Detection of Low-Abundance Protein Phosphorylation by Selective

Jul 20, 2012 - and Jill Barber*. The Michael Barber Centre for Mass Spectrometry, Manchester Interdisciplinary Biocentre (MIB), 131 Princess Street, ...
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Detection of Low-Abundance Protein Phosphorylation by Selective 18 O Labeling and Mass Spectrometry Waleed M. Alghamdi, Simon J. Gaskell,† and Jill Barber* The Michael Barber Centre for Mass Spectrometry, Manchester Interdisciplinary Biocentre (MIB), 131 Princess Street, Manchester M1 7DN, United Kingdom ABSTRACT: Reversible phosphorylation regulates the majority of intracellular networking and pathways. The study of this widely explored post-translational modification is usually challenged by low stoichiometric levels of modification. Many approaches have been developed to overcome this problem and to achieve rigorous characterization of protein phosphorylation. We describe a method for enhanced detection of lowabundance protein phosphorylation that uses selective introduction of 18O label into phosphorylation sites with H218O and mass spectrometric detection. The method was applied to introduce 18O label into bacterially expressed Aurora A kinase phosphorylation sites and resulted in the representation of phosphorylated peptides as doublets or triplets according to the number of phosphate groups. A total of 28 phosphopeptides were observed by this method.

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Accurate determination of the phosphorylation sites of the human proteome has great importance in many other fields such as cancer research and drug discovery. Indeed, it has been estimated that about 30% of research effort in drug discovery is based on targeting protein kinases.6 Analysis of phosphoproteins is, however, limited by a number of difficulties. In the first place, the presence of multiple modification sites in a single protein gives rise to multiple variants differing in both location and overall stoichiometry of phosphorylation. Commonly only a tiny proportion of a protein is phosphorylated in the cell following stimulation. This means that enrichment of phosphoproteins (or phosphopeptides) must generally be achieved prior to detailed analysis. Finally, any whole-cell isolate contains phosphatases, and it is normally necessary to inhibit these during phosphoprotein preparation.1,4,7 Mass spectrometry (MS) is widely used for the analysis of phosphoproteomes. It is a highly sensitive technique, which at first sight makes it especially suitable for the analysis of lowabundance phosphoproteins. However, the yield of ions during MS of phosphopeptides in the standard positive ion mode is usually lower, sometimes dramatically so, than from the nonphosphorylated counterparts. Additionally, the lability of the phosphate group following collision-induced dissociation (CID) during tandem MS can be a problem, reducing the possibility of secure identification of phosphorylated peptides and precise determination of the phosphorylation site.

osttranslational modifications (PTMs) regulate the activity of most cellular proteins. Reversible protein phosphorylation is the most explored and best understood of approximately 200 identified PTMs.1,2 It plays an essential role in controlling and organizing extracellular signaling and intracellular events. Phosphorylation generates conformational changes in targeted proteins.3 The newly shaped molecules are affected in many different ways: they may be activated or deactivated, labeled for destruction, moved from one cellular site to another, or bound to or released from other molecules.4 Kinases, the enzymes responsible for phosphorylation, are encoded by approximately 1.7% of the human genome. The ca. 500 protein kinases are responsible for phosphorylation of 30% of human proteins. The release of phosphate groups from proteins is usually facilitated by phosphatases, and these are much less specific.3,4 In view of the resource invested in reversible phosphorylation by the cell, it is not surprising that many diseases are characterized by faulty phosphorylation patterns. These include some cancers such as papillary renal cancer, chronic myelomonocytic leukemia, and non-Hodgkin’s lymphoma, which are developed as a result of defects in Met receptor kinase, Tel-PDGF receptor kinase, and Alk kinase, respectively.5 Additionally, some pathogenic bacteria exert their effects through deregulation of the phosphorylation system. Yersinia sp., for example, was the cause of many fatal pandemics including the Black Death, which was responsible for killing 25% of the population in Europe in the 12th and 13th centuries. The bacterial toxin responsible for this disaster was found to be a tyrosine phosphatase that caused uncontrolled dephosphorylation.5 © 2012 American Chemical Society

Received: April 19, 2012 Accepted: July 20, 2012 Published: July 20, 2012 7384

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Scheme 1. Two Experimental Designs Developed for 18O Labeling of Aurora A Phosphorylation Sitesa

a

(a) Starter culture is inoculated with Aurora A transformed cells and incubated overnight in light medium. A few microliters of the starter culture is used to inoculate 1 mL of fresh light LB medium. After 3-4 h, when OD600 reaches 0.6, Aurora production is induced with IPTG and the cells are incubated for 10 h. The light medium is then replaced by 1 mL of 18O LB and cells are incubated for another 10 h. (b) Starter culture is prepared as above and 2 mL of light medium is then inoculated with a few microliters of the starter culture and incubated for 3−4 h. This is followed by IPTG induction for 17 h. Light medium is then replaced by 1 mL of 18O LB and cells are incubated for another 10 h.

chromatography (LC)/MS/MS experiments, including T288, which is known to be important for kinase activity.12 Eight of these sites have been confirmed by mass spectrometric phosphopeptide fingerprinting, achieved by detection of a −80 Da shift upon dephosphorylation with hydrogen fluoride.

Fragmentation of phosphorylated peptides in CID is commonly dominated by the signal generated from the neutral loss of phosphoric acid, often leaving insufficient MS/MS data for the determination of peptide sequence. Methods used to overcome this problem include the use of electron transfer dissociation activation, which results in sequence-specific fragmentation and allows preservation of posttranslational modification.8 Recently a novel method for dimetal phosphate ester stabilization using gallium ions was developed and showed improved stability of the peptide−phosphate ester bond, which enhanced sequence information produced by collision-induced dissociation.9 A range of affinity enrichment techniques have been developed to overcome the problems of sample complexity and low abundance of phosphoproteins. Chemical derivatization has alternatively been used for tagging of phosphate groups on either proteins or peptides.7 Veenstra and co-workers10 have attempted to improve the detection of phosphorylated peptides by an 18O labeling method based on γ-[18 O4]ATP. In separate reactions, dephosphorylated peptides and proteins were incubated with a 1:1 mixture of γ-[18O4]ATP and normal (unlabeled) ATP. The phosphorylated products were then subjected to tryptic digestion and mass spectrometric analysis. Two versions of phosphopeptides phosphorylated by labeled and unlabeled ATP were readily detected in the MS spectrum with peak separation of 6.01 Da.10 This method is a successful alternative to 32P labeling for the purpose of pinpointing phosphorylation sites on proteins; it increases confidence and reduces ambiguity in detecting phosphopeptides by mass spectrometry. However, in vitro phosphorylation patterns are not necessarily identical to those occurring in real physiological systems, where other factors may have some influence on phosphorylation patterns. Here, we describe a novel method for 18O labeling of phosphorylated proteins, in order to achieve improved detection of phosphopeptides. The method was applied to Aurora A kinase overexpressed in Escherichia coli and resulted in the selective introduction of 18O label into phosphorylation sites. A total of 28 phosphopeptides were detected from Aurora A labeled samples. Aurora A, a serine/threonine kinase that functions in various stages of mitosis, is autophosphorylated both in human cells and when expressed in recombinant form in E. coli. The deregulation of this kinase contributes to the development of some cancers.11−14 Fifteen phosphorylation sites have been described in human Aurora A on the basis of liquid

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EXPERIMENTAL SECTION Aurora A Gene Sequence and Plasmid. Transformed E. coli cells containing the human Aurora A gene were a gift from Dr. Patrick Eyers (University of Sheffield). The human Aurora A gene was ligated into the BamHI/NotI sites of the vector pET28a, which was transformed into the strain BL21(DE3) pLysS (Novagen).15 The kinase sequence containing the His tag at its N-terminus is shown below. The first amino acid of the Aurora A protein is the D residue in the motif DRSKEN: MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSDRSKENCISGPVKATAPVGGPKRVLVTQQFPCQNPLPVNSGQAQRVLCPSNSSQRVPLQAQKLVSSHKPVQNQKQKQLQATSVPHPVSRPLNNTQKSKQPLPSAPENNPEEELASKQKNEESKKRQWALEDFEIGRPLGKGKFGNVYLAREKQSKFILALKVLFKAQLEKAGVEHQLRREVEIQSHLRHPNILRLYGYFHDATRVYLILEYAPLGTVYRELQKLSKFDEQRTATYITELANALSYCHSKRVIHRDIKPENLLLGSAGELKIADFGWSVHAPSSRRTTLCGTLDYLPPEMIEGRMHDEKVDLWSLGVLCYEFLVGKPPFEANTYQETYKRISRVEFTFPDFVTEGARDLISRLLKHNPSQRPMLREVLEHPWITANSSKPSNCQNKESASKQS H218O Labeling Experiment. Luria broth (LB, 1 mL) containing kanamycin (50 μg·mL−1) in a 15 mL Falcon tube was inoculated with few microliters of glycerol stock and incubated at 37 °C with overnight shaking. About 10 μL of the overnight culture was then subcultured into 1 mL of LB containing the same concentration of kanamycin in a 15 mL Falcon tube. Cell cultures were incubated at 37 °C with shaking at 200 rpm for 3−4 h until OD600 of 0.5−1.0 was achieved. Induction was performed by adding 10 μL of 0.2 mM isopropyl α-D-1-thiogalactopyranoside (IPTG), and the cultures were incubated for either 10 or 17 h at 20 °C (Scheme 1). Cells were then harvested by centrifugation at 10000g for 10 min at 4 °C and resuspended in labeled LB medium, which was prepared by dissolving 10 mg of bactopeptone, 5 mg of tryptone, and 10 mg of NaCl in 1 mL of H218O (Cambridge Isotope Laboratories, Andover, MA) and sterilized by use of 0.22 μm syringe filters 7385

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(Iwaki). Cells were incubated at 20 °C with shaking at 200 rpm for another 10 h in a 15 mL Falcon tube. Cell Lysis. Cells were transferred to 1.5 mL Eppendorf tubes and harvested by centrifugation at 10000g for 10 min at 4 °C. After centrifugation, the supernatant was removed and the cells were resuspended, by gentle vortexing, in 250 μL of room temperature BugBuster protein extraction reagent (Novagen, Nottingham, U.K.) containing 0.2 μL of benzonase (25 u/μL) (Novagen, Nottingham, U.K.). Ethylenediaminetetraacetic acid (EDTA) -free protease inhibitor cocktail tablets were added by dissolving 1/2 tablet in 1 mL of water and adding 50 μL of the solution into the BugBuster reagent. (E. coli does not contain eukaryotic phosphatases, so it was not necessary to use phosphatase inhibitors.) Cells were then incubated with the extraction reagent at room temperature for 20 min. Insoluble debris was removed by centrifugation at 14000g for 20 min at 4 °C, and the supernatant was transferred to a new 1.5 Eppendorf tube. Metal Affinity Resin Purification. Protein isolation was performed by Talon cobalt polyhistidine tag purification (Clontech Laboratories, Inc. Saint-Germain-en-Laye, France). Twenty-five microliters of settled resin bed volume was equilibrated by washing with 400 μL of wash buffer [50 mM tris(hydroxymethyl)aminomethane (Tris) buffer (pH 7.4), 5 M NaCl, 10 mM imidazole, 0.1 mM EDTA, 0.1 mM ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 0.4 mM phenylmethanesulfonyl fluoride (PMSF), and 0.4 mM benzamidine). The wash buffer was removed and the equilibrated resin was then incubated with the cell lysate for about 1 h at 4 °C. The supernatant was allowed to flow through the purification column and the resin was washed four times by adding 400 μL of washing buffer, vortex-mixing, and centrifuging to precipitate the resin and remove the wash buffer. The His-tagged kinase was then eluted by addition of 40 μL of elution buffer (50 mM Tris, 0.3 M NaCl, 10 mM imidazole, 0.1 mM EDTA, and 0.1 mM EGTA, pH 7.4) and shaking for about 5 min. This was repeated three times and the eluted fractions were collected and combined. Trypsin Digestion. About 1 μg of purified Aurora A kinase was mixed with 10 μL of ammonium bicarbonate solution (50 mM) and vortexed. To reduce disulfide bonds, 5 μL of tri(carboxyethyl)phosphine (TCEP) (4 mM in 25 mM ammonium bicarbonate solution) was added. The sample was mixed thoroughly and heated at 60 °C for 30 min, and then it was transferred to ice for 5 min and 5 μL of iodoacetamide (alkylation reagent) stock solution (55 mM, prepared in 25 mM ammonium bicarbonate) was added. The sample was stored in the dark at room temperature for 30 min. In order to quench any excess iodoacetamide, 5 μL of dithiothreitol (DTT, 10 mM prepared in 25 mM ammonium bicarbonate) was added to the mixture. Finally, trypsin was added to the solution with a protein:trypsin ratio of 50:1. Digestion was allowed to proceed overnight at 37 °C. LC MALDI ToF/ToF Analysis. Tryptic peptides were separated by nanoflow LC (Agilent 1100) as follows: preconcentration and washing were carried out on a C18 trap column (5 mm × 0.3 mm i.d., PepMap, Agilent, Lakeside, U.K.) [2% CH3CN and 0.09% trifluoroacetic acid (TFA), flow 30 μL·min−1, 20 min at 40 °C] followed by peptide separation on a Zorbax 300SB C-18, 75 μm 15 cm reverse-phase capillary column (Agilent, Lakeside, U.K.) at a flow rate of 300 nL·min−1; solvent A, 2% CH3CN and 0.09% TFA; solvent B, 80% CH3CN and 0.09% TFA) with a 95 min gradient of 8−

42% solution B in 90 min and 42−95% B in 5 min (at room temperature). The column eluent was collected as droplet-sized fractions on an Anchorchip 800/384 matrix-assisted laser desorption ionization (MALDI) target plate (Bruker Daltonix, Bremen, Germany) by Probot spotting robot (Dionex, Surrey, U.K.). The LC eluent was premixed with matrix before spotting. The matrix was prepared by mixing 3.74 mL of 95% CH3CN and 0.1% TFA, 180 μL of 90% CH3CN saturated with 0.3 mg·mL−1 α-cyano-4-hydroxycinnamic acid (Sigma, Poole, U.K.), 40 μL of 10% TFA, and 20 μL of 100 mM NH4PO4. Fractions were collected (beginning 40 min after injection) every 25 s (125 nL/fraction). In total, about 200 fractions were collected in each separation. MALDI ToF/ToF (time-of-flight) analysis was performed on an Ultraflex II ToF/ToF mass spectrometer (Bruker Daltonik, Bremen, Germany) with an automated run. The laser operated at 100 Hz repetition rate, and 1000−2000 laser shots were accumulated per spectrum. Acquisition and analysis of data were performed by use of DataAnalysis software (Bruker, Bremen, Germany). LC/MS and MS/MS Analyses via a Q-ToF Global Instrument. Peptide separation was performed with an Ultimate 3000 capillary LC system (Dionex, Surrey, U.K.) on a 75 μm 15 cm reverse-phase capillary column at a flow rate of 200 nL·min−1. The Ultimate 3000 is fitted with a built-in oven that was set to 30 °C. By use of a distal-coated fused silica Picotip emitter (New Objective, Inc., Woburn, MA) with a capillary voltage of 2200−2800 V, the HPLC eluate was sprayed into the Z-spray nanoelectrospray source of QToF Global (Waters, Manchester, U.K.). Data were acquired by use of MassLynx version 4.0 (Waters, Manchester, U.K.). LC/MS data were acquired by data-dependent acquisition in survey scan mode with a mass range of 400−2000 m/z, a scan time of 2.4 s, and an interscan time of 0.1 s; acquisition was in survey scan mode over the mass range of 400−2000 m/z. Spectra were acquired with a scan time of 2.4 s and an interscan time of 0.1 s. The capillary voltage was typically set at 1200− 2000 V for nanospray capillaries. The data acquired from LC electrospray ionization (ESI) QToF MS/MS were converted by Proteinlynx (MassLynx 4.0, Waters) to peak list files (.pkl). The file contained the m/z of the precursor ion, the m/z of the corresponding fragment ions, the charge state of these ions, and their respective signal intensities. The generated pkl files were submitted to MASCOT searches. Data were searched by an in-house MASCOT algorithm (http://msct.smith.man.ac.uk/mascot/ )16 against relevant databases. Trypsin was specified as the proteolytic enzyme, and carbamidomethyl of cysteine was selected as fixed modification. Variable modifications include deamidation of asparagine and glutamine, oxidation of methionine, and phosphorylation of threonine, serine, and tyrosine residues. A mass accuracy of 100−200 ppm was specified, and monoisotopic masses were used. The accuracy of identification was judged by the MOWSE (molecular weight search) score and expected value. The probability of correct identification of a peptide sequence increased as the MOWSE score increased and the “expect” value decreased. Where the software indicated the presence of a phosphopeptide, this was confirmed by manual inspection of the MS/MS spectra.

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RESULTS Labeling of Phosphorylated Peptides with H218O. In the initial experiment, E. coli cells were incubated in label-free 7386

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Figure 1. MALDI ToF/ToF MS data of tryptic digests of isolated Aurora A samples. (a) Control sample from light medium. (b) 18O-Labeled sample: Starter and subcultures were prepared in light medium. Cells were then transferred into H218O LB medium at OD600 = 0.6 and incubated for 5−10 min before overnight induction. (c) Control sample prepared by using H218O LB in all phases. The complex envelopes shown in panels b and c are attributed to extensive incorporation of 18O label into the Aurora A backbone. Using light medium in the starter and subculture phases in b resulted in similar levels of data complexity compared to control sample c.

medium for 3−4 h until OD600 reached about 0.5. The cells were then transferred and induced in medium based on H218O. The mass spectra from this sample were, however, extremely complex (see Figure 1). The extent of labeling indicates that 18 O has been incorporated not just into the phosphate groups but into the protein backbone. The phosphorylated peptides could not be identified from these data because of the huge shifts in all masses. The labeling state of the phosphorylation sites (i.e., the number of 18O atoms incorporated into the phosphate groups) could not therefore be identified. In order to identify the heavy versions of phosphorylated peptides obtained from 18O-labeled Aurora A, these were now mixed with a sample of unlabeled Aurora A peptides and analyzed by LC/MS/MS. Peptides with the same sequence were, as expected, eluted at the same time regardless of their labeling state. Figure 2 shows an example of a phosphorylated peptide with m/z 996 +2 that was coeluted with its corresponding 18O-labeled peptide with m/z 1007. This was further confirmed by comparing the retention times of labeled and unlabeled samples. Finally a new list of the masses of the 18 O-labeled peptides was generated (Table 1). Two approaches were now adopted in order to identify the labeling state of the 18O-labeled phosphorylated peptides. We attempted to reduce data complexity by manipulation of growth conditions, and we also used a range of mass spectrometric analysis methods to try to analyze selectively the peptides of interest. Changing the culture conditions (by adding the labeled water concurrently with induction of Aurora A, by adding unlabeled amino acids to the medium, by culturing cells in minimal medium, or by reducing the induction period to 3 h) was unsuccessful in reducing the incorporation of 18O into the peptide backbone; no significant difference was observed in the resulting peptide isotope envelope (data not shown). Applying a variety of mass spectrometric approaches to achieve more selective analysis of phosphopeptides (including neutral loss scanning on an LTQ Orbitrap XL, product ion scanning in negative mode on QQQ or Q-TOF instruments, and manipulating the precursor ion selector in MALDI TOF/ TOF or Q-TOF; data not shown) was equally unsuccessful. These initial experiments were designed so that cells synthesized the Aurora A kinase in the presence of 18O label. Judging from the observed extent of 18O incorporation, this

Figure 2. Identification of 18O-labeled phosphorylated peptides from Aurora A tryptic digest isolated from medium based on H218O. The upper panel represents data acquired from a 1:1 mixture of light and 18 O-labeled Aurora A. The data show the 47 VLCPSNSpSQRVPLQAQK63 phosphorylated peptide with m/z 996.49+2 and its corresponding 18O-labeled homologue with average m/z 1007. Peptides were coeluted regardless of their labeling state. The data from this experiment were confirmed by comparison with the 18 O-labeled sample analyzed under the same conditions (lower panel). The data were further confirmed by comparing retention times of peptides from the sample mixture with those from 18O-labeled sample.

appeared to cause the accumulation of 18O-labeled amino acids, which were then utilized as building blocks for Aurora A synthesis (Figure 3b). This is the classical pulse−chase protocol in which label is introduced initially and then followed by the normal isotope. In this case the reverse experiment was indicated, in which induction takes place in label-free medium to allow the light version of the kinase to accumulate, before chasing with 18O label. When this experiment was performed (Figure 3c), Aurora A kinase with a 16O backbone but labeled with three 18O atoms in the phosphate was clearly detectable. This sample shows a peak at m/z 2561, corresponding to the phosphorylated peptide VLVTQQFPCQNPLPVNpSGQAQR with no isotopic label. At m/z 2567 there is a partner 7387

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Table 1. List of 18O-Labeled Phosphorylated Peptides Identified by Mixing Light and Labeled Samples and Analyzed by LC/MS site

peptide

m/z unlabeled

m/z labeled (most intense signal in envelope)

S54 S104 T82 T28 T28 S10 S51 S41

VLCPSNSpSQRVPLQAQK SKQPLPpSAPENNPEEELASK QLQApTSVPHPVSRPLNNTQK R.VLVpTQQFPCQNPLPVNSGQAQR.V R.VLVpTQQFPCQNPLPVNSGQAQR.V R.SKENCIpSGPVKATAPVGGPK.R R.VLCPpSNSSQRVPLQAQK.L R.VLVTQQFPCQNPLPVNpSGQAQR.V

996.52+2 749.04+3 765.70+3 1281.11+2 854.41+3 693.04+3 664.66+3 854.44+3

1007.32 756.42 772.42 1298 862.45 700.35 672.35 862.45

Figure 3. LC MALDI ToF/ToF data of the tryptic Aurora A digest. (a, top panel) Control sample prepared in light medium. The data show the singly phosphorylated peptides VLVTQQFPCQNPLPVNpSGQAQR with m/z 2561.34+1 and RVLVpTQQFPCQNPLPVNSGQAQR with m/z 2717.42+1. (b, middle panel) Data acquired from control sample prepared in H218O showing the corresponding phosphorylated peptides with average m/z 2584.94 +1 and 2742.90+1. The isotopic complexity resulted from random incorporation of 18O label into the peptide backbone. (c, bottom panel) Spectra of the sample prepared according to the reverse 18O-labeling protocol. The starter and subcultures and induction were performed in light medium. Cells were then transferred into 18O medium for 10 h. Peptides corresponding to incorporation of P18O3 were detected at 2567.26+1 and 2723.33+1.

corresponding to the same peptide incorporating three 18Os (Figure 3c). When cells were induced in light medium, Aurora A accumulated and was phosphorylated with light phosphate groups. We infer that when cells were transferred into 18Olabeled medium, the intracellular phosphate pool rapidly exchanged its oxygens with 18O and heavy phosphate was used to phosphorylate the pre-existing Aurora A, resulting in the doublets as shown in Figure 3c. The bacteria then continued to produce Aurora A after transfer into the 18O medium, incorporating the label into the backbone of the kinase. This is represented by the broad hump on the right side of the spectrum as shown in Figure 3c. LC ESI Q/ToF Analysis. Control tryptic Aurora A samples were further analyzed by LC ESI Q/ToF MS/MS to detect phosphorylated peptides. It was possible to identify 13 phosphorylated peptides representing 10 phosphorylation sites by standard protocols involving submission of the data to a MASCOT search (Table 2). The 18O-labeled sample prepared according to the reverselabeling protocol could, however, be used to identify 28 phosphorylated peptides (Table 3). Phosphorylation was confirmed by the detection of doublets corresponding to the incorporation of light and heavy forms of phosphate in the case of single phosphorylation events (Figure 4). Doubly phosphorylated peptides showed triplets corresponding to the incorporation of two light, one light/one heavy, and two heavy phosphate groups (Figure 5), although the signal corresponding to the peptide bearing two heavy phosphate groups was

Table 2. Identified Phosphorylated Peptides of Aurora A Tryptic Digest Control Sample Analyzed by LC ESI Q/ToFa

a

m/z

phosphopeptide

805.28+2 638.60+4 765.76+3 766.10+3 906.40+3 693.02+3 749.02+3 1122.88+2 664.69+3 691.34+3 996.52+2 854.75+3 1281.17+2

IADFGWpSVHAPSSR QKQLQATpSVPHPVSRPLNNTQK QLQApTSVPHPVSRPLNNTQK QLQATSVPHPVpSRPLNNTQK RVLVpTQQFPCQNPLPVNSGQAQR SKENCIpSGPVKATAPVGGPK SKQPLPpSAPENNPEEELASK SKQPLPpSAPENNPEEELASK VLCPSNpSSQRVPLQAQK VLCPSNpSpSQRVPLQAQK VLCPSNSpSQRVPLQAQK VLVTQQFPCQNPLPVNpSGQAQR VLVpTQQFPCQNPLPVNSGQAQR

A total of 13 phosphorylated peptides were identified.

weak and overlapped with the envelope due to peptide containing 18O in the backbone. Incorporation of P18O3 has successfully detected phosphorylated peptides with low intensities and therefore a lesser chance of identification based on characteristic fragment ions observed during tandem MS. The detection of doublets and triplets is clear evidence of the incorporation of single and double phosphate groups. Although it was not possible to identify the exact site of phosphorylation due to lack of MS/MS data, an 7388

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Table 3. 18O-Labeled Aurora A Tryptic Phosphorylated Peptides Analyzed by LC ESI-Q/ToF, Showing Incorporation of P18O3a peptide GSDRSKENCISGPVK GSHMASMTGGQQMGR GSHMASMTGGQQMGR GSHMASMTGGQQMGRGSDR GSHMASMTGGQQMGRGSDR HPNILRLYGYFHDATR LVSpSHKPVQNQK QKQLQATSVPHPVSRPLNNTQK QKQLQATSVPHPVSRPLNNTQK QKQLQATSVPHPVSRPLNNTQKSK QLQApTSVPHPVSRPLNNTQK QLQATSVPHPVSRPLNNTQK QLQATSVPHPVSRPLNNTQK QLQATSVPHPVSRPLNNTQK RVLVpTQQFPCQNPLPVNSGQAQR SKENCISGPVKATAPVGGPK SKENCISGPVKATAPVGGPK SKQPLPpSAPENNPEEELASK SKQPLPSAPENNPEEELASK TATYITELANALSYCHSK TATYITELANALSYCHSK TTLCGTLDYLPPEMIEGR VLCPpSNpSSQRVPLQAQK VLCPSNSpSQRVPLQAQK VLCPSNSpSQRVPLQAQK VLCPSNSSQR VLVpTQQFPCQNPLPVNSGQAQR VLVTQQFPCQNPLPVNpSGQAQR

light m/z

heavy m/z

+2

900.38+2 811.32+2 819.31+2 690.25+3 716.89+3 706.26+3 484.24+3 853.11+3 1279.14+2 1362.17+2 767.71+3 762.03+3 1142.56+2 1151.08+2 908.45+3 695.00+3 1042.01+2 751.01+3 1126.02+2 711.57+3 1063.86+2 1058.88+2 1039.48+2 666.65+3 999.48+2 617.26+2 1284.11+2 856.40+3

897.34 808.31+2 816.30+2 688.23+3 714.90+3 704.26+3 482.24+3 851.10+3 1276.18+2 1359.16+2 765.72+3 760.04+3 1139.56+2 1148.08+2 906.43+3 692.99+3 1039.00+2 749.00+3 1123.01+2 709.57+3 1063.84+2 1055.83+2 1036.47+2 664.64+3 996.48+2 614.25+2 1281.10+2 854.40+3

indication of the peptide sequence and possible sites of phosphorylation was achieved by cross-matching with an in silico tryptic digest of phosphorylated Aurora A. Insights into Aurora A Phosphorylation Kinetics in E. coli. In order to achieve better understanding of Aurora A phosphorylation kinetics, a time course analysis was performed. Four samples were prepared according to protocol a (Scheme 1) except that the final incubation in heavy medium was varied, with the values 0, 2, 10, and 21 h. Table 4 shows the ratios of heavy and light signal intensities as observed at the different time points for one representative peptide, 64 LVpSSHKPVQNQK75. Because there are no eukaryotic phosphatases in E. coli, the pattern is that light protein (with no 18O in the backbone) is phosphorylated with light phosphate during incubation in H216O and with heavy phosphate during incubation in H218O. The H/L ratio increases upon transfer to heavy water and then plateaus in response to either saturation of the phosphorylation site or cell death. Differences in the H/L ratio can reflect differences in the order of phosphorylation. Figure 6 shows the incorporation of 18O-labeled phosphate at pT288 and pS41 after 10 h of incubation in heavy water. For both phosphorylation sites, the two signals making up the doublets are the same shape, indicating that these are true doublets. T288 is important in regulating the activity of Aurora A17 and the data were searched explicitly for evidence of a peptide containing this residue. Figure 6a shows the spectrum of the peptide 286RTpTLCGTLDYLPPEMIEGR304. Among all the identified sites, T288 showed the lowest H/L ratio (0.58), which indicates that the majority of phosphate incorporation takes place into the site during the incubation period in the light medium. Further, the nonphosphorylated peptide was not detected, suggesting that phosphorylation was essentially complete by the end of the experiment. This finding is consistent with the biological importance of this site; phosphorylation is required for Aurora A activity and is therefore expected to be an early event. In contrast, S41 showed the highest H/L ratios among all identified phosphorylation sites (H/L 1.23, Figure 6b). High H/L ratios could be an indication of late or slow phosphorylation as most of the phosphate is incorporated

a

Peptides shown in boldface type were selected by data-dependent analysis for fragmentation, and site of phosphorylation was confirmed. The remaining peptides were not selected for fragmentation because of their low intensities. Phosphorylation of these peptides was confirmed by the presence of doublet or triplet signals for singly and doubly phosphorylated peptides, respectively. Peptides were crossmatched with an in silico digest of phosphorylated Aurora A, but the site of phosphorylation was not assigned because of lack of MS/MS data.

Figure 4. LC ESI-Q/ToF data of Aurora A tryptic phosphorylated peptide 98SKQPLPpSAPENNPEEELASK117 with m/z 1123.01+2. (a, top panel) Control sample showing the light signals of both peptides. (b, bottom panel) Sample prepared according to the reverse 18O-labeling protocol. The sample shows doublets separated by 3.00 Da as a result of the double charge of the peptide and representing incorporation of P18O3 group. 7389

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Figure 5. LC ESI Q-ToF data of Aurora A doubly phosphorylated tryptic peptide 80VLCPpSNpSSQRVPLQAQK96 with m/z 1036.47+2. (a, top panel) Control sample showing light version of the doubly phosphorylated peptide. (b, bottom panel) 18O-Labeled sample prepared according to the reversed protocol, showing triplet signal corresponding to the incorporation of 2PO3 at m/z 1036.47+2, PO3/P18O3 at m/z 1039.48+2, and 2P18O3 at m/z 1042.50+2. The species in which both phosphate groups are 18O-labeled is of low abundance and overlaps with the envelope caused by 18O labeling of the peptide backbone.

To overcome these limitations and to enhance detection of phosphorylated peptides by mass spectrometry, several approaches have been developed. Generally, these approaches can be classified into affinity enrichment and chemical derivatization methods; mass spectrometric techniques have also been optimized for protein phosphorylation analysis.1 These methods provide complementary data. Each of these methods has both advantages and disadvantages and provides identifications that differ from other methods. Thus there is no method of choice for protein phosphorylation analysis and there is a continuing need for the development of new methods to overcome the limitations of currently available methods. Recently, a novel approach based on in vitro generation of protein phosphorylation sites by use of γ-[18O4]ATP and normal (unlabeled) ATP was developed.6 The phosphorylated peptides were then observed by mass spectrometry, as doublets separated by 6.02 Da. The main advantage of this method is that it provides a definite identification of phosphorylated peptides, avoiding the false positive identification accompanying many other methods. The labeling reaction, however, has to be done in vitro; phosphoproteins undergo dephosphorylation followed by rephosphorylation in the light and heavy mixture of ATP. In the present study we employ an in vivo 18O-labeling method, avoiding the in vitro dephosphorylation/rephosphorylation step. The method has been successfully applied to labeling the phosphorylation sites of Aurora A kinase overexpressed in E. coli. Phosphorylated peptides were readily observed by mass spectrometry as doublets or triplets separated by 6 Da. Following LC/MS/MS analysis of an unlabeled control sample searched against a human taxonomy from the Swissprot database using MASCOT and manual peptide mass fingerprinting, 13 phosphorylated peptides representing 10 phosphorylation sites were identified. As mentioned earlier, the probability of false positive identification of phosphorylated peptides from database searching is one of the main drawbacks of this approach. The labeled sample confirmed the

Table 4. Heavy to Light Ratios (H/L) of the Phosphopeptide 64 LVpSSHKPVQNQK75 a time of incubation in heavy medium (h)

ratio of intensities of signals (H/L)

0 2 10 21

0 0.56 1.67 1.57

a

Light m/z was 1444.76+1 and heavy m/z was 1450.78+1. Ratio of the corresponding nonphosphorylated peptide to the sum L + H is also shown. Signal intensities were used to calculate the ratios. Samples were incubated in 18O medium for 2, 6, 10, and 21 h.

during the incubation period in labeled medium. This phosphorylation site is not reported to be important in Aurora A in vivo.

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DISCUSSION Mass spectrometry has been widely applied for the analysis of proteins in proteomic research. Its high sensitivity and accuracy make it the method of choice for such analysis. The analysis of phosphorylated proteins is, however, especially challenging. Although it is estimated that about 50% of mammalian proteins are phosphorylated, the modification is seldom stoichiometric; furthermore, phosphorylated peptides are commonly detected with poorer sensitivity than their nonphosphorylated counterparts. Moreover, phosphorylated serine and threonine residues tend to lose their phosphate groups as a result of activation by CID or MALDI methods. This results in a fragmentation pattern that is usually dominated by the neutral loss ion, with low backbone fragmentation impeding accurate and efficient analysis of the acquired data. Database searches of mass spectra of phosphorylated peptides2 require variable modifications on Ser, Thr and Tyr residues to be specified. This significantly expands the size of the database against which raw data are searched, leading to higher chances of false positive detections. 7390

dx.doi.org/10.1021/ac301038u | Anal. Chem. 2012, 84, 7384−7392

Analytical Chemistry

Article

Figure 6. (a, top panel) LC ESI Q-ToF data of Aurora A doubly charged tryptic phosphorylated peptide 286RTpTLCGTLDYLPPEMIEGR304with m/z 1151.51. H/L was calculated to be 0.56, indicating that most of the phosphorylation occurred during incubation in the light medium. (b, bottom panel) LC Q-ToF ToF data of Aurora A doubly charged tryptic phosphorylated peptide VLVTQQFPCQNPLPVNpSGQAQR with m/z 1281.08. H/L was calculated to be 1.23, indicating that most of the phosphorylation occurred in the heavy medium.

identification of these peptides as phosphopeptides, as 16O/18O doublets were detected. In addition, the labeled sample revealed another 15 phosphorylated peptides not previously identified. Because their signals were relatively weak, many of the newly identified phosphorylated peptides were not selected for fragmentation- hence, the identity and exact site of phosphorylation were not confirmed. However, the m/z values of the observed doublets and triplets were cross-matched with an in silico tryptic digest of phosphorylated Aurora A, and a list of possible phosphorylated peptides was generated (Table 3). The selectivity of this method increases the probability of identifying all the occurrences of a given phosphorylation site in a sample including peptides containing missed cleavages; this is important for applications such as absolute quantification of phosphorylated peptides. The selectivity is achieved despite the loss of signal intensity due to labeling; both the doublets due to 18 O-phosphate labeling and the envelopes due to incorporation of 18O into the protein backbone have the effect of reducing the signal intensity. Identification of the labeled phosphorylated peptides in this study was performed manually. Although this approach is time-consuming compared with database searches, it permits greater sensitivity when a major criterion for a “hit” is the presence of a doublet. Preliminary analysis of Aurora A phosphorylation kinetics by the 18O-labeling method showed variation in the regulation of kinase phosphorylation sites. T288, the well-recognized biological active site, appears in phosphorylated form at early stages following Aurora A synthesis, while phosphorylation of S41 occurs later in the experiment. This may reflect the importance of phosphorylation at T288 relative to phosphor-

ylation at S41, which is not known to have a specific biological function. We anticipate that the protocol will be quite robust for the study of phosphorylated proteins overexpressed in E. coli. However, the successful application of this approach relies on protein synthesis being faster than phosphorylation. The 10 or 17 h time delay between induction and the change of medium to H218O was optimized for Aurora A overexpressed in E. coli and could require modification if the method is to be applied for other phosphoproteins. This study shows a proof of concept of the possibility of selective 18O labeling of protein phosphorylation sites using Aurora A overexpressed in E. coli. There is no apparent reason (other than cost) why the method should not be applied to more physiologically relevant systems, in, for example, mammalian cells. Each phase of the protocol (Scheme 1) will require optimization. Because mammalian cells contain phosphatases, phosphate groups will undergo turnover, and time courses will be especially informative. Future directions could focus on applying this method to in vivo phosphorylated proteins with the purpose of enhancing their detectability. Furthermore, the application could be extended to explore the kinetics of phosphoproteins by monitoring the rate of label incorporation into phosphorylation sites in normal and abnormal cellular states. In conclusion, a sensitive, novel method of selective 18O labeling of phosphorylation sites based on H218O has been developed. The method has been applied successfully for the labeling of Aurora A phosphorylated sites overexpressed in E. coli. 7391

dx.doi.org/10.1021/ac301038u | Anal. Chem. 2012, 84, 7384−7392

Analytical Chemistry

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Article

AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected]. Present Address †

Queen Mary University of London, London E1 4NS.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS W.M.A. was supported by King Abdulaziz City for Science and Technology. We are grateful to Dr. Patrick Eyers for providing Aurora A construct.

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