Anal. Chem. 2005, 77, 1957-1962
Analysis of CheA Histidine Phosphorylation and Its Influence on Protein Stability by High-Resolution Element and Electrospray Mass Spectrometry Mathias Wind,†,‡ Ansgar Wegener,§ Roland Kellner,§ and Wolf D. Lehmann*,†
Central Spectroscopy, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany, and Merck KGaA, Global Technologies TRB, 64271 Darmstadt, Germany
A combination of electrospray mass spectrometry (ESIMS) and element mass spectrometry (ICPMS) with phosphorus detection was used to characterize histidine phosphorylation (His-48) of the chemotaxis protein CheA quantitatively. The phosphorylation at His-48 was found to be responsible for a stabilization of the protein. For this investigation, the acceptor domain and the kinase domain of the bacterial chemotaxis protein CheA were recombinantly expressed as single proteins. Using in vitro kinase assay conditions, the acceptor domain CheA-H was phosphorylated by the kinase domain CheA-C. The degree of histidine phosphorylation was determined by nanoelectrospray mass spectrometry of intact CheA-H, and was found to be limited to a maximum value of ∼50%. The site specificity of CheA-H phosphorylation was controlled by nanoESI-MS/MS of the [M + 16H]16+ ion of intact (pHis)-CheA-H and allowed localization of the pHis residue to the region between residues 32 and 86, containing candidates His-48 and His-67, for which His-48 phosphorylation has been described. Analysis of the tryptic digest of in vitro histidine-phosphorylated CheA-H by capillary chromatography coupled to ESI-MS and to ICPMS with phosphorus detection revealed a truncated (pHis)-CheA-H protein as the only phosphorus-containing analyte. Since the truncated (pHis)-CheA-H in the digest was found to exhibit a higher degree of phosphorylation than could be generated by in vitro phosphorylation without trypsin treatment, it is concluded that histidine phosphorylation at His-48 strongly interferes with structural properties of the CheA-H domain in particular with respect to proteolytic degradation by trypsin. Reversible protein phosphorylation1 is the most abundant posttranslational protein modification and represents an essential principle in the regulation of numerous cell functions.2 The phosphomonoesters pSer, pThr, and pTyr (estimated relative * To whom correspondence should be addressed. Tel.: 0049-6221-424563. Fax: 0049-6221-424554, E-mail:
[email protected]. † German Cancer Research Center (DKFZ). ‡ Current address: Basilea Pharmaceutica Ltd., 4005 Basel, Switzerland. § Merck KGaA. (1) Cohen, P. Nat. Cell Biol. 2002, 4, E127-E130. (2) Manning, G.; Plowman, G. D.; Hunter, T.; Sudarsanam, S. Trends Biochem. Sci. 2002, 27, 514-520. 10.1021/ac040140h CCC: $30.25 Published on Web 02/18/2005
© 2005 American Chemical Society
abundances 90:9:1) are in the focus of many protein phosphorylation studies, whereas the phosphoamides pHis, pLys, and pArg3 and the phosphoric acid mixed anhydride pAsp4 are less intensively studied. So far, phosphohistidine has been preferably observed in two-component systems of prokaryotes, which are composed of a sensor histidine kinase with autophosphorylation activity and a response regulator,5 to which the phosphate group is transferred to a particular Asp residue. The exclusive occurrence of these two-component systems in prokaryotes has put histidine into focus as a potential target for antimicrobial agents.6 Recently, histidine phosphorylation has also been increasingly observed in higher life forms7 particularly in connection with phosphatetransfer reactions, and it has been correlated to a variety of cellular functions and dysfunctions, including cancer.8 For phosphorylation site pinpointing occurring at the hydroxy amino acids serine, threonine, and tyrosine, numerous techniques based on mass spectrometry have been developed and successfully applied.9 This is not the case for the phosphoric acid amides and mixed anhydrides. One reason for this is their limited stability under acidic conditions. Since analytical protocols usually contain acidic conditions at some step, phosphoamide and phosphoanhydride species are poorly visible using standard procedures. These chemical stability problems are augmented by the instability of phosphohistidine peptides when analyzed by positive ion MALDI10,11 or ESI10 mass spectrometry. Protonated molecules of pHis-peptides show an abundant loss of HPO3, so that their MS and MS/MS spectra essentially show ions of the unmodified peptide. Negative ions of pHis peptides are more stable compared to their protonated counterparts, a phenomenon that was also demonstrated in connection with the specific enrichment of pHis peptides achieved by a Cu(II) IMAC procedure.11 (3) Matthews, H. R. Pharmacol. Ther. 1995, 3, 323-350. (4) Lewis, J. R.; Brannigan, J. A.; Muchova, K.; Barak, I.; Wilkinson, A. J. J. Mol. Biol. 1999, 294, 9-15. (5) Klumpp, S.; Krieglstein, J. Eur. J. Biochem. 2002, 269, 1067. (6) Matsushita, M.; Janda, K. D. Bioorg. Med. Chem. 2002, 10, 855-867. (7) Besant, G. T.; Tan, E.; Attwood, P. V. Int. J. Biochem. Cell Biol. 2003, 35, 297-309. (8) Steeg, P. S.; Palmieri, D.; Ouatas, T.; Salerno, M. Cancer Lett. 2003, 190, 1-12. (9) McLachlin, D. T.; Chait, B. T. Curr. Opin. Chem. Biol. 2001, 5, 591-602. (10) Medzihradsky, K. F.; Phillipps, N, J.; Senderowicz, L.; Wang, P.; Turck, C. W. Protein Sci. 1997, 6, 1405-1411. (11) Napper, S.; Kindrachuk, J.; Olson, D. J. H.; Ambrose, S. J.; Dereniwsky, C.; Ross, A. R. S. Anal. Chem. 2003, 75, 1741-1747.
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Figure 1. Analysis of a tryptic digest of an incubation of the histidine kinase CheA-C with its substrate CheA-H (molar ratio 1:10) in the presence of ATP. (A) Analysis by µLC-ESI-MS, base peak chromatogram. (B, C) Identical µLC analysis, but coupled to ICPMS with 31P detection. Only one late-eluting phosphoprotein compound is detected.
In view of this situation, it is not surprising that analytical methodologies for investigation of histidine phosphorylation are underdeveloped with respect to the established and presumed biological impact of this phenomenon. In this study, we therefore applied a novel combination of analytical methods: we investigated histidine phosphorylation in a recombinant in vitro system derived from the bacterial chemotaxis protein A (CheA)12 by a combination of reversed-phase liquid chromatography, element mass spectrometry with phosphorus detection, and electrospray MS/MS.
Table 1. Tryptic Peptide Annotation of the Recombinant CheA-H Construct As Displayed in Figure 5 tryptic peptide
residue
tryptic peptide
residue
T1 T2 T3 T4 T5 T6
3-39 40-45 46-51 52-77 78 79-95
T7 T8 T9 T10 T11
96-106 107-124 125-131 132-139 140-147
EXPERIMENTAL SECTION Recombinant Expression of Kinase Subdomains. The bacterial histidine kinase CheA became available in the form of two functional domains, namely, CheA-H and CheA-C, as described recently.13 In Vitro Phosphorylation and Sample Treatment. Autocatalytic phosphorylation of CheA-C and phosphotransfer onto
CheA-H was achieved in a homogeneous reaction.14 Aliquots containing phosphorylated CheA-H were subsequently digested for MS studies using trypsin.15 In short, phosphorylated CheA-H was incubated overnight at 37 °C with modified bovine trypsin, sequencing grade (Roche Diagnostics, Mannheim) in 100 mM
(12) Bilwes, A. M.; Alex, L. A.; Crane, B. R.; Simon, M. I. Cell 1999, 96, 131141. (13) Swanson, R. V.; Schuster, S. C.; Simon, M. I. Biochemistry 1993, 32, 76237629.
(14) Levit, M.; Liu, Y.; Surette, M.; Stock, J. J. Biol. Chem. 1996, 271, 3205732063. (15) Wind, M.; Wegener, A.; Eisenmenger, A.; Kellner, R.; Lehmann, W. D. Angew. Chem., Int. Ed. 2003, 42, 3425-3427.
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Figure 2. ESI mass spectrum of the µLC peak at 46.5 min (Figure 1A). (A) Original spectrum; (B) deconvoluted spectrum. CheA-H with a degree of phosphorylation of ∼75% is identified.
NH4HCO3, pH 8.0 at a substrate-to-enzyme ratio of ∼50:1. The digest was desalted using C18 micropipet tips (Waters, ZipTip) using the standard desalting protocol recommended by the manufacturer. Element Mass Spectrometry. Inductively coupled plasma (ICP) mass spectrometry was performed using a sector field instrument type Element 2 (Thermo Finnigan, Bremen, Germany) with medium mass resolution (4000), which is sufficient for interference-free detection of 31P. A microflow nebulizer (Microflow PFA 100, Elemental Scientific, Omaha, NE) with a low-volume spray chamber (PFA Spray Chamber for Microflow, Elemental Scientific) was used as a µLC-ICPMS interface. The outlet capillary of the LC was coupled directly to the nebulizer. ESI-Mass Spectrometry. Electrospray mass spectrometry was performed on a Q-Tof 2 instrument (Micromass, Manchester, U.K.), which was equipped with the nanoflow ESI source. For the static glass capillary option (nanoESI), the spray voltage was ∼+1 kV. Spray capillaries were prepared in-house using a micropipet puller type P-87 (Sutter Instruments, Novato, CA) and coated with a semitransparent film of gold in a sputter unit. Argon was used as collision gas for the tandem MS analyses. Samples were desalted using a variant of the standard ZipTip procedure (Waters, Milford, MA), which avoids acidic incubations before the elution. Pure water was used for desalting and washing. For elution, acetonitrile/water/formic acid 0.5:0.5:0.01 was chosen. For µLC-ESI-MS, a spray voltage of +3.1 kV was applied. Capillary Liquid Chromatography. µLC System. For capillary LC, a dual-syringe solvent delivery system (type 140B, Applied
Biosystems, Foster City, CA) was used. Samples were injected using a 5-µL stainless steel sample loop. Chromatographic separations were performed on a Vydac C18 column (0.3 mm × 250 mm, 5 µm, 300 Å, Grom, Herrenberg, Germany). The standard gradient used was 0-5 min 5% B isocratic; 5-50 min 5-100% B linear. The mobile phase A was water/trifluoroacetic acid 100:0.065 (v/ v) and B was acetonitrile/water/trifluoroacetic acid 80:20:0.05 (v/ v/v). The total solvent flow was 140 µL min-1 using a split ratio of ∼1:35 to achieve a flow of ∼4 µL min-1 over the column. RESULTS AND DISCUSSION µLC-ICPMS and µLC-ESI-MS of a Tryptic Digest of CheA-H. Two domains of the bacterial chemotaxis protein CheA were recombinantly expressed, namely, the kinase domain CheA-C (257-513, average mol wt 29 300) with histidine kinase activity and the domain CheA-H (3-137, average mol wt 16 400) containing the established phosphohistidine site at His-48. Both proteins were mixed at a molar ratio of CheA-C to CheA-H of ∼1:10 in an in vitro phosphorylation assay (for details, see Experimental Section). After ∼1 h, the mixture was subjected to digestion by trypsin, and in order to spot phosphopeptides, the digest was analyzed by reversed-phase µLC-ICPMS and phosphorus detection.16,17 A list of the expected tryptic peptides of CheA-H (see Figure 5) is summarized in Table 1. (16) Wind, M.; Edler, M.; Jakubowski, N.; Linscheid, M.; Wesch, H.; Lehmann, W. D. Anal. Chem. 2001, 73, 29-35. (17) Wind, M.; Kelm, O.; Nigg, E. A.; Lehmann, W. D. Proteomics 2002, 2, 15161523.
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Figure 3. Analysis of CheA-H by nanoESI MS after in vitro phosphorylation by CheA-H and ATP, (A) Original mass spectrum of CheA-H, the asterisk indicates the molecular ion group with charge state 16+, which was selected for collision-induced dissociation (see Figure 4); (B) deconvoluted mass spectrum of CheA-H showing ∼40% of p-CheA-H; C, stability of p-CheA-H in 50% ACN/1% formic acid.
In addition to the 31P isotope, 32S was also monitored in the same run to recognize sulfur-containing peptides15 and to eventually determine the degree of phosphorylation for phosphopeptides/proteins containing Met or Cys.18 To characterize the digest components also by their mass, an aliquot of the sample was analyzed by µLC-ESI-MS using the same chromatographic system. The results are summarized in Figure 1. Figure 1A shows the total ion current of the µLC-ESI-MS analysis indicating the elution of numerous peptides. Figure 1B represents the 31P trace of the µLC-ICPMS run showing only two phosphorus peaks eluting near the end of the LC separation. The corresponding ESI-MS data identified the first phosphorus containing peak at ∼41 min retention time as a low molecular weight non-peptide organic compound. The second peak in the phosphorus trace corresponds to a larger, protein-like compound. The original ESI spectrum is shown in Figure 2A and in its deconvoluted form in Figure 2B. This protein exhibits two signals, (18) Wind, M.; Wesch, H.; Lehmann, W. D. Anal. Chem. 2001, 73, 3006-3010.
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differing by ∼80 Da, which were assigned as the nonphosphorylated and singly phosphorylated forms of CheA-H, which has lost the two most C-terminal tryptic peptides (for the sequence; see Figure 5). These C-terminal peptides are not part of the natural sequence of CheA but are due to the in vitro technology used for expression and purification of the domains. No other large proteins or peptides could be detected. The degree of phosphorylation of this truncated pHis-CheA-H protein as derived from the ESI spectrum was ∼75%. This high degree of phosphorylation was confirmed by the P/S ratio measured in µLC-ICP analysis (see Figure 1C) as described recently.18 In the µLC-ESI-MS run, the tryptic peptides representing almost the entire protein sequence could be found except for the His-tag containing tryptic peptide (T11). Peptide T5 represents a single amino acid, and this residue was found in the tryptic peptide T4-5. Incomplete tryptic cleavage is often observed at two vicinal basic residues. Surprisingly, no phosphorylated peptides were observed, although the presence of pHis in CheA-H
Figure 4. NanoESI-MS/MS of the [M + 16H]16+ ion of p-CheA-H. (A) “Complete” tandem mass spectrum; (B) region below the selected precursor ion; (C) region above the selected precursor ion. The most intense b and y ions are annotated, which allows one to spot the phosphorylation site to the region between residues 32 and 86 (see Figure 5 and discussion in the text).
after in vitro phosphorylation could be detected by different methods (see below). NanoESI-MS Analysis. This unexpected finding that no phosphorylated peptides were observed in the digest of phosphorylated CheA initiated analysis of (pHis)-CheA-H without digestion. The product of the in vitro phosphorylation assay was desalted and measured by nanoESI-MS. All sample preparation was done with respect to stability of phosphohistidine (see Experimental Section). The MS spectrum is shown in Figure 3A. Figure 3 shows a typical result of the in vitro kinase assay, with a phosphorylation degree of CheA-H of ∼40%. Even if prolonged incubation times were applied, the phosphorylation degree could not be increased above this value. The stability of (pHis)-CheA-H was tested by incubation in 50% aqueous acetonitrile and 1% FA. Aliquots were taken from this solution after different incubation times and the degree of phosphorylation determined by nanoESI-MS. The results are shown in Figure 3C. After 1 h, the degree of phosphorylation decreased from an initial value of ∼40% to ∼10%. This instability in dilute acid is charac-
teristic for a pHis residue. For a phosphorylation at Ser, Thr, or Tyr, a dephosphorylation would not be expected under these conditions. NanoESI-Tandem MS. Since the phosphorylated residue could not be pinpointed by analysis of the tryptic digest of pHisCheA-H, nanoESI tandem MS of the intact phosphoprotein was performed. Figure 4 shows the MS/MS spectrum of the [M + 16H]16+ ion (at m/z 1033.4) of the pHis-CheA-H domain with a molecular weight of 16 522 (see Figure 3B). A detailed assignment of the fragment ions observed is presented in Table 2. Fragment ions are annotated and confirm the phosphorylation in an area between the residues 30 and 84, in which the established phosphorylation site His-48 is located. The location of the fragment ions with respect to the CheA-H sequence are schematically annotated to illustrate the significance of the b and y ions observed (see Figure 5). The fragmentation behavior of the highly charged molecular ion of CheA-H corresponds well to the expectation. Abundant Analytical Chemistry, Vol. 77, No. 7, April 1, 2005
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Table 2. Observed and Calculated m/z Values of the MS/MS Sequence Ions Detected by Collision-Induced Dissociation of the [M + 16H]16+ Ion of Phosphorylated CheA-H, As Displayed in Figure 4a m/z ion
z
observed
calculated
y37 y31 y32 y46 y33 y34 y48 y35 y36 y50 y37 y45 y46 y47 y55 y48 y56 y49 y57 y59 y60 y116 b27 y116 b28 b29 b84 b85 b86 b87 b108 b88 b30-108 b95 b85 b86 b87
6+ 5+ 5+ 7+ 5+ 5+ 7+ 5+ 5+ 7+ 5+ 6+ 6+ 6+ 7+ 6+ 7+ 6+ 7+ 7+ 7+ 13+ 3+ 12+ 3+ 8+ 8+ 8+ 8+ 8+ 7+ 8+ 7+ 7+ 7+ 7+ 7+
687.6 707.3 736.7 741.4 754.1 768.3 778.2 782.5 805.5 815.0 825.0 846.0 864.8 886.2 899.1 907.7 915.2 929.0 936.1 968.7 985.0 1007.9 1064.4 1091.7 1102.1 1144.8 1190.9 1205.3 1219.4 1233.5 1241.1 1247.7 1282.3 1393.4 1409.7 1425.9 1446.8
687.77 707.40 736.83 741.54 754.25 768.46 778.29 782.68 805.70 815.33 825.12 846.10 864.96 886.31 899.14 907.83 915.30 929.19 936.33 968.80 984.96 1007.97 1064.57 1091.88 1102.25 1144.96 1191.20 1205.34 1219.49 1233.75 1241.19 1247.90 1282.43 1393.56 1409.86 1426.02 1447.05
a The numbering of the recombinant sequence (Figure 5) starts with amino acid residue 3 of CheA-H, and this residue is considered as residue 1 in the numbering of the b and y sequence ions. This results in a difference of two counts between the corresponding numbering schemes. For extraction of the average mass values, the MS/MS spectra were smoothed by a smooth window channel of 30. A shift between calculated and observed m/z values of 100-150 mDa is observed, which is compatible with externally calibrated Q-Tof 2 data.
fragmentation is observed on the N-terminal side of proline and on the C-terminal side of aspartate. Lipophilic regions exhibit continuous series of fragment ions, whereas basic residues interrupt these series and suppress the fragmentation in their neighborhood. Phosphorylation on histidine in CheA-H was definitively detected. Unfortunately, the tandem MS spectrum of the intact protein could not distinguish between the residues His-48 and His67. The protein is in vitro phosphorylated to a degree of ∼40% (see Figure 3B). After the digest, the nonphosphorylated protein is totally cleaved to its tryptic peptides. From the phosphorylated protein, only a small C-terminal part is cleaved off (T10 and T11
1962 Analytical Chemistry, Vol. 77, No. 7, April 1, 2005
Figure 5. Sequence of CheA-H (145 amino acids, average mol wt 16442.4) and annotation of the MS/MS fragment ions as displayed in Figure 4. The lower diagram explains the location of the internal b ions within the sequence of CheA-H. The numbering of the recombinant sequence starts with amino acid residue 3 of CheA-H, and this residue is considered as residue 1 in the numbering of the b and y sequence ions. This results in a difference of two counts between the two numbering schemes.
peptide of the construct). After µLC, the degree of phosphorylation of the residual truncated protein is ∼75% (see Figure 2B). In view of the acid instability of histidine phosphorylation and the acidic separation conditions in µLC separation, we conclude that the degree of phosphorylation of the nondigested intact protein is even higher than 75%. Thus, our observations are compatible with the simplified conclusion that the nonphosphorylated form of CheA-H is degraded completely under the digest conditions applied, whereas pHis-CheA-H is not digested. Therefore, we further conclude that phosphorylation at His-48 changes most of CheA-H to a structure that cannot be digested by trypsin, except for a small C-terminal part. CONCLUSIONS The possibilities to evaluate phosphorylation at His with slightly modified standard procedures of mass spectrometric protein analysis is demonstrated. The application of electrospray mass spectrometry to an intact small pHis-phosphoprotein and the additional use of element mass spectrometry and phosphorus detection have proven to be highly advantageous for the reliable detection of the degree of phosphorylation at histidine. ACKNOWLEDGMENT We are indebted to Claudia Kubis and Annette Bu¨ttner for excellent technical assistance.
Received for review July 19, 2004. Accepted December 9, 2004. AC040140H
