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Mass Spectrometric Resolution of Reversible Protein Phosphorylation in Photosynthetic Membranes ofArabidopsis thaliana

2001; Elsevier BV; Volume: 276; Issue: 10 Linguagem: Inglês

10.1074/jbc.m009394200

1083-351X

Alexander V. Vener, Amy C. Harms, Michael R. Sussman, Richard D. Vierstra,

Algal biology and biofuel production

The use of mass spectrometry to characterize the phosphorylome, i.e. the constituents of the proteome that become phosphorylated, was demonstrated using the reversible phosphorylation of chloroplast thylakoid proteins as an example. From the analysis of tryptic peptides released from the surface ofArabidopsis thylakoids, the principal phosphoproteins were identified by matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry. These studies revealed that the D1, D2, and CP43 proteins of the photosystem II core are phosphorylated at their N-terminal threonines (Thr), the peripheral PsbH protein is phosphorylated at Thr-2, and the mature light-harvesting polypeptides LCHII are phosphorylated at Thr-3. In addition, a doubly phosphorylated form of PsbH modified at both Thr-2 and Thr-4 was detected. By comparing the levels of phospho- and nonphosphopeptides, the in vivo phosphorylation states of these proteins were analyzed under different physiological conditions. None of these thylakoid proteins were completely phosphorylated in the steady state conditions of continuous light or completely dephosphorylated after a long dark adaptation. However, rapid reversible hyperphosphorylation of PsbH at Thr-4 in response to growth in light/dark transitions and a pronounced specific dephosphorylation of the D1, D2, and CP43 proteins during heat shock was detected. Collectively, our data indicate that changes in the phosphorylation of photosynthetic proteins are more rapid during heat stress than during normal light/dark transitions. These mass spectrometry methods offer a new approach to assess the stoichiometry of in vivo protein phosphorylation in complex samples. The use of mass spectrometry to characterize the phosphorylome, i.e. the constituents of the proteome that become phosphorylated, was demonstrated using the reversible phosphorylation of chloroplast thylakoid proteins as an example. From the analysis of tryptic peptides released from the surface ofArabidopsis thylakoids, the principal phosphoproteins were identified by matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry. These studies revealed that the D1, D2, and CP43 proteins of the photosystem II core are phosphorylated at their N-terminal threonines (Thr), the peripheral PsbH protein is phosphorylated at Thr-2, and the mature light-harvesting polypeptides LCHII are phosphorylated at Thr-3. In addition, a doubly phosphorylated form of PsbH modified at both Thr-2 and Thr-4 was detected. By comparing the levels of phospho- and nonphosphopeptides, the in vivo phosphorylation states of these proteins were analyzed under different physiological conditions. None of these thylakoid proteins were completely phosphorylated in the steady state conditions of continuous light or completely dephosphorylated after a long dark adaptation. However, rapid reversible hyperphosphorylation of PsbH at Thr-4 in response to growth in light/dark transitions and a pronounced specific dephosphorylation of the D1, D2, and CP43 proteins during heat shock was detected. Collectively, our data indicate that changes in the phosphorylation of photosynthetic proteins are more rapid during heat stress than during normal light/dark transitions. These mass spectrometry methods offer a new approach to assess the stoichiometry of in vivo protein phosphorylation in complex samples. mass spectrometry collision-induced dissociation electrospray ionization immobilized metal affinity chromatography liquid chromatography light-harvesting chlorophyll a/b complex II matrix-assisted laser desorption/ionization mass over charge ratio post-source decay photosystem time-of-flight N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine The reversible phosphorylation of specific proteins participates in the regulation of virtually all aspects of cell physiology and development. The extent of its importance is illustrated by the hundreds of conventional protein kinases and phosphatases detected in various eukaryotic genomes (1Hunter T. Plowman G.D. Trends Biochem. Sci. 1997; 22: 18-22Abstract Full Text PDF PubMed Scopus (403) Google Scholar, 2Plowman G.D. Sudarsanam S. Bingham J. Whyte D. Hunter T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13603-13610Crossref PubMed Scopus (230) Google Scholar, 3Adams M.D. et al.Science. 2000; 287: 2185-2195Crossref PubMed Scopus (4807) Google Scholar). Whereas, serine, threonine, and tyrosine residues are the typical targets of these kinases, phosphorylation of at least six other amino acids is feasible, potentially expanding even further the dimensions of this post-translational modification (reviewed in Ref. 4Vener A.V. Biosystems. 1990; 24: 53-59Crossref PubMed Scopus (4) Google Scholar). Despite the importance of this pool of phosphorylated proteins, our understanding of its depth and breadth remains sketchy. One barrier has been the lack of methods to define en masse the "phosphorylome,"i.e. the subset of proteins in the proteome that become modified in vivo by phosphorylation. Precise characterization of the phosphorylome will be essential to fully understand how proteins are activated or inhibited, encouraged to interact with other components in the cell, and selected for rapid degradation. Certainly, the dynamic and transient nature of many protein phosphorylation reactions underscores the difficulties of resolving the complete phosphorylome for a given organism. Nevertheless, the identification of even just the principal cellular phosphoproteins under distinct physiological conditions should bring significant biological insights. The most common method for analysis of the phosphorylome involves the use of radioactive labeling either in vivo or in vitro. However, uneven uptake of the label in complex multicellular organisms, the large pools of endogenous free phosphate, and the presence of pre-existing bound phosphate often limit conclusions. Phosphoamino acid antibodies have been exploited recently but their use is restricted to tailor-made immunological applications and because these antibodies cannot detect the nonphosphorylated form they are unable to determine stoichiometry. More recently, mass spectrometry (MS)1 has been applied to analyses of protein phosphorylation (5Carr S.A. Huddleston M.J. Annan R.S. Anal. Biochem. 1996; 239: 180-192Crossref PubMed Scopus (332) Google Scholar, 6Annan R.S. Carr S.A. Anal. Chem. 1996; 68: 3413-3421Crossref PubMed Scopus (302) Google Scholar, 7Annan R.S. Carr S.A. J. Protein Chem. 1997; 16: 391-402Crossref PubMed Scopus (100) Google Scholar, 8Resing K.A. Ahn N.G. Methods Enzymol. 1997; 283: 29-44Crossref PubMed Scopus (56) Google Scholar, 9Neubauer G. Mann M. Anal. Chem. 1999; 71: 235-242Crossref PubMed Scopus (230) Google Scholar, 10Oda Y. Huang K. Cross F.R. Cowburn D. Chait B.T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6591-6596Crossref PubMed Scopus (940) Google Scholar). This highly sensitive technique offers the advantage of scanning complex mixtures for phosphoproteins that became modified in vivo. Moreover with appropriate considerations, we show here that MS can also be used to provide estimates of the phosphorylation state of specific proteins. To demonstrate the utility of MS, we have applied this technique to the analysis of the major phosphoproteins in the chloroplast thylakoid, the membrane containing the photosynthetic light reactions of photosystem (PS) I and II, light-harvesting chlorophyll a/b proteins (LHCII), cytochrome b·f complex, and the ATP synthase (11Bennett J. Steinback K.E. Arntzen C.J. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 5253-5257Crossref PubMed Scopus (257) Google Scholar, 12Allen J.F. Biochim. Biophys. Acta. 1992; 1098: 275-335Crossref PubMed Scopus (726) Google Scholar). Multiprotein complexes within the thylakoids are responsible for light-driven oxidation of water with concomitant release of oxygen and the production of energy and reducing potentials. During these reactions, reversible phosphorylation is thought to play critical roles in (i) the redistribution of excitation energy between PSI and II via modification of LHCII (11Bennett J. Steinback K.E. Arntzen C.J. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 5253-5257Crossref PubMed Scopus (257) Google Scholar, 12Allen J.F. Biochim. Biophys. Acta. 1992; 1098: 275-335Crossref PubMed Scopus (726) Google Scholar, 13Vener A.V. Ohad I. Andersson B. Curr. Opin. Plant Biol. 1998; 1: 217-223Crossref PubMed Scopus (123) Google Scholar), and (ii) the maintenance of the PSII by controlling the turnover of its reaction center polypeptides (14Rintamaki E. Kettunen R. Aro E.M. J. Biol. Chem. 1996; 271: 14870-14875Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 15Andersson B. Aro E.M. Physiol. Plant. 1997; 100: 780-793Crossref Google Scholar, 16Barber J. Nield J. Morris E.P. Zheleva D. Hankamer B. Physiol. Plant. 1997; 100: 817-827Crossref Google Scholar, 17Kruse O. Zheleva D. Barber J. FEBS Lett. 1997; 408: 276-280Crossref PubMed Scopus (64) Google Scholar, 18Baena-Gonzalez E. Barbato R. Aro E.-M. Planta ( Basel ). 1999; 208: 196-204Crossref Scopus (99) Google Scholar). Studies in spinach and pea using 32P labeling and phosphoamino acid antibodies showed that a number of proteins are phosphorylated, including threonine residues at or near the N termini of LHCII (19Michel H. Griffin P.R. Shabanowitz J. Hunt D.F. Bennett J. J. Biol. Chem. 1991; 266: 17584-17591Abstract Full Text PDF PubMed Google Scholar) and PSII polypeptides, the D1 and D2 reaction center proteins, chlorophyll-binding protein CP43 (20Michel H. Hunt D.F. Shabanowitz J. Bennett J. J. Biol. Chem. 1988; 263: 1123-1130Abstract Full Text PDF PubMed Google Scholar), and peripheral polypeptide PsbH (21Michel H. Bennett J. FEBS Lett. 1987; 212: 103-108Crossref Scopus (79) Google Scholar). However, given the limitations of 32P labeling and immunoassays, it remains unclear how important chloroplast protein phosphorylation is to the normal function and regulation of the photosynthetic light reactions. In vitro studies using32P labeling have suggested that a number of thylakoid proteins are extensively phosphorylated in the light (22Bennett J. Nature. 1977; 269: 344-346Crossref Scopus (288) Google Scholar) by a kinase controlled by the photosynthetic electron transport chain (12Allen J.F. Biochim. Biophys. Acta. 1992; 1098: 275-335Crossref PubMed Scopus (726) Google Scholar, 23Vener A.V. Van, K.an P.J. Gal A. Andersson B. Ohad I. J. Biol. Chem. 1995; 270: 25225-25232Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 24Vener A.V. Van, K.an P.J.M. Rich P.R. Ohad I. Andersson B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1585-1590Crossref PubMed Scopus (249) Google Scholar), and dephosphorylated in the dark by phosphatase(s) that are not light sensitive (12Allen J.F. Biochim. Biophys. Acta. 1992; 1098: 275-335Crossref PubMed Scopus (726) Google Scholar, 25Silverstein T. Cheng L. Allen J.F. FEBS Lett. 1993; 334: 101-105Crossref PubMed Scopus (55) Google Scholar). More recent studies using phosphothreonine antibodies questioned the magnitude of this phosphorylation by showing that some thylakoid phosphoproteins remain phosphorylated even in dark-adapted plants (26Rintamaki E. Salonen M. Suoranta U.M. Carlberg I. Andersson B. Aro E.M. J. Biol. Chem. 1997; 272: 30476-30482Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 27Rokka A. Aro E.M. Herrmann R.G. Andersson B. Vener A.V. Plant Physiol. 2000; 123: 1525-1536Crossref PubMed Scopus (117) Google Scholar, 28Ebbert, V., Demmig-Adams, B., Adams, W. W., Mueh, K. E., and Staehelin, L. A. (2000) Photosynth. Res., in press.Google Scholar). Furthermore, the maximal phosphorylation of LHCII only occurs at low light and is drastically decreased at higher irradiations (26Rintamaki E. Salonen M. Suoranta U.M. Carlberg I. Andersson B. Aro E.M. J. Biol. Chem. 1997; 272: 30476-30482Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). Phosphorylation of spinach LHCII was also found to increase in darkness upon exposure of leaves to heat shock (27Rokka A. Aro E.M. Herrmann R.G. Andersson B. Vener A.V. Plant Physiol. 2000; 123: 1525-1536Crossref PubMed Scopus (117) Google Scholar). Here, we report the identification of the major phosphoproteins in the thylakoid membranes from Arabidopsis thaliana and map their phosphorylation sites using both matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and electrospray ionization (ESI) MS. Measurements of the phosphorylation level of each protein revealed that changes in the steady state phosphorylation during normal light/dark growth cycles may be less extensive and slower than previously thought and may be more significant and rapid during a response to stress (e.g. heat shock). The MS techniques described here should be applicable for many analyses involving complex mixtures of phosphoproteins. A. thaliana ecotype Columbia-0 was grown at 21 °C either in soil or on 0.7% (w/v) agar containing one-half strength MS media (Life Technologies, Inc.). Plants were irradiated with 100 μmol m−2 s−1 of white light provided by fluorescent lights with a photoperiod of 16-h light/8-h dark. Chloroplasts and thylakoids were prepared from 3-week-old plants. Chloroplasts were extracted from the leaves and purified by Percol gradient centrifugation according to Ref. 29Rensink W.A. Pilon M. Weisbeek P. Plant Physiol. 1998; 118: 691-699Crossref PubMed Scopus (74) Google Scholar. To isolate thylakoids, the chloroplasts were resuspended in 7 ml of 10 mm sodium phosphate (pH 7.5), 5 mm MgCl2, 5 mm NaCl, and homogenized 10 times in a Potter grinder. The homogenate was diluted to 30 ml with the same buffer and thylakoids were collected by centrifugation for 5 min at 4000 ×g. For direct preparation of thylakoids, 5 g of leaves were homogenized with a Polytron (Brinkmann PT 10/35) in 25 ml of ice-cold extraction buffer, containing 300 mm sorbitol, 50 mm sodium phosphate (pH 7.5), 5 mmMgCl2, 10 mm NaF. The suspension was filtrated through four layers of Miracloth and centrifuged for 3 min at 1500 × g. The pellet was resuspended in 7 ml of lysis buffer (10 mm sodium phosphate (pH 7.5), 5 mmMgCl2, 1 0 mm NaF) and homogenized 10 times in a Potter grinder. The suspension was diluted to 30 ml with the lysis buffer and centrifuged for 5 min at 4000 × g. The pellet was resuspended in 3 ml of the extraction buffer with the Potter grinder and layered on the top of a sucrose step gradient, containing (bottom to top): 10 ml of 1.8 m, 10 ml of 1.3m, and 10 ml of 0.5 m sucrose in 50 mm sodium phosphate (pH 7.5) and 10 mm NaF. After centrifugation in a swinging bucket rotor for 15 min at 5000 × g, the thylakoid fraction was collected from the 1.3m, 1.8 m sucrose interface. Thylakoids were diluted to 25 ml with extraction buffer and collected by a 5-min centrifugation at 4000 × g. The thylakoid pellet was resuspended in 1 ml of 25 mmNH4HCO3 (pH 8.0), 10 mm NaF, and pelleted again using a microcentrifuge. Chloroplasts (0.2 mg of chlorophyll) were gently resuspended in 1 ml of 20 mmTricine (pH 8.0), 330 mm sorbitol, 6.6 mmMgCl2, 1 mm Na2HPO4 and incubated at 22 °C. Phosphorylation was induced by a 10–20 min irradiation with 100 μmol m−2 s−1 of white light and terminated by addition of 10 ml of ice-cold 20 mmTricine (pH 8.0), 5 mm Na4EDTA, 10 mm NaF. Thylakoids were prepared from chloroplasts as described above. When isolated thylakoids were used, they were resuspended in 20 mm Tricine (pH 8.0), 100 mmsorbitol, 5 mm MgCl2, 1 mm ATP and irradiated as described above. Isolated thylakoids were washed twice with 25 mm NH4HCO3 (pH 8.0), 10 mm NaF by centrifugation and resuspension in the same buffer to a concentration of 1.3–1.5 mg of chlorophyll/ml. The suspension was incubated with sequencing-grade modified trypsin (Promega) (8 μg of trypsin/mg of chlorophyll) at 22 °C for 90 min. The digestion products were frozen, thawed, and clarified at 14,000 ofg. The supernatant containing released thylakoid peptides was collected. Similar peptides were collected from spinach thylakoids as described (30Vener A.V. Rokka A. Fulgosi H. Andersson B. Herrmann R.G. Biochemistry. 1999; 38: 14955-14965Crossref PubMed Scopus (87) Google Scholar). Phosphopeptides were affinity enriched from the thylakoid peptide fraction by chromatography with immobilized Fe(III) or Ga(III) columns (31Andersson L. Porath J. Anal. Biochem. 1986; 154: 250-254Crossref PubMed Scopus (644) Google Scholar, 32Posewitz M.C. Tempst P. Anal. Chem. 1999; 71: 2883-2892Crossref PubMed Scopus (786) Google Scholar). Typically, columns containing 50 μl of chelating Sepharose Fast Flow (Amersham Pharmacia Biotech) beads were washed with 0.3 ml of water, 0.3 ml of 0.1% (v/v) acetic acid, charged with 0.3 ml of 0.1m FeCl3 or GaCl3, and washed with 0.5 ml of 0.1% (v/v) acetic acid. Thylakoid peptides (0.2–0.3 ml) were mixed with an equal volume of 20% acetic acid and loaded onto the columns. After washing twice with 0.2 ml of 0.1% (v/v) acetic acid, bound phosphopeptides were eluted with 300 μl of either 20 mm sodium phosphate buffer (pH 7.0) (for Fe(III) (31Andersson L. Porath J. Anal. Biochem. 1986; 154: 250-254Crossref PubMed Scopus (644) Google Scholar)), or 20 mm of nonbuffered Na2HPO4 (for Ga(III) (32Posewitz M.C. Tempst P. Anal. Chem. 1999; 71: 2883-2892Crossref PubMed Scopus (786) Google Scholar)). Synthetic phosphopeptides included: APRTpPGGRR; CDGVTTKTpTAGTPD, CDGVTTKTpFAGTPD, LIPQQSpINEAIK, DRHDSGLDSpNKDE, DRHDSpGLDSpNKDE, CDRHDSpGLDSpNKDE, and GRPRTTSpFAE (where Tp and Sp indicate phosphothreonine and phosphoserine, respectively). To obtain the corresponding dephosphopeptides, 0.25 nmol of each phosphopeptide were dissolved in the phosphatase buffer (25 mmNH4HCO3 (pH 8.0), 10 mmMgCl2, 2 mm dithiothreitol) and incubated for 2 to 24 h at 37 °C with the addition of 1 to 5 units of alkaline phosphatase (New England Biolabs, Beverly, MA). The extent of dephosphorylation was monitored by MALDI-TOF MS. Samples were prepared by mixing 1–2 μl of each mixture with 1–2 μl of α-cyano-4-hydroxycinnamic acid dissolved in 70% (v/v) acetonitrile with 2% (v/v) trifluoroacetic acid. One μl of final mixture was spotted on the target. Linear and reflector mass spectra were recorded using Biflex III MALDI-TOF mass spectrometer (Bruker, Billerica, MA) operated in delayed extraction mode using an accelerating voltage of 19 kV. Spectra were calibrated externally. Post-source decay (PSD) spectra were recorded using Bruker's FAST procedure. Peptide mixtures were separated on 5 μm of C18 MetaChem 150 × 1.0-mm column at a flow rate 20 μl/min. A gradient of 0.1% (v/v) formic acid in water (A) and 0.1% (v/v) formic acid in acetonitrile (B) was distributed as follow: 0% B in first 3 min; 0–20% B in 3 to 20 min; 20–70% B in 20 to 105 min; 70–99% B in 105 to 115 min. The online detection with positive/negative-ion mode switching was performed using an API 365 triple quadrupole MS with a standard ionspray source (Applied Biosystems/MDS Sciex, Foster City, CA). Each 5.2-s positive-ion scan in the m/z range from 320 to 1800 was followed by a 0.7-s pause for polarity switching and 1.5-s single ion-monitoring of negative 79 and another 0.7-s pause to return to positive-ion mode. In the positive-ion mode, ion source, orifice, and ring voltages were set at 5 kV, 12 V, and 140 V, respectively, to minimize fragmentation. However, in some cases a small amount of nonphosphorylated ion species was generated from the phosphopeptide by partial skimmer-induced fragmentation (see Fig. 3 C, for example). In the negative-ion mode, the ion source, orifice and ring voltages were set at −4.5 kV, −200 V, and −300 V, respectively, to maximize the phosphoryl-79 signal. For skimmer collision-induced dissociation (CID), the ion source, orifice, and ring voltages were set at 5 kV, 95 V, and 200 V, respectively, to maximize peptide fragmentation. Sequencing of high performance liquid chromatography-purified peptides was performed by tandem MS/MS using conditions recommended by Applied Biosystems/MDS Sciex (Foster City, CA). Previous studies with spinach and pea chloroplast thylakoids showed that the primary sites for phosphorylation involve polypeptide regions exposed to the outer surface of the membranes (30Vener A.V. Rokka A. Fulgosi H. Andersson B. Herrmann R.G. Biochemistry. 1999; 38: 14955-14965Crossref PubMed Scopus (87) Google Scholar, 33Bennett J. Eur. J. Biochem. 1980; 104: 85-89Crossref PubMed Scopus (156) Google Scholar). To enrich for these regions, thylakoid membranes were purified from chloroplasts isolated from Arabidopsis leaves and then "shaved" with trypsin to release surface-exposed peptides from the various constituent proteins (Fig.1 A). Although a variety of proteins were digested, several proteins and/or protein domains that we presume were protected by the membrane remained intact. LC-ESI MS analyses of the released fraction revealed approximately a thousand major peptides liberated by this protease treatment. By using the characteristic decomposition products of phosphopeptides following the breakdown of phosphoryl-peptide linkages as a signature (5Carr S.A. Huddleston M.J. Annan R.S. Anal. Biochem. 1996; 239: 180-192Crossref PubMed Scopus (332) Google Scholar, 6Annan R.S. Carr S.A. Anal. Chem. 1996; 68: 3413-3421Crossref PubMed Scopus (302) Google Scholar, 8Resing K.A. Ahn N.G. Methods Enzymol. 1997; 283: 29-44Crossref PubMed Scopus (56) Google Scholar), numerous phosphopeptides were identified. We enriched for phosphopeptides by immobilized metal [Fe(III) and/or Ga(III)] affinity chromatography (IMAC) (31Andersson L. Porath J. Anal. Biochem. 1986; 154: 250-254Crossref PubMed Scopus (644) Google Scholar, 32Posewitz M.C. Tempst P. Anal. Chem. 1999; 71: 2883-2892Crossref PubMed Scopus (786) Google Scholar). Because the binding specificity and elution properties of Fe(III) and Ga(III) IMAC differ, both were used to isolate a range of phosphopeptides (see Table I). Although nonphosphorylated peptides were also present in the eluted fractions, the partial purification of phosphopeptides by IMAC greatly simplified their identification and initial analysis.Table IPhosphorylation sites of the major phosphopeptides from Arabidopsis thylakoidsPeptide sequencePeptide massEnriched by IMAC withParent proteinFe(III)Ga(III)amuAc-TpAILER823.4++D1Ac-TpIALGK723.4+−D2Ac-TpLFNGTLALAGR1354.6++CP43Ac-TpLFDGTLALAGR1355.6++CP431-aCPH43 represents the N-terminal peptide of CP43 protein with deamidated Asn-4.TpVAKPK722.4+−LHCIIAc-RKTpVAKPK1048.8+−LHCII1-aCPH43 represents the N-terminal peptide of CP43 protein with deamidated Asn-4.ATpQTVEDSSR1172.8+−PsbHATpQTpVEDSSR1252.6+−PsbH1-bPsbH represents the doubly phosphorylated N-terminal peptide of PsbH protein bearing a phosphate at both Thr-2 and Thr-4.The + and − indicate the type of IMAC that allowed enrichment of the phosphopeptide as determined by subsequent MS sequencing.1-a CPH43 represents the N-terminal peptide of CP43 protein with deamidated Asn-4.1-b PsbH represents the doubly phosphorylated N-terminal peptide of PsbH protein bearing a phosphate at both Thr-2 and Thr-4. Open table in a new tab The + and − indicate the type of IMAC that allowed enrichment of the phosphopeptide as determined by subsequent MS sequencing. During MALDI-TOF-MS, phosphopeptides lose phosphoric acid as H3PO4 (98 Da) and HPO3 (80 Da) with the concomitant production of metastable ions (6Annan R.S. Carr S.A. Anal. Chem. 1996; 68: 3413-3421Crossref PubMed Scopus (302) Google Scholar). Whereas both the metastable and parent phosphopeptide ions arrive at the detector simultaneously and thus produce one coherent signal in the linear mode, they generate separate signals in the reflector mode, with the metastable ions arriving sooner than the parent ion. The appearance of this metastable ion is especially evident for peptides containing phosphoserine and phosphothreonine, which generate intense daughter signals upon losing H3PO4 (98 Da) (6Annan R.S. Carr S.A. Anal. Chem. 1996; 68: 3413-3421Crossref PubMed Scopus (302) Google Scholar). In our experimental settings, these metastable ions actually appeared as ions 86 m/z rather than 98 m/zsmaller than the parent ions (Fig. 1 B). This difference was a result of the metastable ion flying out of focus from the ion mirror, a phenomenon that also led to a broad ion signal that is characteristic of an ion lacking isotope resolution (Fig. 1 B). From the analysis of a series of synthetic phosphopeptides (see "Experimental Procedures"), we found that all generated this metastable ion peak in the reflector mode regardless of the amino acid sequence or position of the phosphoserine or phosphothreonine residue. 2A. V. Vener, A. Harms, and R. D. Vierstra, unpublished data. In fact, doubly phosphorylated peptides produced two metastable ions in the reflector mode, 86 m/z and 172 m/zsmaller than the parent ion. We could reliably detect these metastable ions with as little as femtomole amounts of these synthetic phosphopeptides, indicating that they could be detected with high sensitivity. Thus, we exploited the presence of metastable signal in the reflector mode at −86 m/z and its unique shape as a reliable indicator for phosphopeptides. Using this metastable ion signature, numerous phosphopeptides were detected by MALDI-TOF MS in the IMAC-enriched fractions of peptides released from thylakoids of light-adapted plants. As an example, Fig. 1shows the MALDI-TOF MS detection of a single ion cluster at 1355.6m/z in the linear mode (Fig. 1 C) that behaved as two ion clusters in the reflector mode, one for the parent ion at 1355.6 m/z and another for the metastable ion at 1269.6 m/z, which was 86m/z smaller and devoid of isotope resolution (Fig. 1 B). By this approach we detected eight phosphopeptides abundant in the IMAC-enriched fractions (Table I). In several cases, we also detected these phosphopeptides in the crude trypsin hydrolysates before IMAC enrichment. As shown in Fig. 1,D and E, the metastable 1269.6m/z ion for the 1355.6 parent ion was readily detected in the reflector mode but absent in the linear mode. The thylakoid phosphopeptides identified by MALDI-TOF MS were sequenced using MALDI-TOF PSD, ESI MS/MS, and LC with online ESI-skimmer CID MS. MALDI-PSD MS of the 1355.6 m/z phosphopeptide identified its sequence as Ac-TLFNGTLALAGR (Fig.2 A). A search of theArabidopsis protein sequence data base revealed that this sequence belonged to the chloroplast-encoded CP43 subunit of PSII, assuming that the first 14 amino acids of the initial translation product were removed and the resulting N-terminal threonine residue was acetylated. Because the phosphate moiety is readily lost from phosphopeptides during MALDI-TOF-MS, we used complementary ESI-CID MS sequencing to unambiguously identify the phosphorylation site(s). The most efficient method was the ESI-skimmer CID MS when used online with LC separation of IMAC-enriched peptides. Fig. 2 B shows the mass spectrum containing mostly y (C-terminal) andb (N-terminal) ion fragments of the N-terminal phosphopeptide from CP43. The fragmentation pattern was consistent with the N-terminal threonine being both N-acetylated andO-phosphorylated. The LC with online ESI-skimmer CID MS also revealed the presence of an unexpected isoform of CP43 in which the amino acid at position 4 was aspartic acid not asparagine (Fig. 2 C). This aspartate isoform was found in all thylakoid preparations isolated from plants under a variety of conditions and comprised ∼15% of the total CP43 pool. Given that the Arabidopsis CP43 gene encodes asparagine at this position (34Sato S. Nakamura Y. Kaneko T. Asamizu E. Tanaka S. DNA Res. 1999; 6: 283-290Crossref PubMed Scopus (393) Google Scholar), it is likely that this isoform was created by a deamidation reaction, the nature of which is currently unknown. By similar analysis, we determined the sequence of the seven other phosphopeptides and identified the corresponding proteins in theArabidopsis sequence data bases (Table I). Besides CP43, the D1 and D2 proteins of the PSII reaction center were identified and found to also contain an N-terminal threonine that was bothN-acetylated and O-phosphorylated. We identified two phosphopeptides that corresponded to the mature LHCII polypeptides phosphorylated at the Thr-3 (Table I). One represented the expected tryptic fragment (TPVAKPK) whereas the other was two amino acids longer and N-acetylated (Ac-RKTPVAKPK). The second form likely represented an incomplete digestion product caused by the phosphate at Thr-3 blocking trypsin cleavage after Lys-2. The PsbH protein of PSII was phosphorylated at Thr-2. Notably, we also found a doubly phosphorylated form of this peptide containing a second phosphate bound to Thr-4 (Fig. 2 D and Table I). The doubly phosphorylated form of PsbH was also detected in spinach thylakoids, suggesting that its presence is widespread in higher plants.2 To study the phosphorylation state of these principalArabidopsis thylakoid proteins in different physiological conditions, we developed an ESI MS method to compare the levels of the phospho and nonphospho forms for each. First, we determined the LC elution positions of the eight phosphopeptides present in the complete tryptic peptide mixtures without IMAC enrichment. This LC separation did not completely resolve these complex mixtures but did make spectrometric identification of separate peptide ions in each fraction possible (Fig. 3 A). Following LC, the peptides were detected online by ESI-MS and their masses were determined from full scan data in the positive-ion mode. Phosphopeptides were concurrently identified by switching to the negative-ion mode every 6 s; this mode led to peptide fragmentation and production of ions of −79 m/z, which are diagno