Extensive Phosphorylation Flanking the C-Terminal Functional

Extensive Phosphorylation Flanking the C-Terminal Functional Domains of the Measles Virus Nucleoprotein Emmanuel J. F. Prodhomme, Fred Fack, Dominique Revets, Patrick Pirrotte, Jacques R. Kremer, and Claude P. Muller* Institute of Immunology, Laboratoire National de Sante´ and Centre de Recherche Public-Sante´, 20A rue Auguste Lumie`re, L-1011 Luxembourg, Grand-Duchy of Luxembourg Received May 5, 2010

The measles virus nucleoprotein (vNP) is the first and most abundant protein in infected cells. It plays numerous important roles including the encapsidation of genomic viral RNA and the transcription of viral proteins. Intricate interactions with host cell proteins rely on the structural integrity of its functional domains. Although some of these functional domains are known, their structural features are still poorly understood. Here we identified multiple isoforms of measles vNP by two-dimensional differential gel electrophoresis (2D-DIGE) and 2D Western blot. These isoforms were further analyzed by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF)/TOF using MS (PMF) and MSMS (PSD) and electrospray ionization (ESI)-ion trap using LC-ESI-ion trap MS1, MS2 (neutral loss), MS3 (phosphosite). Both recombinant NP (rNP) and vNP were R-acetylated at the N-terminus. After tryptic or chymotryptic digestion, phosphopeptides were enriched and nine phosphorylation sites were identified and localized in the rNP, seven of which were also phosphorylated in vNP, probably by casein kinase 2. The phosphosites were all found within the intrinsically unstructured C-terminal domain. They clustered around functional domains involved in transcription and replication, as well as in sequences interacting with host-cell proteins. This underlines the importance of these post-translational modifications. Keywords: Paramyxoviridae • measles virus • nucleoprotein • post-translational modification • phosphorylation, mass spectrometry

Introduction Measles virus (MV) is a member of the Paramyxoviridae within the order Mononegavirales, a family of viruses that contains important human (mumps, parainfluenza, or respiratory syncytial virus) and veterinary (rinderpest, Newcastle disease or Sendai virus) pathogens.1 Measles virus causes a severe fever rash disease with frequent respiratory and other complications in infants and young children. Despite extensive vaccination campaigns the disease is not fully controlled and outbreaks continue to occur even in vaccinated populations. It remains the main cause of child mortality in developing countries and still accounts for approximately 200 000 annual deaths worldwide.2 MV is an enveloped RNA virus with a single-stranded, negative-sense, nonsegmented genome encoding the nucleoprotein (NP), the phosphoprotein (P), the matrix, fusion, hemagglutinin, and the large protein. The P gene encodes two extra accessory proteins, the C and V protein, by using alternative initiation codons or by site-specific RNA editing.3,4 The NP is the first and most abundant protein in infected cells, and its main function is to package the genomic RNA into a helical protein-RNA complex termed nucleocapsid, with a stoichiometry of one NP molecule for six nucleotides.5,6 In the * Corresponding author. E-mail: [email protected]. Phone: +352 490604 220. Fax: +352 490686.

5598 Journal of Proteome Research 2010, 9, 5598–5609 Published on Web 10/06/2010

cytoplasm of an infected cell, the NP is used by the viral RNA polymerase complex containing the large and the P protein, as a template for both the replication and encapsidation of the viral genome, as well as the transcription to mRNAs encoding the viral proteins.7,8 NP induces interferon9 and modulates other cellular genes; it has also been implicated in MV associated immune suppression.10 This protein is 525 amino acids long and can be divided into the N-terminal domain (400 aa), responsible for RNA binding and nucleocapsid formation, and a disordered C-terminal tail (125 aa), accessible on the nucleocapsid surface. Post-translational modifications (PTM) are important for protein activity. The NP is the prototype of a protein undergoing extensive PTMs including phosphorylation11-13 and limited proteolytic C-terminal truncation.14 Phosphorylation of NP was shown to play an important role in activation of transcription of viral mRNA and/or genome replication. In order to better understand the functions and the structural basis of NP interactions with viral and host cell proteins, we characterized its phosphorylation sites using a comprehensive mass spectrometry (MS) approach.

Materials and Methods MV Protein Extraction and Fluorescent Labeling. Vero cells were grown to subconfluency at 37 °C in RPMI 1640 (Gibco, Grand Island, NY, U.S.A.) supplemented with penicillin (100 10.1021/pr100407w

 2010 American Chemical Society

Phosphorylation of Measles Virus Nucleoprotein IU/mL), streptomycin (1 pg/mL), 5% fetal calf serum (or alternatively 2% Ultroser, Gibco), 2 mM glutamine, 50 pM 2-mercaptoethanol, and infected with MV (Edmonston strain, ATCC VR-24) at a multiplicity of infection of 0.3. On day 5, the supernatant was harvested. Cell debris were removed by centrifugation at 1500g and filtration through a 0.45 µm filter. The MV was concentrated and purified by ultracentrifugation.15 Concentrated MV particles were dialyzed against TBS 1× pH 7.5 for 3 × 2 h in a mini dialysis unit with a size cutoff of 3500 Da (Slide-a-lyzer, Pierce) at 4 °C in the presence of phosphatase and protease inhibitors (Roche Phosstop and Complete). After precipitation with 8 vol of acetone, proteins were pelleted by centrifugation at 13 000 rpm for 1 h at 16 °C in an Eppendorf centrifuge. The pellet was resuspended in a DIGE compatible lysis buffer (7 M urea, 2 M thiourea, 30 mM tris-HCl, pH 8.5, 2% ASB14). Total protein was quantified (2D Quant, GE Healthcare) and labeled with CyDyes (GE Healthcare) on lysine side chains using a dye/protein ratio ensuring minimal labeling. Two-Dimensional (2D) Gel Analysis of MV Proteins. Fluorescence labeled MV protein extracts (10 µg) were separated by isoelectric focusing (IEF) on 7 cm IPG strips of a pH range 4-7 (GE Healthcare). After IEF, the strips were equilibrated for 15 min in DTT and iodoacetamide buffers, before they were transferred onto a gradient ZOOM-IPG gel (Invitrogen) for the second dimension separation in MES buffer. Electrophoresis (Novex tanks) was continued for 30 min at 150 V/250 mA until the bromphenol blue had left the gel. For higher resolution up to 30 µg of MV protein was separated on 11 cm IEF strips (Biorad) and 4-12% Bis-Tris 2D gels (Criterion, Biorad). Gels were briefly washed in cold double distilled water (DDW) before scanning on a Typhoon 9400 flat bed fluorescence scanner with a lateral resolution of 100 µm, using the excitation and emission wavelengths recommended for the fluorescent dyes (Cy2, 491/ 509 nm). Prior to protein transfer, the images of the gel were scanned for comparison with the membrane image after Western blotting. 2D Western Blotting. After fluorescence scanning, the 2D gels (8 × 10 cm) were transferred to low-fluorescence PVDF membranes (GE Healthcare) using a semidry blotter (PHASE, Germany) at a current of 0.8 mA/cm2 and 7-9 V. The membranes were then washed in water for 10 min, dried for 1 h at 37 °C or overnight at room temperature, and either scanned or used for staining with antibodies. The membranes were blocked for 120 min in a washing buffer (TBS, 0.3% Tween 20) containing 2% milk. After appropriate washes, the monoclonal antibody BNP49 (0.125 µg/mL; recognizing the epitope Q111-E126)16 a Cy3-conjugated antimouse IgG (GE Healthcare, 1/5000 dilution) was added for 1 h. After three final washes, the membrane was rinsed in TBS without Tween 20 and dried before fluorescence scanning. 2D Gel Analysis of Recombinant NP (rNP). Recombinant nucleoprotein (rNP), derived from the MV Halle strain,17 was produced in insect cells (Sf9) infected by the NP-recombinant baculovirus AcNPVNP (a generous gift from Dr. F. Wild, Lyon, France) as described by Ravanel et al.18 The Halle and Edmonston virus strains used here differ only by three amino acids (LI78V, D25IN, A290D) in the C-tail of the protein (aa 400-525). Following ultracentrifugation on a cesium chloride gradient, rNP was affinity purified using a HiTrap NHS activated column (GE Healthcare) coupled with the NP specific mAb BNP49. After 2D electrophoresis (see above) of rNP (1-10 µg of protein), the gel was fixed for 1 h in a 50% methanol/10% acetic acid

research articles solution, washed in cold water, and stained overnight with the postelectrophoretic fluorescent protein stain Sypro Ruby (Invitrogen). 2D Gel Analysis of Recombinant NP (rNP) after Enzymatic Dephosphorylation. rNP (5.0 µg) was dephosphorylated by alkaline phosphatase (Roche), labeled with Cy3, and separated by 2D electrophoresis. Cy2 labeled BSA (75 ng) was added as an internal standard for the overlay with the untreated rNP. Preparation of MV-NP and rNP for Mass Spectrometry. MV protein extracts were separated by 4-12% Nu-PAGE (Novex, Invitrogen) under reducing conditions and aligned with Sypro Ruby stained rNP as a marker. Unstained proteins bands were excised from the gel, and the viral NP (vNP) was recovered by electroelution using D-Tube dialyser (Novagen). Concentrated rNP was used in solution without further fractionation. rNP (5-20 µg) or gel-purified vNP was resuspended in 20 µL of 50 mM NH4HCO3 (pH 8) and a 50/1 mass ratio of trypsin (recombinant porcine proteomics grade trypsin, Roche) or chymotrypsin (bovine sequencing grade chymotrypsin, Roche) was added; the digestion was carried out at 37 °C for 4 h. No reduction or alkylation was needed as the N protein contains only one cysteine. Phosphopeptide Enrichment and Dephosphorylation. Tryptic or chymotryptic phosphopeptides were enriched using a TiO2 coated stationary phase of a phosphopeptide enrichment kit (Phos-Trap, PerkinElmer) or an IMAC (ferric ions) phosphopeptide enrichment kit (Magnetic Phosphopeptide Enrichment Kit, Clontech) packed in a C4-reverse phase tip (Omix, Varian). Trapping and elution were performed with the PhosTrap kit buffers or with the Magnetic Phosphopeptide Enrichment kit buffers following the manufacturer’s instructions. Samples were then dried in a speed-vac and resuspended for further analysis by matrix-assisted laser desorption (MALDI) or electrospray ionization (ESI). For dephosphorylation, a tryptic digest of 2.5 µg of rNP was treated with 1 unit of alkaline phosphatase (rAPid alkaline phosphatase, Roche) in 5 µL of 50 mM NH4HCO3 (pH 8) at 37 °C for 1 h. MALDI-TOF MS and MS/MS Analysis of Peptides. Digested peptides were directly spotted onto a MALDI target plate (polished steel 384 target plate, Bruker) and 0.3 µL of the matrix solution (5 mg/mL of alpha-cyano-4-hydroxycinnamic acid, HCCA, Bruker and 1 mg/mL of 2,5-dihydroxybenzoic acid (DHB) Bruker in 50% ACN containing 0.1% TFA) was added to each spot according to the dried droplet method (Bruker manufacturer’s instructions). After an on-target washing step with 10 mM ammonium phosphate solution containing 0.2%TFA, the tryptic peptides mixtures were analyzed using a MALDI-TOF/TOF instrument (Ultraflex I, Bruker). Before each analysis, trypsin-digested bovine serum albumin was used for external calibration according to the manufacturer’s instructions. Peptides of interest were identified by peptide mass fingerprints (PMF) and by comparison with the MASCOT 2.0 database (Matrix Science, UK). The peak mass list was created using Flex Analysis 2.4 with the following parameters: signal-to-noise ratio of 3, a quality factor of 50, a window of analysis between 600 to 4000 m/z, an exclusion of classic contaminants (keratin, matrix cluster ions, sodium adducts, trypsin autolysis peaks), and a maximum number of selected peaks of 200. The mass list obtained was used for query in the virus subset of the MSDB 20060908 database (320 448 sequences), with the following parameters: charge state of +1, mass tolerance 150 ppm (necessary for phosphopeptide assignment), variable modificaJournal of Proteome Research • Vol. 9, No. 11, 2010 5599

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Figure 1. 2D analysis of NP. (A) 2D gel of partially purified MV protein extracts prelabeled with Cy2 (8 × 10 cm gel). (B) Detection of NP by 2D fluorescence Western blot using mAb BNP49 and a Cy3 labeled secondary antibody (same gel as in A). (C) Zoom on viral NP after high-resolution 2D gel (11 cm) and Western blot as in B, or (D) of recombinant NP after in gel staining with a postelectrophoretic fluorescent stain.

tions including phosphorylation on S, T, and Y, and tolerance for two missed cleavages. For the peptide 445-458 containing the residue S450 the peak-list for MS/MS data analysis was generated using Flex Analysis 2.4 (signal-to-noise ratio of 12 and a relative intensity threshold of 0%). The MS/MS database search (BioTools 3.0) was performed with a tolerance of three missed cleavages in the virus subset of the MSDB 20060908 database (320 448 sequences), the mass tolerance for precursor ions was set at 0.2 and 0.5 Da for fragment ions and the cutoff score was set to the default MASCOT threshold of 5%. NanoLC-ESI-IonTrap Analysis of Peptides. Digested peptides were analyzed on an HPLC-Chip/XCT Ultra Ion Trap 6340 (Agilent Technologies). Peptides were first desalted and separated by reverse phase using a nano HPLC-Chip featuring a 40 nL enrichment column and 43 mm × 75 µm analytical column packed with Zorbax 300SB C18 5 µm before being analyzed in an XCT Ultra Ion Trap via the HPLC-Chip/MS Cube interface (Agilent Data Analysis 3.4). The following multisegmented elution gradients were used for fractionation: 0% B-5 min, 0-25% B-15 min, 25-70% B-12 min, 70-100% B-3 min, hold 100% B-2 min for a total run of 37 min using as mobile phase mixtures of A (0.1% formic acid and 3% acetonitrile (ACN) in water) and B (0.1% formic acid and 10% water in ACN) at 400 nL/min. Mass spectra were acquired in the positive ion mode applying a data-dependent automatic switch between survey scan and tandem mass spectra (MS2) acquisition. Samples were analyzed with a “top 5” method, acquiring one ESI MS scan in the mass range of m/z 200-2000 followed by MS2 of the five ions with the highest MS signal. Dynamic exclusion was used with exclusion after two spectra and a release after 30 s. The XCT Ultra Ion Trap was used in MS3 mode and a neutral loss in MS2 was used as criteria to select candidates for MS3 analysis. NP peptides were identified by MS/MS ion search (raw MS/ MS data from multiple peptides) using MASCOT 2.0 (Matrix Science, UK) allowing for two missed cleavages in the virus subset of MSDB 20060908 database (320 448 sequences). The mass tolerance for precursor ions was set at 1.2 and 0.6 Da for fragment ions, and the cutoff score was set to the default MASCOT threshold of 5%. The identified NP (VHNZMH/gi: 127901/P10050) was digested in silico (PeptideMass, ExPASy Proteomics Tools) for future peak attribution. For MS/MS analysis, the fragments were assigned manually from in silico 5600

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fragmentation (ProteinProspector V5.2.2 - MS product from University of California San Francisco) using Y, b, and internal fragments with a maximum charge state set to +3 and a fragment tolerance of 300 ppm. MS3 data were acquired selectively in case of a neutral loss of 49 for double charged species and 32.6 for triple charged ions during MS2. Capillary voltage was set to 2100 V, nebulizer gas to 60 psi, dry gas to 4 L/min, dry temperature to 350 °C. Finally, the trap capacity was limited to 5 × 105 ions and the ions scanned between 200 and 1700 m/z at a speed of 8100 m/z per sec.

Results Proteins of MV particles purified from an infected cell culture supernatant and purified rNP were separated by 2D gel electrophoresis (7 cm, pH 4-7). Even after sucrose gradient purification the 2D gel of the MV particles (Figure 1A) showed a large number of contaminating proteins from the culture supernatant. NP was identified by 2D Western blot using mAb BNP49 and a Cy3-labeled secondary antibody (Figure 1B) as multiple confluent spots with a broad charge distribution. The detection of NP was more sensitive by Western blot than by fluorescence of prelabeled proteins, which showed only the most abundant NP protein spots. In addition to the intact form, C-terminally truncated species were detected at a higher PI (Supporting Information 1B). Our results confirmed the tryptic hypersensitive site after the position R413 (Supporting Information 2),12 near the interface between the globular N-terminal domain and the C-tail of the protein (Figure 2). Concerning the recombinant NP (rNP), in addition to the full-length protein, two C-terminally truncated forms (after the positions G433 and Q499) were observed by MALDI TOF in linear mode (Supporting Information 1A-1C). In a fluorescence Western blot of an 11 cm 2D gel up to eight fully resolved protein spots of full size viral NP were detected (Figure 1C). rNP showed a similar spot pattern with a similar charge distribution (Figure 1D). The two truncated forms did not exhibit the multiplicity of intense spots of the full-length protein indicating that most of the post-translational modifications are located in the C-terminus. The NP was further analyzed by mass spectrometry for post-translational modifications in order to explain this spot multiplicity. Phosphorylation of rNP. Different phosphorylation sites were identified by combining several mass spectrometry ap-

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Phosphorylation of Measles Virus Nucleoprotein

After digestion, phosphopeptides were enriched with a selfmade TiO2 enrichment tip19 or by IMAC (see Materials and Methods). The comparison of both IMAC and TiO2 enrichment techniques showed (Supporting Information 5, 6 and Table 1) that all phosphopeptides that were detected by IMAC were also detected by TiO2. In addition, the TIO2 enriched more overlapping phosphopeptides and fewer (and less abundant) copurifying unphosphorylated peptides. As a result of these observations, peptides enriched by TiO2 were further used for the final localization of the phosphosites. Phosphorylated and unphosphorylated peptides were discriminated on the basis of their sensitivity to alkaline phosphatase which removed most of the enriched peptides (Figure 3). In addition, the peptides sensitive to the alkaline phosphatase activity were further selected as a potentially phosphorylated candidate on the basis of an 80 Da (HPO3) mass difference compared to in silico predicted NP peptides. The phosphorylations were also confirmed by a metastable loss of phosphoric acid (H3PO4) corresponding to a 98 Da mass decrease during MS/MS. Phosphopeptides were also identified in ESI-ion trap-MS/MS by a neutral loss of 49 and 32.6 Da for double and triple charged peptides, respectively. The sensitivity to alkaline phosphatase treatment of the protein was also demonstrated by 2D gel (Supporting Information 7). At least six spots were visible before treatment with the enzyme. After treatment the number of spots was reduced to two main spots of increased intensity and a third weak spot. These two to three remaining spots can be explained by an incomplete phosphatase reaction, which is confirmed by MALDI-TOF MS (Figure 3B,D) performed on the same sample. Panels B and D show that several phosphopeptides (e.g., 1674.41 and 2789.32 Da) have not been fully dephosphorylated. Thus, the 2D gel (Supporting Information 7) and the MS analysis of the same sample are in full agreement, although we cannot totally exclude that another NP species comigrates with monophosphorylated NP.

Figure 2. Position of the phosphorylated sites in the C-terminal tail of rNP. (A) The underlined sequence fragments were not covered by tryptic or chymotryptic peptides. Hypersensitive tryptic site.12 (B) Peptides used for the positioning of phosphosites identification.

proaches. After tryptic and chymotryptic digestion, the purified recombinant protein was analyzed by MALDI-TOF/TOF and NanoLC-ESI-IonTrap. An example of MALDI PMF and LCNanoESI MS is shown in the Supporting Information 3 and 4. The MALDI PMF peak list of trypsin digested NP showed that the vast majority of peaks can be attributed to NP peptides. Protein identity was confirmed with sequence coverage of 95% (obtained by compiling all MALDI and ESI data). Table 1. Tryptic Phosphopeptides of Measles Virus Nucleoproteina

enrichment method

site

peptide enzyme

S425 414-431 trypsin S450 445-458 445-452 449-458 T471 464-489

sequence

TiO2

IMAC

QAQVSFLHGDQSENELPR

x x x x x x x x x x x x x x x x x x

x

trypsin trypsin trypsin trypsin

GEARESYRETGPSR GEARESYR ESYRETGPSR AAHLPTGTPLDIDTASESSQDPQDSR

T477 464-489 trypsin

AAHLPTGTPLDIDTASESSQDPQDSR

464-490 trypsin

AAHLPTGTPLDIDTASESSQDPQDSRR

S479 464-489 trypsin


464-490 trypsin S481 464-489 trypsin

AAHLPTGTPLDIDTASESSQDPQDSRR AAHLPTGTPLDIDTASESSQDPQDSR



464-490 trypsin S505 498-521 trypsin

AAHLPTGTPLDIDTASESSQDPQDSRR LQAMAGISEEQGSDTDTPIVYNDR


LQAMAGISEEQGSDTDTPIVYNDR

498-525 trypsin LQAMAGISEEQGSDTDTPIVYNDRNLLD a

x x x x x

x x x

x x x x x

MS method

m/z

MALDI ESI MALDI MALDI MALDI MALDI ESI MALDI ESI MALDI ESI MALDI ESI MALDI MALDI ESI MALDI ESI MALDI MALDI ESI MALDI ESI MALDI

2135.13 1059.9 1674.86 1047.51 1261.63 2949.46 984.2 2869.5 957.6 3025.63 1009.1 2789.51 930.1 2945.63 2869.47 957.7 2949.46 984.4 3105.61 2770.42 924.5 2690.41 897.9 3145.79

Mr (expt) Mr (calc) 2134.13 2117.8 1673.86 1046.51 1260.63 2948.46 2949.6 2868.5 2869.8 3024.626 3024.3 2788.51 2787.3 2944.63 2868.47 2870.1 2948.46 2950.2 3104.61 2769.42 2770.5 2689.41 2690.7 3144.79

2133.99 2117 1673.76 1046.46 1260.56 2948.26 2948.26 2868.26 2868.26 3024.36 3024.36 2788.26 2788.26 2944.36 2868.26 2868.26 2948.26 2948.26 3104.36 2769.2 2769.2 2689.2 2689.2 3144.44

phospho-rylation state mono di mono mono mono tri tri di di di di mono mono mono di di tri tri tri di di mono mono mono

Phosphorylated residues are shown in bold.

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Figure 4. Analysis of phosphopeptide 414-431 (QAQVSFLHGDQSENELPR). (A) MALDI MS spectrum of the unphosphorylated peptide (2055.04 Da) and the corresponding phosphorylated ion (2135.04 Da) (B) MALDI MS spectrum illustrating the spontaneous reaction of the N-terminal glutamine residue into pyroglutamate (structures shown) resulting in a loss of 17 Da.

Figure 3. MALDI-TOF MS spectra of TiO2-enriched tryptic NP peptides (A) before and (B) after enzymatic dephosphorylation using alkaline phosphatase. (C, D) Zoom of the boxed phosphopeptide rich region in A and B. Only masses of phosphopeptides and their amino acid positions are shown. Peptide 120-143 (2743.96 in panel D; 2744.02 in panel C) served as the internal control.

On the basis of these MSn data, 9 phosphorylation sites were identified in 12 tryptic phosphopeptides accounting for various miscleavage sites of the enzyme (Table 1). Four of the nine sites were further confirmed in a chymotryptic digest. In agreement with the 2D gel pattern (Figure 1C and Supporting Information 1B), all phosphorylation sites were found between S425 and S510 within the C-tail of the protein (Figure 2). Among the phosphosites, two were observed before enrichment (S425 and S510) (Supporting Information 3D,E and Figure 4), while the other phosphosites were visible only after enrichment of phosphopeptides. Phosphorylation of S425. The peptide molecular ion 414-431 with the sequence QAQVSFLHGDQSENELPR has two potential 5602


serine phosphorylation sites. The N-terminal glutamine residues, which can spontaneously cyclize to pyroglutamate with a mass loss of 17 Da, represent an additional complication. The resulting monocharged peptide has a mass of 2055.0 Da as M + H, of 2135.0 Da as a monophosphate, of 2037.9 Da as a pyroglutamate (Figure 4) and of 2118.0 Da with both modifications. The spectrum in Figure 4A was obtained from a nonenriched, freshly prepared sample to minimize pyroglutamate formation. Incidentally, the phosphopeptide 414-431 is isobaric with the nonphosphorylated peptide 355-371, but the pyroglutamate carrying peptide 414-431 has no isobaric equivalent in the tryptic digestion excluding possible misinterpreting of fragmentation spectra. Thus, to locate the phosphorylation site, a fragmentation spectrum was generated by CID using the nano-ESI ion trap on the pyro-Q phosphorylated peptide. The fragmentation spectrum was generated in MS3 by selection of the pyro-Q dephosphorylated parent ion in MS2 obtained in turn from the pyro-Q phosphorylated peptide detected in MS1 (Figure 5). The fragmentation spectrum of unphosphorylated peptides was generated in MS2. The obtained fragment y7+ ion of m/z 826,5 corresponds to a Nterminal dehydro-alanine resulting from the loss of phosphoric acid from phosphoserine 425 (Figure 5C) typical in CID fragmentation. Fragment ions can be unequivocally assigned to both the phosphorylated and nonphosphorylated peptides (Figure 5C,D), which demonstrate the phosphorylation at S425 by differences of 18 Da in the y series of ions starting from y7+. Phosphorylation of S450. A phosphorylated serine was localized in position 450 in three different tryptic peptides (Table 1). The phosphorylation site was identified in peptide


Figure 5. Phosphoserine S425 on peptide 414-431 (Q*AQVSFLHGDQSENELPR). Comparison of levels of fragmentation between the phosphorylated and unphosphorylated peptide using the ESI ion trap MS. (A) MS of phosphorylated peptide, (B) MS2 of phosphorylated peptide with neutral loss, (C) MS3 of phosphorylated peptide, (D) MS2 of unphosphorylated peptide. The y7+ ion assigns the phosphorylation to the residue S425. (Q*: pyroglutamic acid).

445-458 on the basis of a pseudo MS3 fragmentation spectrum (MALDI-TOF/TOF). This pseudo MS3 spectrum represents a combination of an in-source LID fragmentation and a PSD fragmentation. The in-source LID fragmentation of the parent ion y14+ (1674.833 Da) generated the dephosphorylated peptide species (Figure 6A), which was in turn selected as a parent ion for a PSD fragmentation. The peptide 445-458 has four potential phosphorylation sites (S450, Y451, T454, S457). The classical y and b ion series plus both the y-Pi and b-Pi ion series were obtained. The mass difference of 167 Da corresponding to a phosphoserine between y8+ and y9+ proved that S450 was phosphorylated (Figure 6B). This was also confirmed by the 98 Da mass difference between y9+ and its corresponding dehydroalanine (y9-Pi)+ expected from the loss of phosphoric acid. Multiple Phosphorylation Site on Peptides 464-489 and 464-490. Both nanoESI-MS and MALDI-TOF MS suggested at least four phosphorylations between position 464 and 490 (Figure 7). Using several di- and triphosphorylated isoforms

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Figure 6. Phosphoserine S450 on peptide 445-458 (GEARESYRETGPSR). MALDI-TOF/TOF LID and PSD fragmentation illustrating the fragmentation of the phosphorylated peptide. (A) MS2 of phosphorylated peptide illustrating the neutral loss. (B) combined pseudo MS3 spectrum and LID spectrum of the phosphorylated peptide. On the basis of the combined spectrum, the mass difference of 167 Da corresponding to a phosphoserine between y8+ and y9+ demonstrates that S450 is phosphorylated.

of these peptides, five phosphorylation sites including two phosphorylated threonine and three phosphorylated serine were identified in position T471, T477, S479, S481, and S482 of both overlapping tryptic peptides (464-489 and 464-490). Neutral losses of phosphate in MS2 were found in mono-, di-, and triphosphorylated peptides all with a 3+ charge. The complete set of these spectra presenting a neutral loss is shown in the Supporting Information 8A-E for the tryptic peptides. The extracted ion chromatograms corresponding to these above phosphorylated peptides are presented in the Supporting Information 9. The presence of several levels of phosphorylation distributed among five phosphorylatable positions generates several isobaric peptides for each level of phosphorylation. This explains the broader elution peaks and the apparent loss of resolution for the LC profile of those peptides particularly for the triphosphopeptide (Supporting Information 9). Phosphorylation of T471. The phosphorylation of T471 was observed in a triphosphorylated tryptic peptide (464-489) in MS2 (Figure 8A,B and Supporting Information 10A and 10B). The selected peptide was present in the extracted ion chroJournal of Proteome Research • Vol. 9, No. 11, 2010 5603

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Figure 7. Multiple phosphorylation levels on peptides 464-489 and 464-90 in ESI-MS and MALDI-TOF MS, respectively, showing three (panel A) or four (panel B, C) phosphorylation sites. (A) Analysis of phosphorylated peptide 464-489 by LC-ESI ion trap MS. MS spectra were cumulated over the elution segment of the LC gradient corresponding to phosphorylated states of peptide 464-489. The resulting spectrum shows unphosphorylated, mono-, bi-, and triphosphorylated peptides with a ∆m of 26.6 m/z corresponding to triple-charged species. (B) Analysis of phosphorylated peptide 464-490 by MALDI-TOF MS. The resulting spectrum shows unphosphorylated, mono-, bi-, and triphosphorylated states of peptide 464-490 with a ∆M of 80 m/z. (C) Tri and tetra-phosphorylated states of peptide 464-490 with a ∆M of 80 m/z by Maldi TOF MS.

Figure 8. Phosphothreonine T471 on peptide 464-489 by LCESI ion trap MS2. (A, B) b8 ion showing a mass increased by 80 Da (829.4-749.5 Da) for the phosphopeptide indicates the phosphoresidue. (A) Unphosphorylated peptide and (B) triphosphorylated peptide. The complete spectrum is in Supporting Information 10. 5604


Prodhomme et al. matograms at a retention time of 13.1 min, corresponding to a shoulder in front of the major peak of the isobaric triphosphopeptides (Supporting Information 9). The T471 phosphorylation site was identified by direct comparison of ESI-CID MS2 spectra of unphosphorylated and phosphorylated peptides since no MS3 spectra were obtained. By comparing characteristic y and b ion series in MS2 spectra of a triple charged peptide, we were able to identify unequivocally peptide 464-489. The ESI-CID MS2 spectra of the unphosphorylated and the triphosphorylated form showed that the ion b8+ of m/z 749.6 had disappeared and that its phosphorylated counterpart at m/z 829.4 emerged. Thus the phosphate group was unambiguously assigned to threonine 471. Phosphorylation of T477 and S479. The phosphorylation of T477 and S479 observed in tryptic peptides (464-489 and 464-490) was also confirmed in chymotryptic peptides. As for T471, the T477 phosphorylation site was identified by direct comparison of ESI-CID MS2 spectra of unphosphorylated and phosphorylated peptides. The ESI-CID MS2 spectra of the unphosphorylated and the double phosphorylated form, showed the disappearance of the ion b15+ of m/z 1474.8 and the appearance of its phosphorylated counterpart at m/z 1554.8. Furthermore, the disappearance of the ion y13+ of m/z 1407.8 and the appearance of its double phosphorylated counterpart at m/z 1567.6 also confirmed the phosphorylation in T477 and suggest a second site in S479 (Figure 9A,B and Supporting Information 11B). This second phosphorylation site in position S479 was confirmed in peptide 464-489 by ESI-CID MS2 (Figure 9C,D and Supporting Information 11A) and MALDI TOF PSD fragmentation (data not shown). The ion b242+ showing a 40 m/z difference corresponding to a phosphorylation and the y11+ shift of 80 Da between the mono- and the unphosphorylated peptide assigned the phosphate group unambiguously to serine 479. Phosphorylation of S481 and S482. As described above for the phosphorylation of T477 and S479 the peptides used below are the result of a H3PO4 neutral loss (Supporting Information 8A-E and Figure 9). The peptide 464-489 AAHLPTGTPLDIDTASESSQDPQDSR possesses three threonines and four serines, accounting for seven potential phosphorylation sites. Both T469 and T471 were shown to be unphosphorylated on the basis of a complete series of unmodified b ions up to b13 for each of the selected phosphorylated peptides 464-489 and 469-490 analyzed in MS2. Similarly, a complete series of unmodified y ions was observed up to fragment y7 for each selected phosphorylated peptide 464-489 and up to fragment y8 for each selected phosphorylated peptide 469-490, excluding S488 as a phosphorylated amino acid. The detection of tri- and tetraphosphopeptides 464-490 (Figure 7B,C) phosphorylated in T477 and S479 but not in T469, T471, or S488 suggested that both S481 and 482 were also phosphorylated. The assignment of the third phosphorylation site on peptide 464-489 to S481 was more complicated, and was only possible by using the selected ion of m/z ) 957.7 characteristic of a biphosphorylated peptide (Figure 10B). However, this ion corresponds to several indistinguishable isobaric biphosphopeptides with four potential phosphorylation sites. T477 and S479 are the main phosphorylated sites on this peptide as shown above. Phosphorylations on both S481 and S482 were also confirmed, always in combination with one of the former sites. As the ion y8+ remained unmodified, the position S482 was excluded (Figure 10A,B and Supporting Information 12A).


Figure 9. Phosphothreonine T477 and phosphoserine S479 on peptide 464-489 by LC-ESI ion trap MS2. (A, B) b15 ion assigns the first phosphorylation site to the residue T477: (A) unphosphorylated peptide and (B) diphosphorylated peptide. (C, D) y11 ion places the second phosphorylation site on S479: (C) unphosphorylated peptide and (D) monophosphorylated peptide. In panel B, * corresponds to the biphosphorylated internal fragment DtAsESSQDPQD and ** to LDIDtAsESSQDP. The complete spectrum is in Supporting Information 11.

The ion y9+ showing a 80 m/z difference between the unphosphorylated and the biphosphorylated peptide assigned the phosphate group to serine 481. The fourth phosphorylation site on peptide 464-489 was deduced from a triphosphopeptide (Figure 10C,D and Supporting Information 12B). The ion y8+ showing a 80 m/z difference between the un- and the triphosphorylated peptide placed the phosphate group on S482. Positions S481 and S482 were never found simultaneously phosphorylated. Phosphorylation of S505 and S510. Finally, MALDI-TOF MS analysis after phosphopeptide enrichment on TiO2 revealed two phosphorylated amino acids in peptide 498-521 on the basis of a mass shift from 2610 to 2690 Da and 2780 Da. The presence of two phosphate groups on the chymopeptide 502-518, together with the conserved y ion series, excludes

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Figure 10. Phosphoserine S481 and S482 on peptide 464-489 by LC-ESI ion trap MS2. (A, B) y9 ion shows a 80 m/z difference between the unphosphorylated (A) and the biphosphorylated peptide (B) assigning the phosphate group to serine 481. The y8 ion remains unmodified and therefore excludes the position S482. (C, D) This y8 ion shows a 80 m/z difference between the unphosphorylated peptide (C) and the triphosphorylated peptide (D) placing the phosphate group on S482. The complete spectrum is in Supporting Information 12.

T518 as a potential phosphorylation site. This leaves only four possible amino acid positions for the two phosphorylations within this peptide. As before, the phosphorylated peptide was identified by the presence of a neutral loss in MS2 (Supporting Information 8F). By comparing the ESI-CID MS2 spectra of peptide 498-521 in its unphosphorylated and monophosphorylated form, we observed the disappearance of the b14+ ion of m/z 1417.7 and the apparition of its phosphorylated counterpart at m/z 1497.9 accounting for a phosphorylation in S510 (Figure 11A,B and Supporting Information 13A,B). In Figure 11, the phosphorylation of S510 is also visible on the internal fragment SEEQGSDTDTPIV of m/z 1359.9. On the double phosphorylated peptide 498-521 the b11+ ion of m/z 1158.7 Journal of Proteome Research • Vol. 9, No. 11, 2010 5605

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Prodhomme et al.

Figure 12. Comparison of the tryptic digests of rNP (A) and vNP (B) for the phosphorylated S450 on peptide (445-458) by MALDITOF. The phosphopeptide is not detectable in vNP suggesting that S450 is not phosphorylated.

recombinant protein using the rNP spectra as a template as illustrated in Supporting Information 14. The MS/MS data obtained confirmed the results of the rNP with two notable exceptions in position S450 and T471. After tryptic digestion and phosphopeptide enrichment, the phosphopeptide 445-458 GEARESYRETGPSR was not detectable in the enriched mixture (Figure 12), suggesting that S450 was not phosphorylated in vNP from infected VeroSLAM cells. Also, the peptides 445-452 and 449-458 were not found in their phosphorylated states (data not shown). The characteristic pattern of MS3 fragmentation indicative of a phosphorylation in position T471 was not present in the viral NP and only the positions T477, S479, S481, and S482 were found to be phosphorylated in the two overlapping tryptic peptides (464-489 and 464-490). Figure 11. Phosphoserines S505 and S510 on peptide 498-521 by LC-ESI ion trap MS2. The b14 ion of m/z 1417.7 in the unphosphorylated form (A) disappears and its phosphorylated counterpart at m/z 1497.9 appears in the monophosphorylated form (B) accounting for a phosphorylation in S510. On the double phosphorylated peptide 498-521 (D) the b11+ ion of m/z 1158.7 was shifted by 80 Da compared to the monophosphorylated peptide (C). This assigns the second phosphate group to S505. In panel B, * correspond to the dephosphorylated b11+ ion with S510 present as a dehydro-alanine. The complete spectrum is in Supporting Information 13.

was shifted by 80 Da compared to the mono- and the unphosphorylated peptide. This assigned the second phosphate group to S505 (Figure 11C,D and Supporting Information 13C). Comparative Phosphorylation of rNP and Purified Viral NP. An identical study was performed with purified vNP using the same experimental protocols for protein digestion, phosphopeptide enrichment, and MS analysis. The phosphosites assignment of the viral protein was done by comparison to the 5606


Although quantification of phosphorylation by MS is difficult, a weaker phosphorylation signal of both S425 and S505 in the purified viral NP was observed compared to the recombinant protein. A high-resolution 2D Western Blot (Figure 1C) was used to quantify phosphorylation levels of the vNP. Taking into account the spot size and intensity, 20% of the vNP was found to be unphosphorylated, 24.5% were monophosphorylated, 17.3% di-, 11.8% tri-, 11.3% tetra-, 9.7% penta-, and 5.3% hexaphosphorylated (Supporting Information 15). N-Terminal Acetylation of NP. After endo Lys-C digestion, the purified recombinant NP and the purified viral NP were analyzed by MALDI-TOF/TOF. A peptide molecular ion of 1274.9 Da with the sequence ATLLRSLALFK was identified by MS2 as the sole N-terminal peptide. A complete series of unmodified y ions was observed up to the fragment y9 and a corresponding series of b ions was observed up to b11, all carrying the acetyl modification and showing a mass difference of 42 Da (data not shown). The absences of methionine as well as the acetylation were confirmed by nanoLC-ESI-ion trap analysis for both proteins.

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Phosphorylation of Measles Virus Nucleoprotein a

Table 2. Conservation of Phosphorylated Residues among all Wild-Type and Vaccine Strain of MV phosphorylated amino acid positions (%) amino acid

425

450

471

477

479

481

482

505

510

S T Y S/T/Y

94.7 0.1 0.0 94.8

77.8 4.9 0.0 82.7

0 95.8 0 95.8

0.2 95.2 0.0 95.4

99.0 0.4 0.0 99.4

53.5 7.7 21.3 82.5

59.7 0.0 0.0 59.7

35.3 0.0 0.0 35.3

99.5 0.1 0.0 99.6

a

832 sequences with 2 (inactive genotypes B1, F, and G1) to 175 (genotype H1) sequences per genotype are included.

Discussion Protein spot patterns in a highly sensitive 2D fluorescence Western blot of both the rNP produced in insect cells and vNP obtained from purified virus grown in African Green Monkey kidney cells (Vero) were similar. At least eight isoforms corresponding to various levels of phosphorylation with different isoelectric points were identified. In agreement with this spot pattern a total of nine phosphorylation sites were identified in the rNP and localized using either MALDI-TOF/TOF MS or ESI-ion trap analysis in MS2/ MS3 mode. Two of these phosphorylation sites, S479 and S510, were previously identified by Hagiwara et al.12 In addition, our MS analysis showed that both the recombinant protein and the protein purified from virus had lost their initiator methionine and were R-acetylated on the N-terminal alanine residue.20,21 As in many proteins of eucaryotes, the N-terminal was thus largely protected against proteolytic digestion by exopeptidases.22 Identical phosphorylation sites were identified both in the recombinant and virus particle-derived NP, except for S450 and T471, which were phosphorylated only in the recombinant protein. According to several tools for predicting post-translational modifications23,24 Casein kinase 2 (CK2), which also phosphorylates the MV phosphoprotein,25 was the only kinase family able to phosphorylate all sites identified in the viral protein. The recombinant NP protein was expressed in insect’s cells possessing a different battery of kinases, which may explain the difference of phosphorylation in positions S450 and T471. Since CK2 is a very ubiquitous kinase this raises the interesting question to what extend NP phosphorylation is constitutive, or dynamically regulated at the different stages of the virus life cycle.26 Except for T477 all other phosphorylated amino acids were serines, which corresponds to the ratio of phosphoserine to phosphothreonine of 88 to 12% previously reported from MV-NP digests.11,27 Furthermore, we did not identify any phosphotyrosine, which is consistent with earlier findings that phosphorylation of this residue in NP is restricted to persistently infected neuroblastoma cells.13 41 possible phosphorylation sites were predicted by NetPhos 2.028 for NP, including 28 serines, 6 threonines, and 7 tyrosines. Sixteen predicted sites were located in the C-terminal domain (aa 400-525) of the protein, including 13 serines, 2 threonines, and 1 tyrosine. Considering the known limitations of the CID for phosphosite assignment,29,30 our data indicate that all phosphorylated residues were located within the C-terminal domain of NP and downstream of the hypersensitive proteolytic cleavage site in position 413. Within this domain, two short sequence segments that contained each one a serine (S443, S460, Figure 2) were not covered by MS analyses. Despite phosphopeptide enrichment, a 95% sequence coverage and a combination of sensitive MALDI-TOF and LC-MS instrumentation no phosphorylation was detected in the N-core of the protein.

The extensively phosphorylated C-terminal tail (NTAIL) mediates the interaction of NP with the globular XD domains (aa 459-507) of tetramers of the viral phosphoprotein (P). This interaction involves mainly NP residues 489-506,8,31 conserved among all morbilliviruses. Small angle X-ray scattering confirmed that NTAIL is not structured in solution and the crystal structure of the NTAIL/XD domains shows that the R-helical recognition element (aa 489-499) of NP enters into hydrophobic interactions with the P tetramer.6,7,31,32 Although not directly implicated in contacts with the P, a second region of the C-tail of NP, including aa residues 517-522, shows structural reshuffling when the R helix aa 489-499 is extruded.8 The interaction of NP with P is essential for viral transcription and replication.6,8,33-35 Since it determines the positioning of the MV polymerase complex formed by the large protein and P, on the nucleocapsid structure, charge modifications affecting their interacting surfaces are likely to play an important functional role. While the R-helical recognition element is not phosporylated, the upstream residues T477, S479, S481, and S482 and the downstream residues S505 and S510 form phosphorylation clusters conspicuously surrounding this domain responsible for transcription. In addition, the C-tail mediates also interactions with several host cell proteins. The last amino acids of the NP sequence interacted with the heat shock protein HSP72, which modulates viral RNA synthesis.36-40 Binding of the NP to an uncharacterized nucleoprotein receptor suppressed cell proliferation41 and amino acids 401-420 mediated this interaction. Type I IFN can be induced through direct interaction of NP with interferon regulatory factor 3 (IRF3) even in absence of other MV components.9 Using truncated recombinant NP, it was shown that amino acids 376-523, which include all of the phosphosites identified in his study, are required for this interaction. Phosphorylated amino acids were conserved in 35.3-99.6% of 832 nonredundant NP sequences (Table 2) available in Genbank (28th of August 2008). The most conserved amino acids were S479 (99.0%) and S510 (99.5%), and their phosphorylation has previously been shown to enhance MV transcription and replication.12 All but one of the strains which were mutated in position 479 was vaccine strains. In these cases S was replaced by T (e.g., Schwarz, Moraten42 or P (Shanghai191). Thus, S479 may play a role in pathogenicity and/or attenuation of vaccine strains. Amino acid residues S481 and S482 were conserved in only about half of the strains, but all sequences had a serine in at least one of the two positions. This may indicate that phosphorylation of one of the above two residues is necessary but also sufficient. For all genotypes T477 is conserved in >98% of all MV strains except for genotype D6 strains, more than half of which have replaced this phosphosite by an asparagine. Gombart and colleagues11 suggested that NP with a phosphorylated threonine is preferentially Journal of Proteome Research • Vol. 9, No. 11, 2010 5607

research articles assembled into viral nucleocapsids, but the position of the corresponding phosphothreonine was not identified at the time. Since T477 was the only threonine which we found phosphorylated in the viral NP, and was conserved among 95.3% of analyzed sequences, we conclude that a phosphorylated T477 is critical for nucleocapsid assembly. In conclusion, seven new phosphosites were identified in the recombinant NP, five of which were also phosphorylated in the viral NP, probably by CK2 kinases. All phosphorylation sites clustered around the C-terminal functional domains important for transcription and RNA encapsidation and their conservation pattern in the virus further underline an important role in virus replication and attenuation of vaccine strains. Abbreviations: ESI, electrospray ionization; mAb, monoclonal antibody; MALDI, matrix assisted laser desorption/ ionization; MS, mass spectrometry; MV, measles virus; NP, nucleoprotein; rNP, recombinant nucleoprotein; vNP, viral nucleoprotein; CK2, casein kinase 2; Cy2, Cy2 Bis NHS ester; Cy3, Cy3 Bis NHS ester; Cy5, Cy5 Bis NHS ester; CyDye, Cy2 or Cy3 or Cy5; DDW, double distilled water; IEF, isoelectric focusing; PMF, peptide mass fingerprint; PSD, post source decay; LID, laser induced dissociation; DIGE, differential gel electrophoresis; PTM, post translational modification.

Acknowledgment. We are grateful to F. Bouche, S. Farinelle, and S. Willie`me for technical assistance. This research was partially supported by the Fonds National de la Recherche of Luxembourg (Project FNR/01/02/01A). Supporting Information Available: Analysis of rNP; MALDI-TOF MS analysis of a tryptic controlled digestion of rNP and of trypsin digested rNP; nano LC-ESI analysis of trypsin digested rNP; partial MALDI-TOF PMF spectra of rNP peptides; 2D gel of rNP; nanoESI IT MS/MS (CID) spectra; extracted ion chromatograms; assignment of phosphosite S479 on the viral protein by comparison to the recombinant protein using the rNP spectra as a template; relative quantification of the different phosphorylation levels of the vNP based on the highresolution 2D Western blot. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Griffin, D. E. Measles Virus. In Fields Virology, 5th ed.; Knipe, D. M., Howley, P. M., Eds.; Lippincott Williams & Wilkins: Philadelphia, 2007; Vol. 2. (2) de Vries, R. D.; et al. Measles vaccination: new strategies and formulations. Exp. Rev. Vaccines 2008, 7 (8), 1215–23. (3) Bellini, W. J.; et al. Measles virus P gene codes for two proteins. J. Virol. 1985, 53 (3), 908–919. (4) Liston, P.; Briedis, D. J. Ribosomal frameshifting during translation of measles virus P protein mRNA is capable of directing synthesis of a unique protein. J. Virol. 1995, 69 (11), 6742–6750. (5) Kolakofsky, D.; et al. Paramyxovirus RNA synthesis and the requirement for hexamer genome length: the rule of six revisited. J. Virol. 1998, 72 (2), 891–899. (6) Houben, K.; et al. Interaction of the C-terminal domains of sendai virus N and P proteins: comparison of polymerase-nucleocapsid interactions within the paramyxovirus family. J. Virol. 2007, 81 (13), 6807–6816. (7) Bourhis, J. M.; Canard, B.; Longhi, S. Structural disorder within the replicative complex of measles virus: functional implications. Virology 2006, 344 (1), 94–110. (8) Belle, V.; et al. Mapping alpha-helical induced folding within the intrinsically disordered C-terminal domain of the measles virus nucleoprotein by site-directed spin-labeling EPR spectroscopy. Proteins 2008, 73 (4), 973–988. (9) tenOever, B. R.; et al. Recognition of the measles virus nucleocapsid as a mechanism of IRF-3 activation. J. Virol. 2002, 76 (8), 3659– 3669.

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(40) Zhang, X.; Oglesbee, M. Use of surface plasmon resonance for the measurement of low affinity binding interactions between HSP72 and measles virus nucleocapsid protein. Biol. Proc. Online 2003, 5, 170–181. (41) Laine, D.; et al. Measles virus nucleoprotein induces cellproliferation arrest and apoptosis through NTAIL-NR and NCORE-FcgammaRIIB1 interactions, respectively. J. Gen. Virol. 2005, 86 (6), 1771–1784. (42) Parks, C. L.; et al. Comparison of predicted amino acid sequences of measles virus strains in the Edmonston vaccine lineage. J. Virol. 2001, 75 (2), 910–920.

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Journal of Proteome Research • Vol. 9, No. 11, 2010 5609

Extensive Phosphorylation Flanking the C-Terminal Functional

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