylated peptides to survive until they enter the mass spectrometer. To localize the site of phosphorylation, electron-based fragmentation methods, such as ECD and ETD, should be used as CID results in very facile phosphate-related neutral losses. The developed methods were applied to a tryptic digest of the cytoplasmic part of the histidine kinase EnvZ after in vitro autophosphorylation. The histidinephosphorylated peptide was indeed detected using these LCMS methods. This paper also presents a method for enrichment of histidine-phosphorylated peptides using nonretentive solid-

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Analytical Chemistry, Vol. 79, No. 19, October 1, 2007

phase extraction with polymeric Strata-X material. Using this approach, histidine-phosphorylated peptide analysis can be performed in more complex samples, which could open up a whole new branch of phosphoproteomics. ACKNOWLEDGMENT We thank Christina B. Kirkegaard from the Department of Biochemistry and Molecular Biology, University of Southern Denmark, for preparing the EnvZc DNA construct and Morten Kirkegaard for assistance with the ECD experiments. The FTICR MS instrument was financed by a generous grant from the Danish Basic Research Foundation to the Center for Experimental Bioinformatics. A.J.K. was supported by the Lundbeck Foundation. F.K. is funded by the Danish Research Council (FTP 274-06-0265). O.N.J. is a Lundbeck Foundation Research Professor.

Received for review April 19, 2007. Accepted July 25, 2007. AC0707838