Quantitative Analysis of Protein Complex Constituents and Their

Quantitative Analysis of Protein Complex Constituents and Their Phosphorylation States on a LTQ-Orbitrap Instrument Ce´dric Przybylski,†,‡ Martin A. Ju ¨ nger,§ Johannes Aubertin,| Franc¸ois Radvanyi,| Ruedi Aebersold,§ and Delphine Pflieger*,†,‡ Laboratoire Analyse et Mode´lisation pour la Biologie et l’Environnement, Universite´ d’Evry-Val-d’Essonne, 91025 Evry, France, CNRS UMR 8587, Laboratoire d’Analyse et de Mode´lisation pour la Biologie et l’Environnement, Universite´ d’Evry-Val-d’Essonne, 91025 Evry, France, Institute for Molecular Systems Biology, ETH, Zu ¨ rich, Switzerland and Faculty of Science, University of Zurich, Zurich, Switzerland, and E´quipe Oncologie Mole´culaire, UMR 144 - CNRS, Institut Curie, 75248 Paris, France Received April 30, 2010

Cellular functions are largely carried out by noncovalent protein complexes that may exist within the cell as stable modules or as assemblies of dynamically changing composition, whose formation and decomposition are triggered in response to extracellular stimuli. The protein constituents of complexes often exhibit post-translational modifications such as phosphorylation that can impact their ability to interact with other proteins and thus to form multicomponent complexes. A complete characterization of a particular protein complex thus requires determining both, the identity of interacting proteins and their covalent modifications, in terms of attachment sites and stoichiometry. We have previously developed a protocol which identifies genuine constituents of partially purified protein complexes and concurrently determines their phosphorylation sites and levels in a single LC-MS/MS analysis performed on a MALDI-TOF/TOF instrument (Pflieger, D.; Junger, M. A.; Muller, M.; Rinner, O.; Lee, H.; Gehrig, P. M.; Gstaiger, M.; Aebersold, R. Mol. Cell. Proteomics 2008, 7, 326-346). The method combines fourplex iTRAQ labeling (isobaric tags for relative and absolute quantification) and phosphatase treatment of peptide samples derived from the tryptic digestion of isolated complexes. To test the performances of this method with nanoESI and different peptide fragmentation modes, possibly better suited for the identification of phosphorylated sequences than MALDI-TOF/TOF-MS, we have implemented it on the nanoESI-LTQ-Orbitrap instrument. The model protein beta-casein was used to optimize the conditions with respect to sensitivity and quantitative accuracy: a combination of CID fragmentation in the linear ion trap and Higher energy Collision Dissociation (HCD) appeared optimal to obtain reliable and robust identification and quantification data. The optimized conditions were then applied to identify and estimate the respective levels of phosphorylation sites on the purified, autoactivated tyrosine kinase domain of Fibroblast Growth Factor Receptor 3 (FGFR3-KD) and to analyze complexes formed around the insulin receptor substrate homologue CHICO immunopurified from Drosophila melanogaster cells that were either stimulated with insulin or left untreated. These new analyses allowed us to improve the assignment of the phosphorylation sites of some peptides previously detected by MALDI-TOF/TOF analysis and to identify additional phosphorylated sequences in CHICO and in the insulin receptor. Keywords: protein complexes • phosphorylation site • phosphorylation stoichiometry • LTQ-Orbitrap • iTRAQ • HCD • Insulin Receptor Substrate • CHICO • Fibroblast Growth Factor Receptor 3

Introduction Mass-spectrometry (MS)-based methods have been used to study affinity-purified protein complexes on a large scale and * To whom correspondence should be addressed. Delphine Pflieger, LAMBE UMR CNRS 8587, Universite´ d’Evry Val d’Essonne, Baˆtiment Maupertuis, bd F. Mitterrand, 91025 Evry, FRANCE. Email: delphine.pflieger@ univ-evry.fr. † Laboratoire Analyse et Mode´lisation pour la Biologie et l’Environnement, Universite´ d’Evry-Val-d’Essonne. ‡ CNRS UMR 8587, Universite´ d’Evry-Val-d’Essonne. § University of Zurich. | Institut Curie.

5118 Journal of Proteome Research 2010, 9, 5118–5132 Published on Web 07/22/2010

to establish proteome-wide interaction networks in several organisms.1-4 These studies provided an impressive picture of the organization of protein modules within cells. Yet only a static representation was thus obtained, and usually no information was collected on the post-translational modifications (PTMs) decorating the proteins that constitute complexes. To distinguish true protein interactors from contaminants interacting nonspecifically with the affinity matrix in such experiments, frequently a list of “background proteins” that copurify with multiple complexes is generated, and these proteins are then subtracted from the proteins identified from a particular 10.1021/pr1003888

 2010 American Chemical Society

Quantitative Analysis of Protein Complex Constituents

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complex, on the grounds of their apparently low specificity. However, some proteins may be contaminants of certain complexes and genuine interactors of baits in the case of other purified modules. Moreover, the composition of protein complexes can vary depending on the cellular environment (external stimuli, etc.) and can be correlated with dynamically induced or removed PTMs, among which phosphorylation has been shown to be particularly relevant.5-7 Methods involving MS have been designed to provide a more in-depth characterization of specific protein complexes partially affinitypurified from total cellular extracts. The core principle of these methods consists of using semiquantitative MS to distinguish the proteins truly associated with a bait from the possibly numerous copurified contaminants; such a differential analysis also monitors changes in the composition of protein complexes.8,9 The sample of interest is most often obtained from cells expressing a tagged version of the targeted protein (bait), whereas the control sample is purified from cells transfected with the tag or from wild type (WT) cells.8,10 Alternatively, the two samples to be compared can be obtained from WT cells and cells submitted to RNA interference against the bait, which allows searching for interactors of endogenous proteins.11 The comparative analyses either rely on a method of differential labeling of the two samples, using for example ICAT,8 SILAC10 or iTRAQ reagents,12 or are performed using a label-free semiquantitative strategy.13 To get a further level of protein complex description, methods were designed to additionally characterize their state of phosphorylation, in terms of sites and relative13 or absolute12 levels. In reference,12 we have described a strategy to determine, in a single LC-MS/MS analysis performed on a MALDI-TOF/TOF instrument, the identity of true protein partners of a given bait and their phosphorylation sites and respective stoichiometries. The procedure similarly estimates the possible quantitative variations of interaction partners and phosphosites upon cell stimulation. It consists of a differential iTRAQ labeling of the proteolytic peptides generated from a sample of interest and from an adequate control sample, combined with the enzymatic dephosphorylation of one-half of each peptide mixture. Using the four iTRAQ reagents, we could thus characterize in particular the protein composition and phosphorylation state of complexes formed around the protein CHICO, the Drosophila melanogaster ortholog of the Insulin Receptor Substrate (IRS), and their variations upon cell treatment with insulin. Here, we adapted and applied the method on the nanoESI-LTQOrbitrap instrument, which could be expected to provide highly complementary results to the MALDI-TOF/TOF and better detection and identification of phosphopeptides. In a first step toward adapting the procedure to the LTQ-Orbitrap instrument, we optimized acquisition parameters to provide high-sensitivity peptide identification and accurate quantitative data. When using an instrument equipped with an additional higher energy collisional dissociation (HCD) cell, peptide identification can be obtained by either classical CID in the linear ion trap (LIT) or by fragmentation in the HCD cell which is followed by detection of the produced fragments in the Orbitrap analyzer.14 Low-mass iTRAQ reporter groups, on which quantification relies, can be detected by Pulsed-Q Dissociation (PQD) in the LIT of stand-alone ion trap instruments15-17 and of the hybrid LTQ-Orbitrap configuration,18 or by HCD in the latter instrument.19,20 When using a LTQ-Orbitrap instrument, either fragmentation type, PQD or HCD, has been estimated to provide better quantification in different reports.19,21 In the

scope of our method, we established that the combined acquisition of a CID scan in the LIT and an HCD scan, from every selected precursor, provided the largest number of peptide identifications and very robust quantitative information. These findings are in agreement with other recent reports.19,22,23 Our final analytical procedure consists of two consecutive LC-MS/MS runs, a first exploratory one and a second one targeting probable phosphorylated/dephosphorylated peptide pairs. After being optimized on the test protein beta-casein, the method was applied to the study of the tyrosine kinase domain (KD) of the fibroblast growth factor receptor 3 (FGFR3) to address more specifically the case of phosphotyrosine modification, and of the complexes centered on the Drosophila protein CHICO, for direct comparison with our previous MALDI-TOF/TOF data. While HCD conditions were optimized to provide high-accuracy quantitative data that are robust over long time periods, we also show the utility of the resulting spectra to confirm phosphosite localization, based on the detection of the immonium ions specific of Tyr or phosphorylated Tyr. The development of our method on the nanoESI-LTQ-Orbitrap also provided more in-depth characterization of the complexes centered on CHICO, in particular with the identification of additional phosphorylated sequences. Residues detected to be (partially) phosphorylated within a peptide sequence are indicated in bold font and with a star: S*. (ST)*: one of the residues in parentheses bears the phosphorylation.

Experimental Procedures Materials. Beta-casein (90% purity, ref C6905), DL-dithiothreitol (DTT), Tris (2-carboxyethyl)phosphine (ref C-4706) and Methyl methanethiosulfonate (MMTS) were purchased from Sigma-Aldrich (St. Louis, MO). HPLC gradient grade acetonitrile and normapur grade formic acid were purchased from VWR (West Chester, PA). All buffers and solutions were prepared using ultrapure water (Milli-Q, Millipore, Bedford, MA). Calf intestinal phosphatase (CIP) had been purchased from Fermentas (ref EF0341, 1 unit/µL). Sequencing-grade modified porcine trypsin (EC 3.4.21.4), Shrimp Alkaline Phosphatase (Cat. 8201) (SAP) and Thermo Sensitive Alkaline Phosphatase (Cat. M9910) (TSAP) were purchased from Promega (Madison, WI). The iTRAQ reagent kit was obtained from Applied Biosystems (Foster City, CA); the iTRAQ buffer (0.5 M triethylammonium bicarbonate, pH 8.5) used for digestion and labeling was part of this kit. ZipTipC18 tips were purchased from Millipore (Billerica, MA). Preparation of the Casein Sample. A solution of beta-casein at 100 µg protein in 100 µL of iTRAQ buffer was heated at 95 °C for 5 min. Five microliters of trypsin at 0.4 µg/µL was added to the sample for overnight incubation at 37 °C. The sample was split into two halves to be labeled with iTRAQ reagents 114 and 115, respectively, following Applied Biosystems instructions. After labeling, the samples were dried in a speedvacuum centrifuge and peptides were resuspended in 100 µL of SAP buffer provided with this enzyme. Hydrolysis of the excess of iTRAQ reagents was allowed to proceed at ambient temperature for 30 min, before adding 2 µL of CIP and 2 µL of SAP to the 115-labeled sample. After incubation at 37 °C for 2 h 15min, samples were heated at 85 °C for 20 min to denature the phosphatases. Both samples were pooled, and the mixture was redigested with 4 µL of trypsin at 0.4 µg/µL, by incubation at 37 °C for 2 h, to proteolyze the phosphatases. Journal of Proteome Research • Vol. 9, No. 10, 2010 5119

research articles Preparation of Chico3 Sample. Affinity-purified protein complexes assembled around the protein CHICO were obtained from Drosophila Kc167 cells stably expressing an HA-tagged version of the protein. Complexes were isolated from cells which were either stimulated with insulin for 7 min or left untreated. The detailed purification procedure and sample treatment before LC-MS/MS analyses were described elsewhere.12 The final obtained sample was named Chico3. Preparation of the FGFR3-Kinase Domain (FGFR3 KD) Sample. The coding sequence of the kinase domain of FGFR3 (FGFR3 KD, amino acid residues 439-806) was subcloned into pFastBacHTc baculovirus expression vector (Invitrogen, Carlsbad, CA). The FGFR3 KD protein was expressed in Sf9 insect cells as a hexa-histidine-tagged protein and purified by affinity chromatography on Ni-NTA beads. The hexa-histidine tag was removed using AcTEV-protease (Invitrogen, Carlsbad, CA) followed by a second affinity chromatography on Ni-NTA beads. To generate phosphorylated FGFR3 KD, the purified protein was incubated with 1 mM ATP, 5 mM MgCl2, 5 mM MnCl2 and 50 mM Tris (pH 7.5) for 1 h at room temperature. Phosphorylated FGFR3 KD was stored at -80 °C until use. Ten microliters of iTRAQ buffer were mixed with an equal volume of purified FGFR3 KD sample. The protein was reduced by incubation for 1 h at 60 °C in the presence of 5 mM DTT and then alkylated by addition of 10 mM MMTS and incubation for 10 min at room temperature. Ten microliters of acetonitrile were added to obtain a final concentration of 40% organic solvent. Digestion was performed by addition of 1 µL of trypsin at 0.4 µg/µL and incubation for 6 h at 37 °C. After evaporation of acetonitrile in a speed-vacuum centrifuge, the sample was thoroughly desalted on a ZipTipC18 column, to remove any residual Tris buffer. After concentrating the sample to 10 µL in a speed-vacuum centrifuge, 20 µL of iTRAQ buffer was added, and the sample was divided into 2 equal parts (15 µL each). For labeling, each iTRAQ reagent (114 and 115) was dissolved in 35 µL ethanol and added to one sample half. Vials were incubated for 1 h at room temperature and then totally dried in a speed-vacuum centrifuge. Twenty microliters of SAP buffer was added to each sample to hydrolyze the remaining iTRAQ reagents (30 min at room temperature). Half a microliter of both SAP and TSAP at 1 U/µL was introduced in the vial containing the 115-labeled sample. Vials were incubated for 2 h 30 min at 37 °C to allow dephosphorylation and then for 15 min at 85 °C to deactivate the phosphatases. The contents of two vials were mixed together, and an additional digestion step with 1 µL of trypsin at 0.4 µg/µL was performed to cleave phosphatases (2 h incubation). Finally, the sample was concentrated down to 20 µL. Reversed Phase-LC-MS/MS Analysis on a LTQ-Orbitrap Instrument. NanoLC-MS/MS analyses were performed on a Dual Gradient Ultimate 3000 chromatographic system (Dionex). Approximately 2 pmol of casein, 2 pmol of FGFR3 KD or the equivalent of 1/9 of affinity-purified Chico sample was injected onto a C18 precolumn (Acclaim PepMap C18, 5 mm length × 300 µm I.D., 5 µm particle size, 100 Å porosity, Dionex). After desalting for 5 min with buffer A (water/acetonitrile/formic acid, 98/2/0.1, v/v/v), peptide separation was carried out on a C18 capillary column (Acclaim PepMap C18, 15 cm length ×75 µm I.D. × 3 µm particle size, 100 Å porosity, Dionex) with a gradient starting at 100% solvent A, ramping to 70% solvent B (water/acetonitrile/formic acid, 10/90/0.1, v/v/v) over 70 min (50 min for the casein sample), then to 100% solvent B over 2 min (held 10 min), and finally decreasing to 100% solvent A in 5120

Journal of Proteome Research • Vol. 9, No. 10, 2010

Przybylski et al. 3 min. The column was finally re-equilibrated with 100% solvent A for 15 min. The LC eluent was sprayed into the MS instrument with a glass emitter tip (Pico-tip, FS360-50-15-CE20-C10.5, New Objective, Woburn, MA). The LTQ-Orbitrap XL mass spectrometer (Thermo-Fisher Scientific) was operated in positive ionization mode. Singly charged species were excluded from fragmentation; dynamic exclusion of already fragmented precursor ions was applied for 90 s, with a repeat count of 1, a repeat duration of 30 s and an exclusion mass width of (5 ppm. The minimum MS signal for triggering MS/MS was set to 500. In all scan modes one µscan was acquired. The Orbitrap cell recorded signals between 400 and 1600 m/z in profile mode with a resolution set to 15 000 in MS mode. During MS/MS scans, CID and MSA fragmentation and detection occurred in the linear ion trap analyzer in centroid mode. HCD fragmentation was performed with an activation time of 30 ms, in profile mode, with a resolution set to 7500. The automatic gain control (AGC) allowed accumulating up to 3 × 105 ions for FTMS scans, 105 ions for FTMSn scans and 104 ions for ITMSn scans. Maximum injection time was set to 500 ms for both FTMS and FTMSn scans and 100 ms for ITMSn scans. Additional information on MS acquisition methods is provided in the results section. When comparing the performances of MS2 and MSA for the identification of casein phosphopeptides, 1 µscan was acquired in all cases. MSA scan was triggered on the fragment corresponding to the heaviest mass loss among 32.67, 49, 65.33, 98, 130.67 and 147 Da (( 0.5 Da) on the condition that the fragment had an intensity >25 and belonged to the 5 most intense fragments. The precursor selection window was 2 Da in MS2 and 3 Da in MSA. Database Searches and Relative Quantification. Raw data files were processed using the software Bioworks 3.3.1 SP1 to obtain Mascot-compatible .MGF files. No grouping of CID MS2 scans obtained on the same selected precursor was performed; this allowed us to assess the variation in precursor mass measurement accuracy over different scans. Database searches were performed using the Mascot server v2.2.1 with the following parameters. For the casein sample: database Swiss-Prot (release version 54.7) restricted to mammalian taxonomy; enzymatic specificity, semitryptic with one allowed missed cleavage; possible phosphorylation of S, T and Y residues; 5 or 10 ppm tolerance on precursor masses, as indicated in the text; 0.8 Da tolerance on fragment ions in CID mode and 0.02 Da in HCD mode; fragment types taken into account were those specified in the configuration “ESI-trap”. For the FGFR3 KD sample: same as those utilized for casein, except that all cysteines were alkylated with MMTS, methionine could be oxidized and the tolerance on precursor masses was set to 10 ppm. For the Chico3 sample: same as for FGFR3 KD, except that Swiss-Prot was restricted to the Drosophila melanogaster taxonomy and oxidation of methionine residues was not considered. Phosphorylated sequences identified in FGFR3 KD and in CHICO were carefully checked by comparing the two first peptide matches assigned by Mascot to each MS2 or MSA spectrum. These matches usually differed in terms of phosphosite localization. As described by Tweedie-Cullen et al.,24 a normalized delta score was calculated as the score difference between the first and second match, divided by the first match score. When this delta score was below 0.4, MSn spectra were visually inspected to search for y/b discriminating ions and validate


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Figure 1. (a) Procedure of sample preparation used to compare the protein composition of two samples and characterize their phosphorylation sites. (b) When one sample is analyzed according to the workflow utilizing iTRAQ reagents 114 and 115, the following iTRAQ reporter ion patterns can be generated during MS/MS fragmentation of different precursor ions: (A) a nonmodified peptide is unaffected by the phosphatase treatment and will thus give rise to iTRAQ reporter groups 114 and 115 of equal intensities. (B) In the case of a phosphopeptide, in vitro dephosphorylation leads to the disappearance of the iTRAQ 115-labeled precursor; if dephosphorylation is complete, only the iTRAQ 114 signal is observed in the MS/MS spectrum. (C) Correspondingly, when the dephosphorylated peptide ion is fragmented, the MS/MS spectrum will contain a more intense iTRAQ 115 signal due to the in vitro phosphatase treatment. The intensity of this peak depends on the abundance of the initial phosphopeptide and the yield of the phosphatase treatment. If there is also a detectable iTRAQ 114 signal in the same MS/MS spectrum, this is derived from peptide that was initially nonphosphorylated in the sample and therefore allows conclusions about the level of phosphorylation of the considered protein region.

the most probable phosphosite localization. Remaining ambiguities in site assignment are indicated by parentheses. The areas of iTRAQ reporter groups detected in HCD scans were retrieved using the software Bioworks. A Sequest database search was performed, using a sufficiently large database (usually Swiss-Prot, digested into semitryptic peptides, with possible phosphorylation on S, T and Y residues) to allow matching a theoretical sequence to all HCD spectra. The areas of iTRAQ reporter groups could thus be easily retrieved from all HCD scans, with a tolerance on iTRAQ reporter group masses of (0.009 Da. This SequestQuan information was matched to the peptide sequences identified by Mascot from CID data using a homemade Perl script using the information scan(HCD) ) scan(CID) + 1.

Results The method we have developed to characterize in detail a protein complex in terms of genuine partners and their sites and levels of phosphorylation is outlined in Figure 1. Briefly, to highlight proteins truly enriched in a partially purified protein complex as compared to an adequate control sample, or to detect changes in the complex composition between two

cellular conditions, a semiquantitative analysis of the two compared protein samples is obtained from iTRAQ differential labeling. To determine the phosphorylation sites and levels decorating the protein complex constituents, the protocol additionally involves a treatment with phosphatases of the samples (see ref 12 for full details). More precisely, each protein sample (sample of interest and control, or two samples isolated from cells at different states) is digested by trypsin, split in two halves, for each half to be differentially labeled with iTRAQ reagents 114 and 115 (or 116 and 117). The odd-labeled halves are then treated in vitro with phosphatases, and the peptide mixtures are pooled again to be analyzed by LC-MS/MS on a nanoESI-LTQ-Orbitrap instrument. Specific iTRAQ group signatures are obtained depending on the nature of the fragmented peptide. When considering in more detail the sample labeled with iTRAQ reagents 114 and 115, three distinct iTRAQ group patterns can be observed, which inform on the phosphorylation state of the fragmented sequence (Figure 1b): initially nonphosphorylated in the original protein, intact phosphorylated sequence or peptide dephosphorylated by the action of phosphatases. When a pair of phosphorylated/ dephosphorylated peptides of same sequence is fragmented, Journal of Proteome Research • Vol. 9, No. 10, 2010 5121

research articles it is possible to estimate the level of phosphorylation x in the intact protein at that particular site, using the following equation: x ) (r - 1)/(r - 1 + y) where r is the ratio of iTRAQ reporter group areas measured in the dephosphorylated peptide form (subscript dP): (A(115)dP)/ (A(114)dP); and y is the yield of enzymatic dephosphorylation estimated in the phosphorylated peptide form (subscript P): 1 - (A(115)P)/(A(114)P). Analogous equations apply to peptide samples labeled with iTRAQ reagents 116 and 117. Finally, the relative abundance of a protein between the two compared samples is obtained while calculating the average ratio [A(116) + A(117)]/[A(114) + A(115)] over all peptides identifying that specific protein. The results obtained using the procedure described above are organized in four main parts. First, we optimized the LC-MS/MS analysis conditions on the LTQ-Orbitrap instrument to yield both sensitive peptide identification and accurate iTRAQ-based relative quantification. Second, we assessed the performance of the method on beta-casein as a standard phosphoprotein. Then two additional samples of more biological significance were characterized, namely the kinase domain of human FGFR3 and affinity-purified complexes centered on the Drosophila protein CHICO. 1. Preliminary Remark: Impact of iTRAQ Labeling on Mass Measurement Accuracy. Very high mass measurement accuracy is achieved when ions are analyzed in the Orbitrap cell: precursor ions are commonly detected with a mass tolerance e5 ppm, even with a few-day-old external calibration. However, because iTRAQ reagents are not perfectly isobaric, the mass measured for a given peptide sequence labeled with different tags will slightly vary. If an average mass of 144.1021 u is specified for iTRAQ modification in the database search, the mass of a peptide which actually carries reagent 114 only (and has incorporated one label only) will be underestimated by 3.8 mmu. This represents 3.8 ppm for a total peptide mass of 1000 Da. The same reasoning is valid for 115-labeled peptides. Peptides carrying only one label may thus escape identification when searches are run at 5 ppm precursor mass tolerance. Then, for all analyses aiming at detecting as many phospho- and dephosphorylated peptides (only 114- or 115labeled, respectively) as possible, database searches were performed with 10 ppm mass tolerance. However, for the initial analyses of casein samples which were meant to optimize fragmentation conditions (i.e., mainly the HCD activation value), searches were performed at 5 ppm tolerance in MS mode. We indeed estimated quantification accuracy provided by HCD fragmentation from peptides which were nonmodified in the original proteins (A(115) ≈ A(114)). 2. Optimization of the LC-MS/MS Acquisition Scheme. A peptide sample prepared following the procedure of Figure 1, using either two or four iTRAQ labels, is first submitted to an “exploratory” LC-MS/MS analysis, in such conditions that a maximum of peptide sequences are reliably identified and quantified. We then first sought to establish an optimized LC-MS/MS method to simultaneously obtain most sensitive peptide identification and most accurate relative quantification. Efficient detection of iTRAQ reporter groups, of masses between 114.1 to 117.1 Da, is usually not feasible by classical CID fragmentation in the LIT. The introduced PQD mode allows generating, stabilizing in the LIT and finally detecting small mass fragment ions and is thus suitable for detection of iTRAQ 5122


Przybylski et al. 15,16

labels. However, the optimized parameters are reported to vary significantly from one instrument to another and even need to be redefined after tuning of the LTQ-Orbitrap.15,16,19,21 Alternatively, HCD fragmentation can be performed in the additional octopole collision cell located after the C-trap.14 Nevertheless, HCD fragmentation is less sensitive than CID fragmentation, due to the different detection mode and probably to the extra travel ions need to cover (i.e., from the LTQ to the HCD cell, including C-trap focusing). This implies that HCD fragmentation cannot provide the most in-depth characterization of a peptide sample. We then tested the performance of a method combining CID and HCD fragmentation of peptides by LC-MS/MS analysis of beta-casein, which had been prepared following the procedure of Figure 1. The method was built as 7 successive scans: (i) one MS scan performed in the Orbitrap; MS/MS scans in CID and HCD modes acquired on the most intense ion detected in the previous MS scan and (iii) repeat (ii) on the second and third most intense ions detected in the MS scan. The obtained results are detailed in Supporting Information S1. Briefly, the number of peptides identified from HCD spectra was always lower than that yielded by CID scans which supported our choice of combining CID and HCD scans on all precursors, in agreement with other recent reports.19,23 Besides, varying HCD NCE values in several series of LC-MS/MS analyses run at weeks intervals oriented us toward the NCE of 65% as the optimal value to get the highest intensity iTRAQ reporter groups and low RSD on semiquantitative measurements. The final scheme of exploratory LC-MS/MS analysis thus consisted of one MS scan acquired in the Orbitrap, followed by three pairs of (CID, HCD) scans on the top three precursors, the latter scan being obtained at a NCE of 65%. The exploratory analysis of a sample treated following the protocol of Figure 1 allows identification of a majority of nonmodified peptides showing iTRAQ ratios A(115)/A(114) close to 1, whereas a few may exhibit iTRAQ ratios A(115)/ A(114) significantly above 1 (>2). The latter nonmodified sequences then appear to be probably phosphorylated in the intact proteins. Some of these sequences may also be identified in a phosphorylated form in the exploratory analysis, with corresponding MS/MS spectra showing the characteristic iTRAQ reporter group pattern A(114) . A(115). To conclusively identify these phosphorylated sequences and estimate their modification level, we designed a second LC-MS/MS analysis that would meet the following requirements: (i) systematically acquire fragmentation spectra on the putative phosphorylated counterparts of all the identified sequences showing a A(115)/ A(114) ratio .1, (ii) use optimized fragmentation conditions to obtain most reliable phosphopeptide identifications and (iii) increase the reliability of quantitative measurements on the selected phospho/dephospho peptide pairs. First, to target fragmentation of nonmodified sequences exhibiting high A(115)/A(114) ratios and their putative phosphorylated counterparts, parent mass lists to be specifically fragmented were designed: the m/z values (typically with z ) 2 and 3) of the nonmodified peptides of interest and of all the possible corresponding phosphorylated forms (from 1 to n times phosphorylated, n being the number of S/T residues contained in the peptide) were calculated and imported into the acquisition method. Only ionic species whose m/z ratios matched values of this list at a ( 20 ppm tolerance were allowed to be selected for fragmentation. Second, we asked which LIT scanning mode would provide best phosphopeptide

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Quantitative Analysis of Protein Complex Constituents a

Table 1. Phosphopeptides Characterized in a Targeted LC-MS/MS Analysis of the Beta-Casein Sample

protein

Beta-casein

peptide

RELEELNVPGEIVES*LS*S*S*EESITR VPGEIVES*LS*S*S*EESITR VPGEIVES*LS*S*S*EESITR FQS*EEQQQTED S*EEQQQTEDELQKD S*EEQQQTEDELQKD FQS*EEQQQTEDELQKD FQS*EEQQQTEDELQKD alpha S1 casein DIGS*ES*TEDQAMEDIK DIGS*ES*TEDQAMEDIK QMEAES*IS*S*S*EEIVPNSVEQK VPQLEIVPNS*AEER YKVPQLEIVPNS*AEER alpha S2 casein NTMEHVS*S*S*EES*IISQETYK NANEEEYSIGS*S*S*EESAEVATEEVK EQL(STS)*EENSKK EQLS*TS*EENSKK TVDMES*TEVFTK TVDMES*TEVFTK

z(PO4) z(nonPO4)

/ / / / 3 3 2 3 / / / 2 3 / / 3 2 2 2

3 2 3 2 2 3 2 3 2 3 3 2 3 3 3 3 3 2 3

yield of dephosphorylation yi

0.80 0.80 0.96 0.95 0.95 0.82 1 1 0.93 0.93

0.83 0.83 0.96 0.96 0.96 0.85 1 1 0.96 0.96

0.83 0.83 0.94 0.95 0.94 0.88 1 1 0.97 0.97

iTRAQ ratio A(115)/A(114) ri

inf. inf. 7.71 inf. inf. 8.59 40.5 45.9 inf. 8.20 170 inf. 20.8 16.0 inf. inf. inf. 8.55 24.5

inf. inf. 12.7 inf. inf. 10.8 54.0 48.7 inf. 9.48 161 11.9 23.7 23.3 inf. inf. inf. 11.3 19.0

inf. inf. 9.07 11.7 inf. 10.3 55.1 50.0 inf. 10.3 147 inf. 25.3 18.7 inf. inf. inf. 11.9 15.9

estimated level of phosphorylation xi(%)

100/100/100 100/100/100 87.0/92.1/89.0 100/100/91.4 100/100/100 90.4/92.2/91.8 97.6/98.2/98.3 97.9/98.0/98.1 100/100/100 87.8/89.5/90.3 99.4/99.4/99.3 100/91.9/100 96.1/96.4/96.5 93.8/95.7/94.7 100/100/100 100/100/100 89.0/91.5/91.9 96.2/94.9/93.9

a This method included the acquisition of CID MS2 and MSA scans and three HCD scans on putative pairs of phosphorylated and de-phosphorylated peptides. z: charge state. The charge states of the de-phosphorylated peptide (z(nonPO4)) and the corresponding phosphopeptide ions (z(PO4)) that were considered for estimation of the phosphorylation level are indicated. When a given peptide ion (m/z, z) was fragmented several times, the quantification data relative to the CID spectrum which provided the best identification score was considered. Inf.: infinite ratio (no signal was detected for label 114). yi ) 1 - A(115)/A(114) measured on phosphospecies; ri ) A(115)/A(114) measured on de-phosphorylated species in the three successive HCD scans (i ) 1, 2, 3); xi: phosphorylation level estimated from the pair (yi, ri).

identification, in terms of sequence determination and phosphosite localization. The recent literature appears controversial: when comparing LC-MS/MS methods acquiring either MS2, MS2 + MS3, or MSA scans on complex phosphopeptide samples, either MS2 25 or MSA26 was preferred on the hybrid LTQ-FT or LTQ-Orbitrap instruments. Since our LC-MS/MS analysis specifically targeting the putative pairs of phosphorylated/dephosphorylated peptides did not need to be optimized in terms of cycle time, we decided to combine the acquisition of MS2 and MSA scans on all precursors present in the parent mass list. Third, to improve the reliability of quantification data, three repeated HCD scans were acquired on each selected precursor. In conclusion, the targeted LC-MS/ MS analysis designed to better characterize putative pairs of phosphorylated/dephosphorylated peptides consisted of: MS2, MSA and three HCD scans specifically acquired on all precursors present in the parent mass list incorporated in the acquisition method. 3. Results Obtained on the Beta-Casein Sample with Combined Exploratory and Targeted Analyses. The betacasein sample prepared following the protocol of Figure 1 was analyzed by exploratory and targeted analyses, using the optimized conditions established above. This study aimed to check the ability of our method to identify the known phosphosites and to provide repeatable estimations of phosphorylation stoichiometry. Since the beta-casein protein used was 90% pure, R-S1 and R-S2 caseins were also detected. In a single exploratory LC-MS/MS analysis, as much as 91, 61 and 72% sequence coverage could be reached for these three proteins, respectively. Table 1 indicates the phosphopeptides identified and quantified. As expected, most peptides appeared to be fully phosphorylated in the intact proteins. We provided more digits to characterize phosphorylation levels than is biologically meaningful (for example 98.9%), with the goal to highlight the excellent repeatability of quantitative measurements provided by the three consecutive HCD scans, in agreement with a

previous report.21 In addition, estimations of the phosphorylation levels provided by 2+ and 3+ species of the same sequence are highly coherent. The specific case of the doubly phosphorylated peptide EQLS*TS*EENSKK from RS2-casein is of special interest because it was identified in nonmodified, singly and doubly phosphorylated forms. Strikingly, the singly modified form exhibited a 114-iTRAQ signal, which indicated that part of the original protein bore a unique phosphorylation motif in the considered region. It is difficult to obtain the respective proportions of proteins that are singly and doubly modified at these sites using our method (see Supporting Information S2 for explanations). However, we can estimate that (nearly) 100% of the protein is modified in this sequence, given that ratios ri ) A(115)/A(114) measured on the dephosphorylated sequence are infinite. In addition, when observing the signals detected in MS for the singly and doubly phosphorylated species, the ratio of their respective chromatographic peak areas was around 50-100, thus indicating that the singly modified version was most probably of very minor abundance. 4. Analysis of a Phosphotyrosine-Containing Sample: FGFR3-KD. The previous optimization steps were performed on proteins containing phosphorylated serine residues. In contrast to phospho-serine/-threonine residues, phosphotyrosine (pTyr) residues do not give rise to the loss of phosphoric acid during CID MS/MS. In addition, the presence of pTyr in a fragmented sequence is often associated to a specific immonium ion at m/z 216.043, which usually escapes detection in ion trap analyzers but can be tracked as a marker of pTyr in HCD spectra.14 To study how the method optimized for Ser/ Thr phosphorylation performed with pTyr-containing peptides, we analyzed the kinase domain (KD) of the receptor tyrosine kinase Fibroblast Growth Factor 3 (FGFR3-KD). This protein domain was allowed to autophosphorylate in the presence of ATP and we sought to identify which Tyr residues became modified and to what extent. Journal of Proteome Research • Vol. 9, No. 10, 2010 5123

research articles The trypsinized protein was first analyzed by an initial exploratory LC-MS/MS analysis that allowed the identification of a list of Tyr-containing sequences, some of which were in a phosphorylated state. A sequence coverage of 88% for FGFR3KD could be reached. Second, the sample was submitted to a targeted analysis, to more systematically identify pTyr-containing peptides and estimate their phosphorylation stoichiometry. For this second analysis, we established a parent mass list by combining the m/z ratios (with z ) 2, 3, 4 or 5) of theoretical tryptic sequences and of the few semitryptic peptides that had been identified during the initial exploratory analysis; the list contained both m/z values of nonmodified and phosphorylated sequences and was established while considering up to N sites of phosphorylation, with N being the number of S/T/Y residues in the sequence. These m/z values were then specifically targeted to be successively fragmented by MS2, MSA and by a triplicate of HCD scans. We decided to further complete the targeted analysis with two exploratory analyses, also consisting of the series of MS2, MSA and a triplicate of HCD scans: the latter runs provided a few additional identifications, of a few peptides not considered in the inclusion list (such as with an oxidized methionine or an unanticipated proteolytic cleavage), or possibly not selected in the targeted analysis due to inadequate detection of their ionic signal (see Discussion). Table 2 primarily presents the results provided by the targeted analysis but also shows the additional peptide identifications yielded by the two other runs. The detailed identification and quantification results separately obtained in the three runs are provided as Supplementary Table S1 (Supporting Information). Interestingly, very different phosphorylation levels could be detected, ranging from about 85% for residue Tyr760, down to about 20% for residues Tyr599 and Tyr607. When a given peptide was identified at different charge states, the various ionic species usually provided coherent quantification results. Similarly, the detection of methionine-containing sequences, with this residue in a nonmodified or an oxidized form, yielded consistent quantitative results. We systematically checked that the immonium ion diagnostic for pTyr had been detected in HCD scans of identified pTyrmodified sequences; it is worth noting that its relative intensity within the MS/MS spectra varied greatly, from 12 up to 100%, which illustrated the high dependence of immonium ion formation on the peptide sequence. The detection of the immonium ions corresponding to unmodified Tyr or pTyr at theoretical m/z 136.076 or 216.043, respectively, helped confirm the localization of the phosphorylation moiety. Indeed, the very long sequence spanning residues Val751-Arg805 was detected in three forms: nonmodified, singly and doubly phosphorylated. Whereas Tyr760 appeared clearly nonmodified in the singly modified sequence (from a readable stretch b8-b15), definite positioning of the phosphorylation site between Tyr770 and Ser771 was impossible. The HCD spectra acquired on that species exhibited both immonium ions expected for nonmodified and phosphorylated Tyr residues, which oriented the modification on Tyr770 (Figure 2). The doubly modified sequence was intriguingly interpreted by Mascot as being phosphorylated on residues Tyr760 and Ser764. We validated the phosphorylation on Tyr760 based on the detection of signals at 1090.35 and 1333.42 Da, corresponding to the fragments b9 and b10 that differed by the mass of pTyr (243.07 Da measured). Careful inspection of the MS/MS spectrum obtained on the doubly modified peptide indicated that the positioning of the second phosphorylation moiety on Ser764 5124


Przybylski et al. was based on fragments which were already detected in the MS/MS spectrum acquired on the sequence singly modified at Tyr770. Finally, in the three consecutive HCD scans obtained on the doubly modified peptide, no signal about m/z 136.076 was detected, whereas a clear signal at m/z 216.043 ( 0.001 was systematically detected. We then preferred to conclude that the doubly phosphorylated sequence was modified at both Tyr760 and Tyr770. In terms of phosphorylation levels, the yields of dephosphorylation at both Tyr residues appeared to be equal to 1, and it was only possible to calculate a global modification level over both sites (see Supporting Information S2 for explanations). Because A(115)/A(114) was calculated to be infinite in the nonmodified sequence, it is possible to conclude that the sequence stretch Val751-Arg805 was fully modified (at least singly phosphorylated at Tyr770) in the intact protein. The sequence DVHNLDYYK, which contains the two consecutive Tyr residues of the (auto)catalytic domain, was detected in the two possible singly modified forms (at either residue Tyr647 or Tyr648) and in the doubly modified form. Interestingly, peptide DVHNLDYY*K gave rise to a chromatographic peak whose area was approximately ten times lower than that of peptide DVHNLDY*YK, which probably indicates that Tyr647 is preferentially phosphorylated before the subsequent phosphorylation of Tyr648. Given the high ratio A(115)/ A(114) (around 5-7) observed on the nonmodified peptide DVHNLDYYKK, the vast majority of the protein is phosphorylated within this sequence stretch. It is however problematic to determine the modification levels at both sites, all the more so as the formation of the two tryptic peptides DVHNLDYYK and DVHNLDYYKK complicates quantitative estimations. 5. Analysis of Protein Complexes Formed around the Protein CHICO. When we initially developed the method on the MALDI-TOF/TOF instrument, we analyzed protein complexes assembled around the Drosophila insulin receptor substrate (IRS) ortholog CHICO, which were immuno-purified from cultured cells either stimulated with insulin (sample labeled with iTRAQ reagents 114 and 115) or left untreated (sample labeled with reagents 116 and 117). We sought to detect (i) possible variations in the abundance of CHICO interaction partners, the insulin receptor (dInR) and the two proteins 143-3 epsilon and zeta, and (ii) possible variations in the phosphorylations decorating CHICO. The sample called Chico3 in our previous study was analyzed here on the nanoESI-LTQOrbitrap using the optimized method described above, to compare the new results to the quantitative measurements obtained by MALDI-TOF/TOF, to confirm or invalidate previously identified phosphosites and to evaluate whether additional phosphorylated peptides could be detected. To answer the question whether insulin signaling modified the composition of complexes formed around the bait protein CHICO, sample Chico3 was analyzed by an exploratory LC-MS/ MS analysis: an MS scan in the Orbitrap was followed by pairs of (CID/HCD) scans acquired on the three most intense ions detected in MS. For all identified proteins, the ratio [A(116) + A(117)]/[A(114) + A(115)] averaged over their proteolytic peptides was then calculated (we only considered peptides identified with scores above the Mascot threshold indicative of identity or extensive homology, p ) 0.05). The quantification results are provided in Table 3 and are compared to those previously obtained on the same sample analyzed on the MALDI-TOF/TOF instrument. An increased association of the two 14-3-3 epsilon and zeta proteins upon stimulation with

724

MDKPANCTHDLY*MIMR + Metox

5

4 5 2 3 2 2 2 3 2 2 / 3 4 3 3 3 2 2 3 4 2 3 3 2 2 5

z(non-PO4)

/

/ 49 / /

63 22 37 / 53 31 53 29 44 28 / 17 15 / / 28 30 21 /

17

77

/ 38 / 63

45 52 37 / 70 31 58 26 27 22 / 14 16 / / 29 20 20 /

16

scores PO4

54 (MS2) 37 (MSA) 64 (MS2) 47 (MS2) 54 (MS2) 53 (MSA) 49 (MS2) 56 (MS2) 49 (MS2) 49 (MS2) 38 (MS2) / 32 (MS2) 38 (MS2) 38 (MS2) 42 (MSA) 24 (MS2) 73 (MS2) 68 (MS2) 69 (MS2) 50 (MSA) 46 (MS2) 53 (MSA) 73 (MS2) 44 (MSA) 36 (MSA)

score nonPO4

1.00

/ 1.00 / 1.00

0.92 0.77 0.46 0.86 / 0.95 0.90 0.89 0.90 0.95 0.93 / 0.89 0.94 / / 0.95 0.94 0.77 /

1.00

/ 1.00 / 1.00

0.94 0.78 0.62 0.88 / 0.93 0.89 0.90 0.92 0.95 0.93 / 0.91 0.96 / / 0.95 0.95 0.84 /

yi

1.00

/ 1.00 / 1.00

0.94 0.81 0.48 0.85 / 0.96 0.88 0.90 0.92 0.95 0.93 / 0.92 0.95 / / 0.94 0.96 0.82 /

/

/

1.58 1.79 1.45 1.79 1.43 1.74 1.67 1.37 7.36 4.10 inf.

1.82 1.59 1.43 1.90 1.68 2.12 1.93 1.15 inf. inf inf.

ri

2.17 inf. 1.09 1.19 1.28 1.17 1.44 1.84 1.44 1.44 / 6.17 6.00 6.17

3.72 2.65 1.29 1.30 1.18 1.21 1.38 1.94 1.38 1.38 / 5.56 5.14 5.56

/

1.93 1.55 1.60 1.68 1.53 1.97 1.89 1.31 3.75 3.88 inf.

2.40 3.86 1.19 1.17 1.36 1.23 1.46 1.79 1.46 1.46 / 5.37 5.90 5.37 >45 >37 31 49 47 >53 >48 >13 100 100 100(a)

75 64 27 39 17 >17

xi (%)

>37 >44 32 45 34 >43 >40 >27 86 >76 100(a)

55 100 10 24 24 >15

>48 >35 39 42 39 >49 >47 >23 73 >74 100(a)

60 75 19 26 29 >19

>43 >38 34 45 40 >48 >45 >21 86 >83 100

63 80 19 29 23 >17

mean x

a Same abbreviations as in Table 1 are used. For phosphopeptides, both the Mascot identification scores attributed to the MS2 and MSA scans are successively indicated; for their non-modified counterparts, only the higher score provided by either fragmentation mode is indicated. (a): this peptide was detected in both a singly and doubly phosphorylated forms. Since both phosphospecies were observed to be fully de-phosphorylated by phosphatases (y ) 1 in both instances), it is only possible to estimate a global modification level, which appears to be 100% here (see Supporting Information S2 for calculations). In other words, this sequence is fully modified within the protein.

4

648 647-648 724 724 724

DVHNLDYY*KK DVHNLDY*Y*KK CTHDLY*MIMR MDKPANCTHDLY*M MDKPANCTHDLY*MIMR

760, 770

648 647-648 647

DVHNLDYY*K DVHNLDY*Y*K DVHNLDY*YKK

/ 2 / 4

607 647

GMEY*LASQK GMEY*LASQK + Metox DVHNLDY*YK

2 3 2 / 2 3 2 2 2 3 / 3 3 / / 2 3 4 /

5

z(PO4)

724 760 760 770

599

DLVSCAY*QVAR

PANCTHDLY*MIMR VLTVTSTDEY*LDLSAPF VLTVTSTDEY*LDLSAPFE VLTVTSTDEYLDLSAPFEQY*SPGGQDTP SSSSSGDDSVFAHDLLPPAPPSSGGSR VLTVTSTDEY*LDLSAPFEQY*SPGGQDTP SSSSSGDDSVFAHDLLPPAPPSSGGSR

577

Tyr residue

RPPGLDY*SFDTCKPPEEQLTFK

peptide

Table 2. Phosphotyrosine-Containing Peptides Identified in the Kinase Domain of Human FGFR3a


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Figure 2. Continued

insulin was again clearly observed; a moderately increased association with the insulin receptor was also detected. The analysis on the LTQ-Orbitrap instrument provided relative quantification with a higher number of reliably identified peptides: 12 additional proteins could be identified and quantified with at least 3 different peptides. In particular for the protein CHICO, the numerous identified peptides corresponded to a sequence coverage of 78%. 5126


We next studied the impact of insulin stimulation on the phosphorylation pattern of CHICO. The previous exploratory analysis identified a list of nonmodified sequences for which A(115)/A(114) . 1 and/or A(117)/A(116) . 1, as well as some phosphorylated peptides. An LC-MS/MS analysis targeting all the m/z values (z ) 2, 3) of the corresponding phospho/ dephospho peptide pairs was carried out, consisting of an MS scan in the Orbitrap, fragmentation in the LIT and a triplicate

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Figure 2. CID fragmentation spectra acquired during the LC-MS/MS analysis of the kinase domain of FGFR3 which targeted putative groups of phosphorylated/dephosphorylated sequences. MultiStage Activation (MSA) spectra of three differentially phosphorylated peptide ions of same sequence are shown: (a) MSA spectrum of the nonphosphorylated peptide 751VLTVTSTDEYLDLSAPFEQYSPGGQDTPSSSSSGDDSVFAHDLLPPAPPSSGGSR805 (precursor m/z 1155.3474; z ) 5), (b) MSA spectrum of the monophosphorylated peptide 751 VLTVTSTDEYLDLSAPFEQY*SPGGQ DTPSSSSSGDDSVFAHDLLPPAPPSSGGSR805 (precursor m/z 1464.1753; z ) 4) and (c) MSA spectrum the diphosphorylated peptide 751VLTVTSTDEY*LDLSAPFEQY*SPGGQDTPSSSSSGDDSVFAHDLLPPAPPSSGGSR805 (precursor m/z 1484.1682; z ) 4). b0n is bn-H20, b*n is bn-NH3 and y0n is yn-H20. The insets show the HCD spectra acquired after the MSA scans on the three ionic species, zoomed over the m/z range 100-220 to highlight the detection of iTRAQ reporter groups, as well as the absence or presence of Tyr immonium (m/z 136.075) and pTyr immonium (m/z 216.042) ions.

of HCD scans. The obtained results are presented in Table 4. In most instances, the latter analysis provided phosphorylation levels in good agreement with the results obtained in the MALDI-TOF/TOF-based study. Similar effects were detected upon insulin treatment: increased phosphorylation levels of several peptides were observed, in particular for the sequences predicted by Scansite Motif Scanner (http://scansite.mit.edu/ motifscan_seq.phtml27) to be 14-3-3 protein recognition motifs. Only sequence CDS*LPTR provided here results in clear contradiction with the previous study, possibly due to the contamination of the phosphorylated and nonphosphorylated peptides of interest with coeluting species of close m/z ratios. We visually checked that the ionic signals identifying the pairs of phospho-/dephospho-peptides of CHICO were not more contaminated by other closely eluting species within a window of (2.5 Da (precursor selection width used here) than within (1.5 Da. Reducing the precursor width to 3 Da thus would not have improved the quantification data obtained on CHICO, and would have been detrimental to iTRAQ group areas (data not shown). The phosphosite localization in the peptide NGTLSESSNQTYFGS*NHGLR was corrected by the analyses on the LTQ-Orbitrap instrument (Figure 3): very poor MALDI-TOF/ TOF fragmentation had been obtained on that sequence and the phosphorylation had been placed within the stretch TLSESSNQTY based on the detection of very low intensity peaks. A refined localization of the phosphorylation moiety was deduced from ESI-MS/MS data for the peptide IS*QPELHYASLDLPHCSGQNPAK. Finally, two additional phosphorylated sequences were identified in the present study,

namely TDSSSLTLHATS*QK from CHICO and KS*PTNPNSGIGATGAGNR from the insulin receptor. In the HCD spectra obtained on the latter sequence (in its phosphorylated and nonmodified versions), iTRAQ reporter groups were not systematically detected. Globally, these fragmentation spectra were devoid of high-intensity fragments, probably due to the low amount/low ionization and fragmentation efficiencies of this pair of ionic species. The amount of the insulin receptor in the affinity-purified sample must be definitely much lower than the bait CHICO, as judged by the respective numbers of peptides identifying both proteins, which may explain the poor HCD spectra obtained on the above phospho/nonphospho peptides.

Discussion We have previously developed on a MALDI-TOF/TOF instrument a method to characterize partially purified protein complexes in terms of genuine interaction partners and phosphorylation sites and levels. We describe here how we transferred the method to the nanoESI-LTQ-Orbitrap instrument, by optimizing (i) the acquisition conditions to get highly robust analysis of iTRAQ-labeled peptides and (ii) the use of scanning modes to obtain best characterization of phosphorylated sequences. The final method scheme consists of analyzing a given sample by two LC-MS/MS runs: the first exploratory one acquires pairs of CID and HCD scans to combine sensitive identifications and accurate quantification data; the second one specifically targets m/z ratios corresponding to putative pairs Journal of Proteome Research • Vol. 9, No. 10, 2010 5127

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Table 3. Proteins Identified and Quantified with at Least 3 Peptides by the Exploratory LC-MS/MS Analysis of the Drosophila CHICO Complex (Sample Chico3)a protein

acc. number

noINS/INS Orbitrap (mean ( SD)

N

noINS/INS MALDI-TOF/ TOF (mean ( SD)

Insulin receptor substrate 1 (chico) 14-3-3 epsilon 14-3-3 zeta Insulin-like receptor precursor 60 kDa heat shock protein, mitochondrial Heat shock 70 kDa protein cognate 3 Heat shock 70 kDa protein cognate 4 Heat shock 83 kDa protein Elongation factor 1-alpha Actin-5C Actin-57B Actin-42A Tubulin alpha-1 Tubulin beta-1 chain Pterin-4-alpha-carbinolamine dehydratase Probable prefoldin subunit 2 Probable prefoldin subunit 3 Probable prefoldin subunit 5 rRNA 2′-O-methyltransferase fibrillarin Peroxiredoxin 1 Putative glycogen [starch] synthase 60S ribosomal protein L28 60S ribosomal protein L27a 60S ribosomal protein L13 40S ribosomal protein S2 Ubiquitin Heterogeneous nuclear ribonucleoprotein 27C

Q9XTN2 P92177 P29310 P09208 O02649 P29844 P11147 P02828 P05303/P08736 P10987 P53501 P02572 P06603 Q24560 O76454 Q9VTE5 Q9VGP6 Q9VCZ8 Q9W1V3 Q9V3P0 Q9VFC8 Q9VZS5 P41092 P41126 P31009 P68198 P48809

1.00 ( 0.24 0.57 ( 0.13 0.52 ( 0.12 0.79 ( 0.11 1.10 ( 0.18 1.12 ( 0.16 1.03 ( 0.17 0.93 ( 0.08 0.90 ( 0.14 1.34 ( 0.15 1.34 ( 0.15 / 1.06 ( 0.26 0.99 ( 0.10 0.71 ( 0.15 0.85 ( 0.10 0.96 ( 0.09 1.13 ( 0.62 1.23 ( 0.26 1.12 ( 0.10 1.31 ( 0.09 1.17 ( 0.08 1.34 ( 0.11 / 0.96 ( 0.17 1.06 ( 0.09 1.14 ( 0.11

104 54 30 26 11 8 30 8 8 10 9 / 3 6 13 5 5 4 7 4 3 3 4 / 3 5 3

1.00 ( 0.18 0.51 ( 0.13 0.41 ( 0.11 0.76 ( 0.18 0.98 ( 0.14 / 0.97 ( 0.14 / 0.69 ( 0.08 / / 1.17 ( 0.33 1.27 ( 0.23 0.99 ( 0.12 0.54 ( 0.08 0.74 ( 0.04 / / / / / / / 1.00 ( 0.07 / / /

N

32 18 12 12 6 / 19 / 5 / / 6 3 6 7 3 / / / / / / / 3 / /

a We only considered peptides that were identified with Mascot scores above the threshold indicative of identity/extensive homology (p ) 0.05; scores >29 in the database search conditions used for the LTQ-Orbitrap data); N designates the total number of peptides meeting this criterion.

of phospho/dephospho peptides, and combines MS2, MSA and a triplicate of HCD scans, to yield optimal phosphopeptide identification and reliable quantification. The new method was demonstrated to be suitable to study phosphorylations on S/T/Y residues, to provide repeatable measurements of phosphorylation stoichiometries, and to provide a more in-depth characterization of protein complexes than its MALDI-TOF/ TOF-based version. Performing the experimental strategy on either instrument, MALDI-TOF/TOF or nanoESI-LTQ-Orbitrap, presents advantages and drawbacks. The method applied on an ESI-MS/MS instrument provides more successful identification of multiply phosphorylated sequences (at least doubly modified peptides), which however remain not easily amenable to phosphorylation stoichiometry determination. The high mass accuracy of the LTQ-Orbitrap instrument makes it possible to efficiently target the fragmentation of m/z ratios of interest (m/z values corresponding to the theoretical pairs of phospho/dephosphorylated peptides) in an automated fashion: a parent mass list of selected ionic species can be established and specified in the acquisition method. However, if the charge state of a phosphorylated species could not be determined during the FTMS preview scan, or if its isotopic distribution pattern significantly deviated from the one expected for a peptide, it may not have been fragmented in our method, which used the MonoIsotopic Precursor Selection mode.28 In contrast, using a MALDI-TOF/ TOF instrument, we could fragment putative phosphopeptides even in the absence of any signal detected in MS. Indeed, knowing their theoretical m/z ratios and assuming LC retention times similar to their nonmodified counterparts, we could acquire readable MS/MS spectra in several instances where no 5128


precursor could be detected in MS. HCD fragmentation usually provided iTRAQ reporter groups of reasonably repeatable intensities but exceptionally happened to be devoid of these peaks, for example in the case of low-abundance species; the LTQ-Orbitrap-based method may then only provide the identification of a phosphorylated sequence (due to the high sensitivity of MS/MS scans in the LIT) but no information on the modification level. In contrast, MALDI-TOF/TOF analysis usually provided semiquantitative data even when no sequence could be read. Our method was successfully applied to the characterization of phosphorylated tyrosine residues in the kinase domain of FGFR3. On an analytical point of view, HCD fragmentation appeared very useful to confirm/invalidate the localization of phosphorylation motifs on Tyr residues, based on the detection of immonium ions at m/z 136.076 or 216.043. Our study also contributes to a better understanding of the functional roles of these residues, which are highly conserved within the members of the FGFR family,29 and appears complementary to several reports dealing with tyrosine phosphorylation of FGFRs. We observed the peptides containing the two tyrosines Tyr647 and Tyr648 in large part phosphorylated in FGFR3 KD after in vitro autophosphorylation. These residues in the activation loop are conserved in all FGFRs and their requirement for kinase activity has been shown for FGFR130 and FGFR3.31 In addition, we found tyrosine residues Tyr577 and Tyr760 to bear high phosphorylation levels. Tyr766, corresponding to Tyr760 in FGFR3, has been identified as the major autophosphorylation site in FGFR1.32 Beside the tyrosines in the activation loop, Tyr577 and Tyr760 have been previously identified as the most important tyrosine residues implicated

yes, slightly

yes, slightly yes none / yes, slightly yes (nonPO4) none yes (nonPO4) none yes, slightly (nonPO4) yes, slightly (PO4) none none yes, slightly none

contamination of precursors

score PO4

36 22 97 ND 50 45 83 38 ND 22 66 54 60 50 90

score non PO4

From the insulin receptor S2100 52 77

From CHICO S286-287 20 S300 24 258-260 33 / ND S555 30 T554-S555 35 S319 52 S342 29 S471 13 S471 22 S620 36 / ND / ND S813 33 S905 39

residue

NQ

25/36/25 20/36/23 90/90/90 / 75/74/78 71/74/79 39/57/49 NTY / 81/80/87 19/24/41 >23/10/4 >43/38/53 99/96/98 32/34/27

+INS

NQ

15/0/0 41/0/19 17/32/15 / 50/50/48 43/60/53 NTY NTY / 34/29/33 14/14/31 >62/52/55 >48/39/32 97/97/97 44/41/71

noINS

Chico3 (%)

/

S286 S300 S258-S260 Y501-S505 S555 S555 T307-T315 S342 S471 S471 / S784 S784 S813 S905-S920

residue

/

35 / 38 / 32 24 32 61 35 38 / 13 68 33 /

score PO4

/

49 ( 7 76 ( 10 76 ( 2 >71 ( 5 77 ( 2 83 ( 2 pbm NTY 76 ( 3 89 ( 2 / NTW 43 ( 4 90 ( 3 36 ( 20

/

none 10 ( 13 26 ( 19 >33 ( 11 56 ( 4 65 ( 4 pbm NTY 53 ( 10 48 ( 12 / NTW 53 ( 4 93 ( 1 82 ( 8

noINS

Chico3 (%) +INS

MALDI-TOF/TOF

a Localization of the phosphorylation sites determined from analyses on the MALDI-TOF/TOF and on the nanoESI-LTQ-Orbitrap is indicated in the columns named “residue”. For each peptide, we provide the higher identification score obtained from the exploratory and targeted LC-MS/MS analyses performed on the LTQ-Orbitrap.14-3-3 binding motif: mode 1 and mode 2 motifs that, when phosphorylated, constitute binding sites for 14-3-3 proteins, were predicted by the Scansite algorithm.27 Contamination: contamination of the isotopic distribution of the precursor ion of interest by another ionic species within a 5-Da window. None, no phosphorylation could be detected due to iTRAQ ratios below 1 measured on the de-phosphorylated species. ND, the peptide was not detected. NT, quantification not trusted; more precisely, NTW, the obtained spectrum was too weak to allow identification and/or reliable quantification; NTY, the yield of de-phosphorylation was too low to obtain a reliable estimate of the initial phosphorylation level; NQ, iTRAQ reporter groups were not systematically detected in the three consecutive HCD scans acquired on the same precursor, thus accurate quantification was not possible. Pbm, this peptide was detected in two distinct MALDI spots and its fragmentation provided incoherent quantification data.

/

Mode 1 Mode 1 Mode 2 Mode 2 Mode 2 Mode 2 / / / / / / / / /

SCS*SPHNYGFGR CDS*LPTR (SSS)*ANEASKPINVNVIQNSQNSLELR A(YSVGS)*K STS*APLLSLK KS(TS)*APLLSLK NGTLSESSNQTYFGS*NHGLR HSNS*PTFTMPLR AYS*IGNK AYS*IGNKVEHLK TDSSSLTLHATS*QK LVHSIS*SEDYTQIK KLVHSIS*SEDYTQIK ILQIKS*DSSLISSK IS*QPELHYASLDLPHCSGQNPAK

KS*PTNPNSGIGATGAGNR

14-3-3 binding motif?

peptide

nanoESI-LTQ-Orbitrap

Table 4. Phosphopeptides Identified in the Drosophila CHICO Complex (Sample Chico3)a


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Figure 3. Fragmentation spectra acquired during the targeted LC-MS/MS analysis of the affinity-purified Drosophila IRS ortholog CHICO (sample Chico3). CID MS/MS spectra of a pair of phosphorylated/dephosphorylated peptides of same sequence are shown: (a) MS2 spectrum of the nonphosphorylated peptide 305NGTLSESSNQTYFGSNHGLR324 (precursor m/z 771.6061; z ) 3), (b) MS2 spectrum of the singly phosphorylated peptide 305NGTLSESSNQTYFGS*NHGLR324 (precursor m/z 798.6945; z ) 3). dhA is dehydroalanine and y&n is yn-H3PO4. The insets show the HCD spectra, acquired on the ionic species after CID MS2/MSA scans, and zoomed on iTRAQ reporter ions.

in transformation mediated by activated FGFR3 in vivo and in vitro.33 In the same study, only minimal impact on transforma5130


tion efficiency has been observed for Tyr724 which was detected to be phosphorylated at less than 50% in our study.


research articles

Interestingly, we observed all tyrosine residues with described implication in full-length FGFR3-mediated signal transduction to a large extent autophosphorylated. This suggests that intrinsic kinase activity alone can be sufficient for activation of signal transduction by FGFR3 without requirement of other kinases.

modification; RSD, relative standard deviation; SAP, shrimp alkaline phosphatase; TSAP, thermo sensitive alkaline phosphatase; WT, wild-type.

The analysis of sample Chico3, the very same sample as the one previously characterized on a MALDI-TOF/TOF instrument, allowed comparing the results provided by the two analytical pipelines, including either the latter instrument or the LTQ-Orbitrap. At the protein level, comparable quantitative data were obtained, with a clearly detected increase in 14-3-3 proteins association with CHICO upon cell stimulation with insulin. The higher sensitivity of the LTQ-Orbitrap allowed obtaining measurements based on a higher number of peptides per protein, which strengthened the validity of their identification and quantification. At the peptide level, the present analyses allowed confirming most of the previously identified phosphopeptides and the evolution of their stoichiometry with insulin treatment. In some instances, the new MS/MS data provided more precise/corrected localization of the phosphosites and two previously undetected phosphorylated sequences were identified (TDSSSLTLHATS*QK in CHICO and KS*PTNPNSGIGATGAGNR in the insulin receptor). Both peptides have been detected in a previous large-scale phosphoproteome measurement and cataloged in the PhosphoPep database,34 however without quantitative information under which biological condition the phosphopeptide was observed, or about protein-protein interactions. In all phosphopeptides identified here, the modification was positioned on a Ser residue; for reasons which are not clear, no pTyr-containing sequence was detected. The low cellular abundance of phosphotyrosine (roughly 0.05%) compared to phosphoserine and phosphothreonine35 is unlikely to contribute to this because the analyses were performed on immunopurified CHICO. Maybe these phosphorylations on tyrosine residues occur at such low stoichiometry that we were not able to detect them. So far, others have identified three distinct pTyr-containing peptides from CHICO. In experiments employing the capture of phosphopeptides with immobilized antibodies against pTyr from tryptic digests, peptides containing pTyr64136 as well as pTyr316 and pTyr911 were detected (Bernd Bodenmiller, personal communication). The peptide harboring pTyr911 was also identified in a global phosphoproteome measurement which applied large amounts of starting material and peptide separation by isoelectric focusing, and has been archived in the PhosphoPep database. Tyr411 and Tyr641 have been functionally characterized in the context of insulin signaling,37 however Tyr411 has not yet experimentally been shown to be indeed phosphorylated. Abbreviations: CIP, calf intestinal phosphatase; DTT, dithiothreitol; FGFR3, fibroblast growth factor receptor 3; FTMS scan, MS scan acquired in the Orbitrap cell of the LTQ-Orbitrap instrument; FTMSn scan, MSn (usually MS2) scan acquired in the Orbitrap cell of the LTQ-Orbitrap instrument; HCD, higher energy collision dissociation; INS, insulin; IRS, insulin receptor substrate; ITMS scan, MS scan acquired in the LIT cell of the LTQ-Orbitrap instrument; ITMSn scan, MSn (usually MS2) scans acquired in the LIT cell of the LTQ-Orbitrap instrument; iTRAQ, isobaric tags for relative and absolute quantitation; KD, kinase domain; LIT, linear ion trap; MMTS, methyl methanethiosulfonate; MSA, multistage activation; NCE, normalized collision energy; PQD, pulsed-Q dissociation; PTM, post-translational

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