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Proteomics 2001, 1, 200–206
Review Albert Sickmann, Helmut E. Meyer Institut für Physiologische Chemie der Ruhr Universität Bochum, Germany
Phosphoamino acid analysis Phosphorylation of amino acid residues in proteins plays a major role in biological systems. Often, phosphorylation acts as a molecular switch controlling the protein activity in different pathways as in metabolism, signal transduction, cell division etc. Therefore, identification of phosphoamino acids in proteins is an important task in protein analysis. Since the introduction of high resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) a separation of phosphorylated and dephosphorylated protein species is possible. The identification of phosphorylation sites from preparative 2-D gels need very sensitive mass spectrometry methods and a specific enrichment of the phosphoprotein or -peptide. Keywords: Review / Phosphorylation / Amino acid
Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Identification of phosphoamino acids . . . . . . . 2.1 Identification of phosphoserine, phosphothreonine and phosphotyrosine . . . . . 2.2 Identification of N-phosphates and acylphosphates . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Identification of phosphoamino acids using specific antibodies . . . . . . . . . . . . . . . . . . . . . . . 2.4 Specific enrichment of phosphopeptides and -proteins using immobilized metal ion-affinity chromatography . . . . . . . . . . . . . . . 2.5 Localisation of phosphorylated amino acid residues in peptides and proteins . . . . . . . 2.6 Localisation of O-phosphates . . . . . . . . . . . . . . 2.7 Localisation of Phosphohistidine . . . . . . . . . . . 2.8 Localisation of Acylphosphates . . . . . . . . . . . . 2.9 Phosphocysteine . . . . . . . . . . . . . . . . . . . . . . . . 3. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1 Introduction Phosphorylation of amino acid residues in proteins plays a major role in biological systems. Often, phosphorylation acts as a molecular switch controlling the protein activity in different pathways as in metabolism, signal transducCorrespondence: Albert Sickmann, Institut für Physiologische Chemie, Ruhr Universität Bochum, Proteinstrukturlabor, Gebäude MA 2/143, Universitätsstraße 150, 44780 Bochum, Germany E-mail:
[email protected] Fax: + 49 (0) 234-321 45 54
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tion, cell division etc. Therefore, identification of phosphoamino acids in proteins is an important task in protein analysis. In this paper an overview for phosphopeptide analysis is presented. Detailed protocols and examples for each step of analysis are described in the given references. In special cases contact to the authors is recommended, because sample preparation is the most important step for successful analysis. Described functions of protein phosphorylation are: (A.) The phosphorylation takes part in enzymatic mechanisms. In this case the phosphorylation is a reactive intermediate (most often S- and N-phosphates), e. g. the HPr kinase (histidine residue) in the phosphoenolpyruvate dependent phosphotransferase system (PTS) [1, 2]). (B.) The phosphorylation mediates protein activity. Proteins are phosphorylated by protein kinases e. g. protein kinase A [3] (serine and threonine residues) or different receptor tyrosine kinases [4] (tyrosine residues). (C.) The phosphorylation of aspartate, glutamate and histidine takes part during the sensory transduction in bacterial chemotaxis [5, 6]. Four different types of phosphoamino acid residues are known: (1.) O-phosphates (O-phosphomonoesters) are formed by phosphorylation of hydroxyamino acids such as serine, threonine or tyrosine. The phosphorylation of hydroxyproline or -lysine is yet unknown. (2.) N-phosphates (phosphoamidates) are generated by phosphorylation of the amino groups in arginine, lysine or histidine. (3.) Acylphosphates (phosphate anhydrides) are produced by the phosphorylation of aspartic or glutamic acid. (4.) S-phosphates (S-phosphothioesters) are formed by phosphorylation of cysteine. The chemical stability of phosphorylated amino acids is shown in Table 1. 1615-9853/01/0202–200 $17.50+ .50/0
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Table 1. Chemical Stability of Phosphorylated Amino Acids [5] Stability in Nature of phosphoamino acid
Acid
Alkali
O-Phosphates Phosphoserine Phosphothreonine Phosphotyrosine
+ + +
– 6 +
+ + +
+ + +
N-Phosphates Phosphoarginine Phosphohistidine Phospholysine
– – –
– + +
– – –
– – –
Acylphosphates Phosphoaspartate Phosphoglutamate
– –
– –
– –
– –
S-Phosphates Phosphocysteine
(+)
HydroxylPyridine amine
+
+
+
Reprinted from Martensen [7], with permission.
All O-phosphates are stable under acidic conditions, in the presence of hydroxylamine, and pyridine. The Nphosphates, except for phosphoarginine, are stable under alkaline conditions. All acylphosphates are reactive phosphoamino acids and are labile in acid, alkali, hydroxylamine, and pyridine. Phosphocysteine is moderately stable under all tested conditions.
2 Identification of phosphoamino acids Recently methods for identification of phosphoamino acids have been developed. Very often the major problem is to identify samples containing phosphoamino acids. The method of choice is radioactive labeling with 32P allowing the detection of Cerenkov radiation of the 32P containing samples. Nonradioactive methods often need a specific enrichment of the phosphorylated protein [7] because only a few percent of the whole amount of a protein is usually phosphorylated in vivo and in vitro. Phosphorylation of amino acid residues can also be determined by [31P]-NMR, but this method needs a high amount of sample.
2.1 Identification of phosphoserine, phosphothreonine and phosphotyrosine As shown in Table 1, O-phosphates are stable under acidic conditions. After partial acid hydrolysis of phosphorylated proteins the phosphoamino acids phosphoserine, phosphothreonine and phosphotyrosine can be separated using an ion-exchange HPLC column [8]. A strong anion-exchange column with isocratic elution is used (Fig. 1). Another technique using 2-D separation of phos-
Figure 1. HPLC separation of fluorenylmethoxycarbonyl (FMOC) derivatized phosphoserine, phosphothreonine, and phosphotyrosine (80 pmol each) on a Partisil 10 strong anion exchange (SAX) column at 1.5 mL/min with buffer containing 55% methanol, 1% tetrahydrofuran, and 10 mM potassium phosphate, pH 3.9 (Reprinted from Niedbalski and Ringer [8], with permission).
phoamino acids by thin-layer systems is described by Dulcos et al. [9]. The detection is done by staining with ninhydrine or in case of 32P-labeled phosphoamino acids by autoradiography (Fig. 2). A method for determination of phosphoamino acids in phosphopeptides by capillary electrophoresis is decribed by Meyer et al. [10]. The partially hydrolysed phosphopeptide sample is derivatized by dabsyl chloride, analysed in negative mode and detected at 436 nm. Under these conditions only negatively charged amino acids, like phosphoamino acids or cysteic acid, are detected (Fig. 3).
2.2 Identification of N-phosphates and acylphosphates The identification of N- and acylphosphates is quite difficult. The N-phosphates are extremely labile in acid but relatively stable in alkali. Therefore, amino acid analysis of N-phosphates is possible only after hydrolysis in alkali. Acylphosphates are unstable under acidic and alkaline conditions and show a bell shape of their hydrolysis curve; such phosphoamino acids can only be analysed indirectly [11, 12].
2.3 Identification of phosphoamino acids using specific antibodies A sensitive method to detect O-phosphates in proteins and peptides is Western blot analysis followed by immuno staining. The generation of mono- or polyclonal
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Figure 2. Separation of phosphoamino acids and uridine monophosphate by double chromatography. Samples were chromatographed in isobutyric acid/0.5 M NH4OH (5:3 v/v) in the first dimension (1D) and in 2-propanol/ HCl/water (7:1.5:1.5 v/v) in the second dimension (2D), then revealed by staining or autoradiography. The origin (O) is on the right, at the intersection of the two arrows. Analysis was performed by using cellulose plates from Macherey-Nagel (Dueren, Germany). Shaded spots indicate the position of phosphoamino acids. Average data from three to five experiments are presented (Reprinted from Dulcos et al. [9], with permission).
antibodies against P-Ser, P-Thr and P-Tyr is described by several authors. Antibodies against P-Thr and P-Ser are generally not specific enough to detect one phosphorylated serine or threonine side chain, because this epitope is too small. However, such antibodies are specific for a certain sequence motif containing P-Ser or P-Thr. Anti-P-Tyr antibodies however are specific enough to detect one phosphorylated tyrosine. Some commercially available monoclonal antibodies, e. g. the ‘4G10’ (Upstate Biotechnology, Lake Placid, NY, USA) show good results in detecting P-Tyr peptides and proteins. A method for the specific enrichment of P-Tyr peptides is described by Schnölzer and Stempka (personal communication).
2.4 Specific enrichment of phosphopeptides and -proteins using immobilized metal ion affinity chromatography Immobilized metal ion affinity chromatography (IMAC) was first introduced by Hellferich [13] and Porath [14]. The chromatographic sorbent used in IMAC consists of a chromatographic matrix to which a metal chelating group has been attached by a leash or linkage group. The IMAC technique is commonly used for protein purification due to the fact that histidine, cysteine and tryptophan are adsorbed. An overview of the IMAC technique is given by Kagedal et al. [15]. A further advantage of the IMAC technique is the specific enrichment of phosphoproteins
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Figure 3. Determination of phosphoamino acids in phosphopeptides by capillary electrophoresis. Phosphopeptides are partially hydrolysed with 6 N HCl at 1108C for 2 h followed by derivatization of the released amino acids with dabsyl chloride. The samples are dissolved in 100 mL 70% v/v aqueous ethanol and 30 nL are analysed by capillary electrophoresis. A. Analysis of a standard mixture containing 5 pmol/mL dabsylcysteic acid (CYA), dabsylphosphoserine (S-P), dabsylphosphothreonine (T-P), and dabsylphosphotyrosine (Y-P). Additional signals are hydrolysed reagent (DABS-ONa). B. 1 nmol of the diphosphopeptide ASpYpSA is used for hydrolysis. Dabsylphosphoserine (S-P) and dabsylphosphotyrosine (Y-P) are recovered with a 10% and 24% yield, respectively. C. 1.5 nmol of the diphosphopeptide ATpYpSA is used for hydrolysis. Dabsylphosphothreonine (T-P) and dabsylphosphotyrosine (Y-P) are recovered with a 16% and 14% yield, respectively. D. 1.2 nmol of the phosphopeptide ATYpSA is used for hydrolysis. Dabsylphosphotyrosine (Y-P) is recovered with a 17% yield. Conditions for capillary electrophoresis: electrophoresis buffer: 25 mM sodium citrate, pH 4.2; capillary: fused silica, 72 cm (50 cm to detector), 50 mm ID; injection time (vacuum): 10 s = 30 nL injection volume; units full scale: 0.008 A; polarity: negative; detection: 436 nm. Take notice that under these conditions only negatively charged amino acid derivatives will be detected. Therefore, no signals are obtained from the nonphosphorylated amino acids (from Meyer et al. [10]).
and -peptides [16]. A novel product for nanoscale purification of phosphopetides from peptide mixtures was this year introduced by Millipore. The IMAC ZipTip (Millipore, Bedford, MA, USA) consists of a 10 mL pipette tip with 0.5 mL IMAC material. The group of Hunt [17] demonstrated first results with the identification of phosphopeptides from MHC peptide pools.
2.5 Localisation of phosphorylated amino acid residues in peptides and proteins Before the development of mass spectrometry (MS) for large biomolecules (ESI- and MALDI-MS) the only availa-
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Figure 4. MALDI-PSD spectrum of the phosphotyrosine containing peptide SSGSGSSVADERVDYpVVVGQQK. The single charged parent ion is taken for PSD analysis using an acceleration voltage of 20 kV. The spectrum is recorded in 14 windows with reflectron voltages between 21.6 kV and 0.65 kV. The PSD fragment data of complete band y-ion series easily allows the phosphotyrosine in the primary structure to be assigned.
ble method for the localisation of phosphoamino acids in peptide sequences was Edman degradation. Through the conversion of P-Ser to S-ethyl cysteine [18] or P-Thr to bmethyl-S-ethyl cysteine [19] a positive evidence for P-Ser and P-Thr is possible. P-Tyr is stable during Edman degradation but nearly insoluble in the conventional transfer solvents used in the Edman sequenator, yielding a gap in the sequence course. Applying solid phase sequencing to this problem provides a solution [20]. Since the introduction of MS, the combination of Edman degradation and MS is the most powerful tool for localisation of phosphoamino acid residues in protein sequences.
2.6 Localisation of O-phosphates As shown in Table 1, O-phosphates are stable under acidic conditions. Therefore, the separation of a proteolytic digest containing such phosphopeptides is possible with the common acetonitrile/water/TFA gradient system. The use of completely inert HPLC pumps and columns is strongly recommended, because all kind of phosphopeptides adsorb irreversibly on etched iron surfaces. Using MALDI-PSD for structure determination, the MALDI-
target should be inert (e. g. gold surface). When using steel targets the ‘sandwich’ preparation technique might be helpful. In Fig. 4 the MALDI-PSD spectrum of the phosphotyrosine containing peptide SSGSGSSVADERVDYpVVVGQQK from human Gab-1 is shown. The complete b- and y-ion series allows to determine the structure of the peptide and to localise the phosphotyrosine residue. Further examples for identification of phosphotyrosine are described by Lehr et al. [21, 22]. Phosphoserine or -threonine residues can be localised the same way. However, fragment ion spectra from peptides containing these O-phosphates show in most cases very intensive signals at [M+H]+-80 Da (loss of HPO32-) and [M+H]+-98 Da (loss of H2PO4-) [23, 24]. This behavior makes it sometimes impossible to localize the phosphoamino acid by ESI-MS/MS or MALDI-PSD experiments.
2.7 Localisation of phosphohistidine Phosphohistidine residues are unstable under acidic conditions and therefore a hexafluoroacetone/NH3 gradient system at pH 8.6 is well suited. The common acetonitrile/water/TFA system is also possible for analysis but a
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Figure 5. Deconvoluted ESI mass spectrum of the phosphorylated Glc T protein. Purified GlcT is dissolved in water and separated using a C4 column connected on-line to the electrospray source. The phosphorylated form of the GlcT protein with a measured mass of 33 586 6 5 Da predominates over the unphosphorylated form of the protein (from Knezevic et al. [11]).
fast chromatography is recommended, because the halflife time of phosphohistidine is about 30 min at pH 3. In Fig. 5 a deconvoluted ESI mass spectrum of the histidine phosphorylated protein GlcT is shown [11]. The localisation of phosphohistidine residues in peptide sequences is difficult. Medzihradsky et al. [12] give an example for the localisation of phosphohistidine in a synthetic phosphopeptide (Ac-SFTNPLHpSAAW-NH2, Fig. 6). However, the phosphorylation site cannot be determined to the histidine residue but to the partial sequence LHS. The major signals are derived from a loss of HPO32- and H2PO4- similar to the O-phosphates P-Ser and P-Thr.
2.8 Localisation of acylphosphates A direct identification of acylphosphates is difficult. However, selective reduction of acylphosphate with NaBH4/[3H]NaBH4 to the corresponding alcohol and labeling with tritium is possible. After digestion and separation of tritium-containing peptides, a structure determination can be done by MS. Sanders et al. [5] show the localisation of phosphoaspartate in CheY using NaBH4 derivatisation followed by ESI-MS/MS. The reduction of phosphoaspartate leads to a homoserine residue which can easily be assigned in MS/MS spectra (Fig. 7). An example for the
Figure 6. MALDI-high energy CID spectrum of the histidine phosphate containing peptide Ac-SFTNPLHpSAAW-NH2. DHAP/DHAC was used as matrix. Signals labeled with an asterix represent gas-phase dephosphorylation ions (Reprinted from Medzihradszky et al. [12], with permission).
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Figure 7. Localisation of phosphoaspartic acid. CID mass spectra of the 809 and 823 Da peptides. The molecular ions of interest were allowed to pass through the first mass spectrometer and were then collisionally activated. The resulting fragment ions were analysed by mass to form the daughter ion (MS/MS) spectra. A, the Y sequence and immonium ions obtained from the 823 Da peptide are indicated. B, the Y sequence and immonium ions obtained from the 809 Da are indicated. The spectrum contains all the immonium ions found in the spectrum from the 823 Da peptide with the addition of an ion at m/z 74, which indicates the presence of a homoserine in the peptide (Reprinted from Sanders et al. [5], with permission).
identification of phosphoglutamate is given by Trumbore et al. [25].
tion containing the phosphocysteine peptide was subjected to Edman degradation, allowing the positive identification of the phosphocysteine residue.
2.9 Phosphocysteine An example of the combination of Edman degradation and MS is given by Weigt et al. [26]. The phosphorylated EIIMtl fragment from Staphylococcus carnosus is analysed with ESI-MS. The deconvoluted spectrum of the phosphorylated protein is shown in Fig. 8. After digestion with Glu-C followed by LC-MS analysis of the digest, the frac-
3 Conclusion Using MS combined with Edman degradation the localisation and identification of phosphorylated amino acids as the O, N, S, and acylphosphates is possible. The sensitivity of these methods is in the low picomolar (Edman
Figure 8. Deconvoluted ESI mass spectrum of the phosphorylated EIIMtl fragment, containing phosphocysteine. EIIMtl fragment is dissolved in water and separated using a C4 column connected online to the electrospray source. The phosphorylated form of the EIIMtl fragment with a calculated mass of 20 066 Da predominates over the unphosphorylated form (from Weigt et al. [26]).
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degradation) or subpicomolar (MS) range. To record a MALDI-PSD spectrum of a pure phosphopeptide, commonly less than 100 fmol are needed. In mixtures with unphosphorylated peptides (e. g. after digest) the ionisation rate of the phosphopeptide is impaired and more substance is needed. Therefore, a separation of the mixture or an enrichment of the phosphopeptide is important. Altogether, the identification and localisation of the O, S, N, and acylphosphates in the primary structure is possible, however, for each kind of phosphoamino acid a very special technique is needed. Received August 3, 2000
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