Discovering and validating unknown phospho-sites from p38 and HuR protein kinases in vitroby Phosphoproteomic and Bioinformatic tools
© López et al; licensee BioMed Central Ltd. 2011
Received: 20 April 2011
Accepted: 6 July 2011
Published: 6 July 2011
The mitogen activated protein kinase (MAPK) pathways are known to be deregulated in many human malignancies. Phosphopeptide identification of protein-kinases and site determination are major challenges in biomedical mass spectrometry (MS). P38 and HuR protein kinases have been reported extensively in the general principles of signalling pathways modulated by phosphorylation, mainly by molecular biology and western blotting techniques. Thus, although it has been demonstrated they are phosphorylated in different stress/stimuli conditions, the phosphopeptides and specific amino acids in which the phosphate groups are located in those protein kinases have not been shown completely.
We have combined different resins: (a) IMAC (Immobilized Metal Affinity Capture), (b) TiO2 (Titanium dioxide) and (c) SIMAC (Sequential Elution from IMAC) to isolate phosphopeptides from p38 and HuR protein kinases in vitro.
Different phosphopeptide MS strategies were carried out by the LTQ ion Trap mass spectrometer (Thermo): (a) Multistage activation (MSA) and (b) Neutral loss MS3 (DDNLMS3).
In addition, Molecular Dynamics (MD) bioinformatic simulation has been applied in order to simulate, over a period of time, the effects of the presence of the extra phosphate group (and the associated negative charge) in the overall structure and behaviour of the protein HuR.
This study is supported by the Declaration of Helsinki and subsequent ethical guidelines.
The combination of these techniques allowed for:
(1) The identification of 6 unknown phosphopeptides of these protein kinases. (2) Amino acid site assignments of the phosphate groups from each identified phosphopeptide, including manual validation by inspection of all the spectra. (3) The analyses of the phosphopeptides discovered were carried out in four triplicate experiments to avoid false positives getting high reproducibility in all the isolated phosphopeptides recovered from both protein kinases. (4) Computer simulation using MD techniques allowed us to get functional models of both structure and interactions of the previously mentioned phosphorylated kinases and the differences between their phosphorylated and un-phosphorylated forms.
Many research studies are necessary to unfold the whole signalling network (human proteome), which is so important to advance in clinical research, especially in the cases of malignant diseases.
As with other MAPK pathways, the p38 signalling cascade involves sequential activation of MAPK kinase kinases (MAP3Ks) and MAPK kinases (MKKs) including MKK3, MKK4, and MKK6, which directly activate p38 through phosphorylation in a cell-type- and stimulus-dependent manner [1, 2]. Once activated, p38 MAPKs phosphorylate serine/threonine residues on their substrates, such as transcription factors, cell cycle regulators as well as protein kinases. The p38 signalling pathway allows cells to interpret a wide range of external signals, such as inflammation, hyperosmorality, oxidative stress and respond appropriately by generating a plethora of different biological effects [3–14]. HuR has been implicated in processes such carcinogenesis, proliferation, immune function or responsiveness to DNA damage .
It is of interest to note that numerous HuR-regulated mRNAs encode proteins responsible for implementing five major cancer traits:
(a) Promote cell proliferation (p27, cyclin D, Cyclin E1 or EGF)
(b) Increase cell survival (SIRT1, Mdm2 or p21)
(c) Elevate local angiogenesis (VEGF, Cox-2 or HIF-1alpha)
(d) Invasion and metastasis (Snail, MMP-9, or uPA)
(e) Evasion of immune recognition (TGF-beta).
Moreover, HuR was broadly elevated in cancer tissue compared to the corresponding non-cancer tissues. It has been widely reported that in the general principles of signalling pathways p38 and HuR kinases are modulated by phosphorylation, mainly by western blotting techniques. The phosphopeptides and the specific amino acids in which the phosphate groups are located in these low expressed proteins have not been completely shown as yet [16–22].
The analysis of the spatial and temporal aspects of protein phosphorylation is of great interest for the discovery of functions of specific biological processes. An extensive mass spectrometry-based mapping of the phosphoproteome progresses and computational analysis of phosphorylation has been carried out. Phosphorylation-dependent signalling becomes increasingly important for clinical research and requires improvements for each different sample. In addition, the linear sequence motifs that surround phosphorylated residues have been successfully used to characterize kinase-substrate specificity. To complement phosphoproteomic research, bioinformatics offers a range of methods to analyze and to simulate structural properties of the studied phosphoproteins. Both unphosphorylated and phosphorylated states of a residue can be generated "in silico" and included in the appropriate 3D protein context. After this initial modelling, Molecular Dynamics (MD) techniques can be applied in order to simulate, over a period of time, the effects of the presence of the extra phosphate group (and the associated negative charge) in the overall structure and behaviour of the protein [23–25].
We describe the successful strategy (also used by other scientists [26–28]) for the discovery of 6 unknown phosphorylated peptides from p38 and HuR kinases. Our data comes from advances in MS strategies coupled to different resins (IMAC, TiO2 and SIMAC) that we have applied, coupled to bioinformatics tools (MD simulation). The specific peptides discovered, which are phosphorylated in p38 and HuR protein kinases, are provided. In addition, the specific amino acid assignments of the phosphate groups from the identified phosphopeptides are also presented. Unknown phospho-sites from these kinases in vitro have been discovered for the first time. Our data is supported by previous scientific studies related to these protein phosphorylated kinases.
It has have been reported that p38 and HuR kinases are phosphorylated mainly by western blotting techniques although not showing all amino acids in which the phosphate groups are located. It should be pointed out that the phosphate groups can vary according to the conditions of the sample analysis (see references of p38 and HuR previously mentioned [16–22]). In this study, MSA (multistage activation) compared to DDNLMS3 (neutral loss MS3) gave more information for the suite of phosphopeptides studied when using SIMAC coupled to the ion Trap mass spectrometer. Using bioinformatics MD simulations we have proposed functional variations in both structure and interactions of the previously mentioned phosphorylated-kinases comparing the phosphorylated and un-phosphorylated forms previously described in vitro. Finally, we point out possible developments or alternatives and complementary tools with the intention of providing the community with improved and additional phosphorylation studies of cellular signalling networks, this being such an important issue owing to the fact that if we had complete knowledge of the signalling-networks, many malignant diseases could be more fully understood and thus facilitate drug development for different pathologies. This article also aims to improve the knowledge of p38 and HuR protein kinases by identifying and validating new phosphopetides in vitro, with the knowledge that this is essential to advance in the knowledge of signalling networks (human proteome). These and many other advances will help clinical research investigations, especially in relation to human malignant diseases.
Materials and methods
Statement of ethical approval
This study was conducted in compliance with the international "Declaration of Helsinki." An informed consent about the procedures as well as permission from the Ethical Committee of Carlos III Hospital of Health was obtained. This study adhered to the tenets of the Declaration of Helsinki. (http://www.wma.net/e/policy/b3.htm). (Declaration of Helsinki (1964), Belmont (1978) and agreement of Oviedo (1997) - the basic principles for human and biological samples research studies -) http://www.isciii.es/htdocs/index.jsp).(http://www.madrid.org/cs/Satellite?pagename=HospialCarlosIII, http://www.cnio.es "working links")
Purification and Kinase assay
Recombinant glutathione S-transferase (GST) fusion proteins were expressed in Escherichia coli BL21 (DE3) and purified using standard protocols. p38beta was activated with MalE-MKK6DD (5:1 ratio) in 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 2 mM DTT pH 7.5 and 200 uM ATP for 1 hour at 30°C. Kinase assay were carried out in a buffer A (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 2 μM microcystin, 50 mM NaF, and 10 μM ATP) supplemented with Phosphatase inhibitor cocktail 1 (P2850, 1:100) and Phosphatase inhibitor cocktail 2 (P5726, 1:100) from SIGMA, containing 12 μg of HuR and 500 ng of activated p38 for 30 min at 30°C.
Protein digestion in solution
Proteins (10 μg) were subjected to digestion procedure following the protocol described by Zhao and co-workers with slight variations . Digestion with Lysyl Endopeptidase: the reduced and alkylated sample was incubated at room temperature for 3 h with 1 μg of lysyl endopeptidase/50 μg protein (WAKO). Digestion with Trypsin: the lysyl endopeptidase-digested sample was diluted with 50 mM NH4HCO3 (Sigma) to make a 5 times dilution of urea, since trypsin is not fully active at high concentrations of urea. One microgram of modified trypsin (Promega) was added per 50 μg of lysyl endopeptidase-digested protein and the sample was incubated at room temperature for 16-24 h. The digests were evaporated to about 20 μL in a SpeedVac centrifuge and subsequently 5 μl were used for TiO2, 5 μl for IMAC and 5 μl for SIMAC phosphopeptide enrichments.
Dioxide Titanium phosphoenrichment (TiO2)
Titanium dioxide-microcolumns with a length of ~2 mm were packed in GELoader tips. A small plug of C8 material was stamped out of a 3M Empore C8 extraction disk using an HPLC syringe needle and placed at the constricted end of the GELoader tip. The C8 disk serves only as a frit to retain the titanium dioxide beads within the GELoader tip.
Note that the solvent used for either washing or loading the sample onto the TiO2 microcolumn contains organic solvent (50-80% CH3CN), which abrogates adsorption of peptides to the C8 material. The TiO2 beads were suspended in 80% acetonitrile, 0.1% TFA, and an aliquot of this suspension (depending on the size of the column) was loaded onto the GELoader tip. Gentle air pressure created by a plastic syringe was used to pack the column as described previously. The bound peptides were eluted using 3 μl of NH4OH, pH 10.5. An additional elution step using 0.5 μL of 30% acetonitrile was added to elute peptides, which had remained bound to the C8 membrane plug. The eluents were pooled and acidified using 100% formic acid prior to the desalting step and desalted using Poros-R3 coupled to C18-Disks microcolumns prior to MS analysis [30, 31].
Immobilized Metal Affinity Capture (IMAC) phosphoenrichment
Purification of phosphorylated peptides was performed according to Nuhse and co-workers  and Lee and co-workers with minor changes . Briefly 10 μl of ironcoated PHOS-selectTM metal chelate beads (Sigma) were washed twice in 100 μl of washing/loading solution (0.25 M acetic acid, 30% acetonitrile) and resuspended in 40 μl of washing/loading solution. An aliquot of this solution (20 μl) was incubated with the peptide solution in a total volume of 40 μl of washing/loading solution for 30 min with constant rotating. After incubation, the solution was loaded onto a constricted GELoader tip, and gentle air pressure was used to pack the beads. Subsequently the beads were washed extensively with the washing/loading solution. The bound peptides were eluted using 3 μl of NH4OH, pH 10.5, and desalted using Poros R3 coupled to C18-Disks microcolumn prior to MS analysis.
Sequential Elution from IMAC (SIMAC) phosphoenrichment
For each experiment 10 μl of iron-coated PHOS-selectTM metal chelate beads IMAC (Sigma) were used. The beads were washed twice in loading buffer (0.1% TFA, 50% acetonitrile) as described previously . The beads were incubated with 30 μl of loading buffer and 4 μg of peptide mixture (tryptic digest). The beads were shaken in a Thermomixer (Eppendorf) for 30 min at 20°C. After incubation, the beads were packed in the constricted end of a 200 μl GELoader tip (Alpha Laboratories) by application of air pressure forming an IMAC microcolumn. The IMAC flow-through was collected in an Eppendorf tube for further analysis by TiO2 chromatography (see below). The IMAC column was washed using 20 μl of loading buffer, which was pooled with the IMAC flow-through. The putative monophosphorylated peptides and contaminating non-phosphorylated peptides were eluted from the IMAC column using 10 μl of 1% TFA, 20% acetonitrile, and the possible multiple phosphorylated peptides were subsequently eluted from the same IMAC microcolumn using 40 μl of ammonia water, pH 11.3 (10 μl of 25% ammonia solution (Merck) in 490 μl of ultra-high quality water). The IMAC flow-through and the IMAC eluents were dried by lyophilization. Titanium Dioxide (TiO2) Chromatography after lyophilization, the pooled flow-through and wash from the IMAC microcolumn was enriched for phosphopeptides using TiO2 chromatography. For the complex mixture of the putative monophosphorylated peptide fraction (1% TFA) was also subjected to TiO2 chromatography as described below. A TiO2 microcolumn was prepared by stamping out a small plug of C8 material from a 3M EmporeTM C8 extraction disk (3M Bioanalytical Technologies) and placing the plug in the constricted end of a P10 tip (Eppendorff). The TiO2 beads (suspended in 100% acetonitrile) were packed in the P10 tip where the C8 material prevented the beads from leaking. The TiO2 microcolumn was packed by the application of air pressure. Buffers used for loading or washing of the microcolumn contained 80% acetonitrile to prevent non-specific binding to the C8 membrane and the TiO2 beads. The lyophilized sample was resuspended in 2 μl of 4 M urea and 3 μl of 1% SDS and diluted five times in loading buffer (1 M glycolic acid (Fluka) in 80% acetonitrile, 5% TFA) and loaded onto a TiO2 microcolumn of 5 mm . The TiO2 microcolumn was washed with 5 μl of loading buffer and subsequently with 30 μl of wash buffer (80% acetonitrile, 5% TFA). The phosphopeptides bound to the TiO2 microcolumns were eluted using 50 μl of ammonium water (pH 11.3) followed by elution using 0.5 μl of 30% acetonitrile to elute phosphopeptide bound to the C8 disk. The eluent was acidified by adding 5 μl of 100% formic acid prior to the desalting step.
Desalting the isolated phosphopetides by chromatography reversed phase (RP) using POROs R3 coupled to C18 Disks, prior to MALDI and ESI Mass Spectrometry analysis
Poros Oligo R3 reversed phase material was from PerSeptive Biosystems (Framingham, MA). GELoader tips were from Eppendorf (Eppendorf, Hamburg, Germany) and Alpha Laboratories (Hampshire, UK). Orthophosphoric acid (85%, v/v) was from J. T. Baker Inc. Ammonia solution (25%) was from Merck. 3M Empore C8 disk was from 3M Bioanalytical Technologies (St. Paul, MN). All reagents used in the experiments were sequence grade, and the water was from a Milli-Q system (Millipore, Bedford, MA). The Poros Oligo R3 reversed phase resin (PerSeptive Biosystems) was dissolved in 70% acetonitrile. The R3 beads were loaded onto constricted GELoader tips, and gentle air pressure was used to pack the beads to obtain R3 microcolumns of 2 mm. Each acidified sample was loaded onto a R3 microcolumn. The R3 microcolumns were subsequently washed with 30 μl of 0.1% TFA, and the phosphopeptides were eluted directly onto the MALDI target using 0.5 μl of 20 μg/μl DHB (Fluka), 50% acetonitrile (ACN), 1% phosphoric acid. MALDI-MS analysis was just carried out in order to check there were sufficient eluted peptides to be analyzed by LC-ES-MS, after the microcolumns applied for the isolation, cleaning and concentration of putative phosphorylated peptides. For LC-ESI/MSMS analysis of the phosphorylated peptides originating from the sample, the phosphopeptides were desalted in a similar way; however, the phosphorylated peptides were eluted from the Poros R3 column coupled to C18 using 30 μl of 70% acetonitrile, 0.1% TFA followed by lyophilization. The phosphopeptides were subsequently resuspended in 0.5 μl of 100% formic acid and 10 μl of Buffer A (0.1% formic acid, and 5% ACN) prior to LC-ESI/MSn analysis (see references previously mentioned [30, 31, 35]).
Nano-LC-ESI-MSMS analysis using the LTQ ion Trap mass spectrometer
The nano-LC-MS experiments were performed using a LTQ ion Trap mass spectrometer (Thermo Electron, Bremen, Germany). The sample (5 μl) was applied onto an EASY nano-LC system following protocols from Thermo Company and Protein Research Group of Odense University courtesy. Each elute was then entered into a C18 reverse phase column (100 μm i.d., 10 cm long, 5 μm resin from Michrom Bioresources, Auburn, CA). The peptide mixtures were eluted with a 0-40% gradient (Buffer A, 0.1% formic acid, and 5% ACN; Buffer B, 0.1% formic acid and 95% ACN) over 180 min and were then online detected in LTQ ion Trap- mass spectrometer using a data-dependent TOP6 method. The general mass spectrometric conditions were: spray voltage, 1.85 kV; no sheath and auxiliary gas flow; ion transfer tube temperature, 1900C; 35% normalized collision energy using for MS/MS (MS2). Ion selection thresholds were: 500 counts for MS2. An activation q = 0.25 and activation time of 30 ms were applied in MS2 acquisitions. The mass spectrometer was operated in positive ion mode and a data-dependent automatic switch was employed between MS and MS/MS acquisition modes. For each cycle, one full MS scan in the LTQ ion Trap followed by ten MS2 in the LTQ at 5000 on the six most intense ions. Selected ions were excluded from further selection for 90 s. Maximum ion accumulation times were 1000 ms for full MS scans and 120 ms for MS2 scans. For the pseudo- MS3 method or Multi Stage Activation (MSA), an MSA was triggered if in the MS2 a neutral loss peak at -49, -32.7 or -24.5 Da was observed and that peak was one of the five most intense ions of the MS2 spectrum. To improve the fragmentation of phosphopeptides, multi-stage activation (MSA) in the Xcalibur software was enabled for each MS/MS spectrum. When a neutral loss of 97.97, 48.99, or 32.66 Thomson (Th) was detected, the MSA was applied to further fragment the ions. For the Neutral Loss MS3, MS Conditions were: the NanoMate® 100 was mounted to the Finnigan LTQ, and 5 μL (like for MSA) samples were infused at a rate of approximately 100 nL/min. Mass Spectrometer: Finnigan LTQ ion Trap. Ionization Mode: Nano-electrospray, Ion Polarity: Positive, Spray Voltage: 1.55 kV, Spray Pressure: 0.2 psi., Capillary Temperature: 150°C, Normalized Collision Energies: 20-25% for MSn., Maximum Scan Time: 50 ms., Number of Micro Scans Summed for Each Scan: 2-3. Neutral loss MS3 experiment activated for the loss of 98, 49 and 32.7 (singly, doubly and triply charged phosphopeptides). [Mascot searches (http://proteomicsresource.washington.edu/mascot/search_form_select.html) were carried out by "in-Mascot-house server of Centro Nacional de Investigaciones Oncológicas CNIO, http://www.cnio.es"].
Database searching using an in-house MASCOT server and the validation of the identified phosphopeptides
The Mascot generic format file was produced by the following process: the utilities provided by Thermo Electron and Bioworks first converted Xcalibur binary (RAW) files into peak list (DTA) files, then the programs of merge.pl and merge.bat provided by MASCOT public web merged all DTA files into a Mascot generic format file.
For peptide or protein identification, all the raw data files were processed using BioWorks 3.3.1 (Thermo Finnigan, San Jose, CA) and the derived peak list was searched using the Mascot search engine (Matrix Science, London, UK) against a real and false human IPI protein database (V3.49), respectively. The following search criteria were employed: full tryptic specificity was required; two missed cleavages were allowed; carbamidomethylation (Cys) was set as fixed modification, whereas oxidation (Met), N-acetilation (protein), phosphorylation (STY), and intact phosphorylation (STY) were considered as variable modifications. Initial mass deviation of precursor ion and fragment ions was allowed up to 10 ppm and 0.5 Da, respectively. A peptide identified by Mascot was accepted if it had a peptide score above 20 in all the experiments performed. In addition, each phosphopeptide spectrum assignment was manually validated. All of the potential phosphopeptides were confirmed by manual interpretation of MS/MS and MSA ion spectra using the criteria described by Mann and Jensen , Gruhler and co-workers , and Thingholm and co-workers .
Bioinformatics modelling and molecular dynamics simulations
(a) The aim of this study was to establish, as a routine path, a method for identification and characterization of individual phosphorylated kinases p38 and HuR in vitro using: TiO2, IMAC, SIMAC coupled to MSA and MS3NL on the LTQ ion Trap mass spectrometer (Thermo). Purification and fusion proteins were expressed in Escherichia coli. The kinase assay was carried out incubating with different types of protein phosphatase inhibitors in order to increase the levels of protein-kinases phosphorylation prior to the analysis. In fact, sodium pervanadate, a tyrosine phosphatase inhibitor, coupled to a combination of two phosphatase inhibitor cocktails from Sigma (one cocktail containing serine/threonine phosphatase inhibitors and one containing tyrosine phosphatase inhibitors) was also used. Protein kinases were digested with lysyl endopeptidase and trypsin and subsequently enriched for phosphorylated peptides using TiO2, IMAC and SIMAC phosphoenrichments. The isolated phosphopeptides were desalted, cleaned and analyzed by nano-LC ESI-MS/MS using a Thermo LTQ ion Trap MSMS instrument. All experiments were performed in triplicate. The LC-MS/MS experiments and Mascot database searching resulted in overall significant peptide hits. All the peptides were determined with a mass error of less than 5.5 ppm. A total of 6 phosphopeptides were validated by manual evaluation of the LC-MS/MS data sets obtained from the four triplicated experiments. Of these, 6 were assigned to unique amino acid phosphorylated sequences resulting in the identification of 3 unique proteins across all experiments.
(b) The analysis of the 5 μl (~3 μg) of the sample purified by IMAC and desalted and cleaned by R3/C18 permitted us to obtain 2 unknown phosphorylated peptides when using MSA on the nano-LC-LTQ ion Trap instrument. Both phosphorylated peptides were manually validated and correspond to: R.VLVDQTTGLSR.G and R.SLFSSIGEVESAK.L. Those two phosphopeptides belong to the HuR RNA binding protein gi/1022961 protein. The analysis of 5 μl (~3 μg) of the sample purified by TiO2 and desalted and cleaned by R3/C18 permitted us to obtain 4 unknown phosphorylated peptides when using MSA on the nano-LC-LTQ instrument.
The 3 phosphorylated proteins (HuR, Chain B and p38p) and the 6 phosphopeptides identified and validated (amino acid sequences below the identified proteins) when using SIMAC coupled to MAS by the LTQ ion Trap mass spectrometer are shown in this table.
HuR RNA binding protein gi/1022961
Chain B, Structure Of Appbp-1-Uba3-nedd8-Mgatp-Ubc 12 (c111a), A Trapped Ubiquitin-Like Protein Activation Complex gi/126031226
P38 MAP Kinase gi/1469306
The analysis of the 5 μl (~3 μg) of the sample purified by SIMAC and desalted and cleaned by R3/C18 allowed us to obtain 6 unknown phosphorylated peptides when using MSA on the nano-LC-LTQ ion Trap instrument. The six phosphorylated peptides were manually validated and correspond to: K.DVEDMFSR.F, R.VLVDQTTGLSR.G, K.DANLYISGLPR.T, R.SLFSSIGEVESAK.L which belong to HuR RNA binding protein gi/1022961 and; R.TAVINAASGR.Q which belongs to Chain B, Structure Of Appbp1-Uba3-nedd8-Mgatp-Ubc12 (c111a), A Trapped Ubiquitin-Like Protein Activation Complex gi/126031226, and K.DLSSIFR.G which belongs to p38 MAP Kinase gi/1469306 (Table 1).
Therefore, when using MSA by the LTQ Ion Trap instrument, SIMAC (6 phosphopeptides purified, identified and validated) efficiency is higher than TiO2 (4 phosphopeptides purified and identified) and IMAC (2 phosphopeptides purified, identified and validated) for these protein-kinases studied. It has been described that IMAC easily enriches multiple phosphorylated peptides while TiO2 mono-phosphorylated ones. In fact, SIMAC has been optimized to get the best efficiency from IMAC and TiO2 and complement both in just one method (see reference previously mentioned ). This supports our data.
In any case, we recommend that in order to study kinase phosphorylated protein kinases, combine the three resins (or ever more phosphoenrichments methods) in order to purify as many as possible phosphopeptides . The reason for this is that each sample needs to be optimized and tested with different complementary strategies. The analysis of the 5 μl (~3 μg) of the sample purified by SIMAC and desalted and cleaned by R3/C18 allowed us to get 5 unknown phosphorylated peptides when using Data Dependent Neutral Loss MS3 (DDNLMS3) on the nano-LC-LTQ ion Trap instrument. The five phosphorylated peptides were manually validated and correspond to: K.DVEDMFSR.F, K.DANLYISGLPR.T, R.SLFSSIGEVESAK.L which belong to HuR RNA binding protein gi/1022961; K.DLSSIFR.G which belongs to p38 MAP Kinase gi/1469306 and R.TAVINAASGR.Q which belongs to Chain B, Structure Of Appbp1-Uba3-nedd8-Mgatp-Ubc12 (c111a), A Trapped Ubiquitin-Like Protein Activation Complex gi/126031226 (Table 1).
All our MS analyses were carried out by CID. We hypothesize that combining CID with ETD or ECD fragmentation, it is probable that more and/or complementary data would be obtained according to the methodological study of Navajas and co-workers . ECD occurs only on the peptide backbone - which is an advantage -, and labile phosphate groups are left intact on the resulting c- and z- fragment ions, thus, complementary identification of other specific phosphorylation sites would be enabled [51, 52]. As a result, we recommend using CID to start with, and would recommend switching to ETD, in the event you were not able to determine the phosphorylation site, if you have the possibility of the required instrument [53–57]. The phosphopeptides purified, identified and validated, including also the site-assignments of the phosphate group are illustrated in Table 1.
The efficiency and reproducibility of the phosphopeptide purification and identification when using ~3 μg of protein kinases per each resin and or phosphoenrichment method (SIMAC, TiO2 and IMAC) coupled to R3/C18 and MSA-LTQ ion Trap mass spectrometer is illustrated in Figure 3.
An example of a phospho-site assignment and manual validation of the phosphorylated peptide (VLVDQTTphGLSR) obtained by Mascot analysis is illustrated in Figure 2.
Bioinformatic modelling and molecular dynamics simulations
In the case of HuR, only one of the four phosphorylated residues found (Ser-48) fall into a structure domain of the protein previously crystallized: the dimmerized first RNA recognition motif (see reference previously mentioned ). Thus, only the putative structural effect of the phosphorylated and non-phosphorylated state of Ser-48 could be analyzed through structural bioinformatic tools including Molecular Dynamics (MD) simulation.
These results suggest that the phosphorylation of Ser48 in the first protein RNA recognition motif of HuR has potentially a stabilizing effect, exerting a regulatory role on the biological function of protein by regulating the maintenance of the dimmerized quaternary state, a requirement of the complex prior to RNA binding activity .
Reversible protein phosphorylation plays an important role in the regulation of many different processes, such as cell growth, differentiation, migration, metabolism, and apoptosis. Identification of differentially phosphorylated proteins by means of phospho-proteomic analysis coupled to bioinformatic tools provides insight into signal transduction pathways that are activated in response to, for example, growth factor stimulation or toxicant-induced apoptosis . Four triplicate experiments were carried out in order to identify the phosphopeptides of the protein kinases studied in this article (p38 and HuR). The phospho-site identifications were carried out from pooled and non-pooled assays (inter- and intra-assays) confirming a high reproducibility: the 6 isolated and identified phosphorylated peptides were validated in the four triplicate analyses by Mascot (score >20, and at least 4-y and -b ions continuously matched) also including manually inspection via phosphate-fingerprints of all the spectra. It should be pointed that some spectra showed a better resolution than others. Three different resins (IMAC, TiO2 and SIMAC) were used despite the fact that it is well known that SIMAC has been developed and described as a method to purify more phosphopeptides for high-throughput analysis giving great efficiency (see references previously mentioned [34–36]). But it has also been demonstrated that the analyses of different phosphorylated proteins when using different resins (e.g. TiO2, IMAC or ZrO2) -including SIMAC- are more efficient in a complementary way for specific samples. This is due to the fact that the intrinsic characteristic of each protein (e.g. amino acid composition). In fact, in order to get as many phosphopeptides as possible from the same sample, different tests must be carried out [e.g of other possibilities of phosphoenrichments: Strong cation and anion exchange (SCX and SAX), Calcium phosphate precipitation, Hydrophilic interaction chromatography (HILIC)]. Moreover, phosphoproteomic analysis of kinases implies more difficulties as they usually are low expressed proteins with the disadvantage that the un-phosphorylated form is more abundant than the phosphorylated within the same sample to be analyzed. In addition, IMAC elutes easily multi-phopshorylated peptides than TiO2 and ZrO2. TiO2 and ZrO2 have the capacity of binding strongly the multiple phosphorylated peptides, thus both last mentioned resins retain multi-phosphorylated peptides and do not give -usually- the chance to elute them. We chose TiO2 for our research study instead of ZrO2 although they are of similar characteristics, but further experiments using ZrO2 or other resins or different possibilities of phosphoenrichments (SCX and/orHILIC for e.g) could surely give complementary and interesting data for signalling network research advances. The phosphopeptides found in the analyzed sample corresponding to p38 MAP kinase protein do not include the described phosphorylation sites in the T-loop (pThr-180 and pTyr-182). We hypothesize that the protein was not phosphorylated in those precise sites in the original sample, indicating a particular inactive state of the kinase activity [60, 61]. In addition, we detected unphosphorylated peptides after protein digestions by MS and Mascot analysis. These unphosphorylated peptides were very useful for the assured identification of the studied protein kinases. During the evaluation of the data, we found that the peptide scores of phosphopeptides assigned by Mascot did not always correlate with the quality of the spectra, and for this reason, all fragmentation spectra were manually verified, in order to avoid false positives. A peptide was accepted when at least four consecutive y- and b- ions were assigned to abundant signals in the fragmentation spectra. If this was not the case, then the amino acid sequence was inspected to look for indications of why these y/b-ions were missing. Peptides lacking a C-terminal arginine or lysine residue, due to a C-terminal position in the particular protein sequence, were accepted, when at least four consecutive b-ions had been assigned in the fragmentation spectra. Peptides with a C-terminal lysine residue and an N-terminal arginine residue due to missed cleavage were also accepted, when at least four consecutive b-ions were assigned in the fragmentation spectra. In addition, the spectra were inspected for the presence of proline residues, which usually give rise to intense signals, suppressing other signals in the spectra. A neutral loss of 98 Th due to the loss of phosphoric acid resulting from gas-phase Β-elimination of phosphoserine or phosphothreonine residues together with the presence of dehydroalanine or dehydro-2-amino butyric acid or the presence of intact phosphoserine or phosphothreonine residues in the sequence were used as indicators of a serine or threonine phosphorylated peptide, respectively. The characteristic phosphotyrosine immonium ion at 216.05 Th or the presence of an intact phosphotyrosine residue in the sequence was used for confirmation of tyrosine phosphorylation if it was the case. The analysis by the LC-nano-ESI-LTQ ion Trap and the manual validation of the MS/MS and MSA spectra was performed by EL following the rules of Mann and Jensen (see references [36, 57]). (Table 1, Figure 2, Figure 3).
One of the disadvantages of Electron Capture Dissociation (ECD) is that it has selectivity for disulfide bonds, due to the high radical affinity of the bond. Further analysis related to these protein kinases will be carried out as it preserves the intact information about labile modifications, which are not observed directly when using CID at the same time with the knowledge that the drawback of ETD is less sensitive compared to CID, because of lower ionization efficiency. Thus more complementary data would be available soon. A third phosphorylated protein (Trapped ubiquitin-like protein complex gi/126031226) was isolated and identified during the MS analysis of the p38 and HuR protein kinases. Apparently, this third phosphorylated protein seems to be linked to p38 and HuR kinases. The reason for that could be the kinase assay. The origin of the Ubiquitin-like protein activation complex protein needs to be studied in a deep way, as although p38/MAPK is a fundamental actor for the network connectivity of signalling partners, no data yet clearly implicate gi/126031226 in these processes or interactions (see references [53–59])
MD bioinformatics simulations are often good indicators of potential behaviour of protein-protein complexes; but they are only computational results, but coupled to experimental proteomics data, they give relevant clues. In fact, this study suggests that the phosphorylation of Ser48 in the first protein RNA recognition motif of HuR has potentially a stabilizing effect (Figure 5), exerting a regulatory role on the biological function of protein by regulating the maintenance of the dimmerized quaternary state, a requirement of the complex prior to RNA binding activity (see reference ). In addition, a figure illustrating the 3D position of phosphorylated Ser-279 in the structure of human MAP kinase p38beta (Figure 4) (p38B) indicates clearly that this precise phosphorylation site is located far from the ATP binding site and also far from the T-loop, indicating a different role for this residue, which opens a new research door for this relevant protein kinase [59–61].
Our proteomics studies have demonstrated that there are 6 new phosphopeptides of the protein kinases p38 and HuR during in vitro assays. This was possible by testing different resins coupled to different MS strategies to isolate and identify phosphopeptides. The identified phosphopeptides were manually validated and the phospho-site assignments were also carried out in order to avoid false positives. Recently, several signalling studies for clinical research are ongoing and each sample needs to be tested by different strategies in order to check which one adapts better to your goals, the characteristics of the proteins to be studied, and the possibility of getting complementary data. The experiments carried out showed excellent reproducibility in the four triplicate experiments for the identification and validation of the identified phosphopeptides, also for the protein identifications of p38 and HuR kinases. A Trapped ubiquitin-like protein complex gi/126031226 was also isolated and identified containing one phosphopeptide during this study. The origin of the Ubiquitin-like activation complex protein needs to be studied in a depth as although p38/MAPK is a fundamental actor for the network connectivity of signalling partners, no data yet clearly implicate gi/126031226 in these processes or interactions. Overall, the combination of SIMAC and MSA resulted more efficient to isolate and identify phosphopeptides from p38 and HuR protein kinases. It should be pointed out that when using other phosphoenrichment alternatives (SCX and HILIC) coupled to other MS strategies for this study, we will be able to corroborate even more complementary data related to the results presented.
Bioinformatic studies, using structural tools, of reversible phosphorylation in proteins will allow the generation of useful models for protein-protein contacts at the atomic level. These models can be used as input for sophisticated techniques, as molecular dynamics, that offer the possibility of analyzing the potential effect of the phosphorylation and de-phosphorylation process of each residue in the global structure and behaviour of the protein. In the present work, the molecular dynamics analysis of the phosphorylation of Ser-48 in HuR homodimmer offered a suitable hypothesis of how the phosphorylation state of Ser-48 can regulate the protein mechanism through controlling the homodimmerization arrangement of the corresponding structural domain. Regarding the 3D position of phosphorylated Ser-279 in the structure of human MAP kinase p38, molecular simulation clearly indicates that this precise phosphorylation locus is located far from the ATP binding site and also far from the T-loop, indicating a different role for this residue: this could open a new and relevant research door for this important protein kinase, which has been related as a connectivity-link for different signalling pathways.
List of Abbreviations
- AQUA :
- CID :
- Da :
Dalton (molecular mass)
- DIGE 2-D :
Fluorescence Difference Gel Electrophoresis
- ECD :
Electron Capture Dissociation
- ESI :
Electron Spray Ionization
- ETD :
Electron Transfer Dissociation
- FT-ICR :
Fourier transform-Ion Cyclotron Resonance
- HILIC :
Hydrophilic interaction chromatography
- HPLC :
High-performance liquid chromatography or high-pressure liquid chromatography
- H 3 PO 4 :
- ICR :
Ion Cyclotron Resonance
- IMAC :
Immobilized Metal Affinity Capture
- IT :
- iTRAQ :
Isobaric Tag for Relative and Absolute Quantification
- kDa :
kilodalton (molecular mass)
- LC :
- MALDI :
Matrix-Assisted Laser Desorption/Ionization
- MD :
- MOAC :
Metal Oxide Affinity Chromatography
- Mr :
Relative molecular mass (dimensionless)
- MRM :
Multiple reaction monitoring
- MS :
- MSA :
- MS/MS :
tandem mass spectrometry
- m/z :
Mass to charge ratio
- PID :
- PTM :
- SILAC :
Stable Isotope Labelling with Amino acid in cell Culture
- SIMAC :
Sequential Elution from IMAC
- TiO 2 :
- TOF :
Time Of Flight
- ZrO 2 :
EL PhD is a recipient of a Postdoctoral Fellowship (Programme Clinical Proteomics of Ministerio de Ciencia e Innovación de España (MICINN). IL, MD PhD is a recipient of a Grant from "Fundación Leucemia & Linfoma de España). JL and AF are MD PhD and hold a tenured position at Spanish National Hospitals Carlos III and La Paz respectively. We are very grateful to Biomol-Informatics S.L. (CSIC Bioinformatics Group of Severo Ochoa, Prof. Paulino Gómez-Puertas) for the welcomed and useful discussions. Special thanks Prof. Rune Matthiesen (Institute of Molecular Pathology and Immunology of the University of Porto) who contributed to the publication of this article. We also thank the great discussions of p38 and HuR protein kinases (Prof. Ángel R. Nebreda and Vanesa Lafarga PhD). Thanks also to the Molecular Modelling Group (CSIC-UAM), and Prof. Keith Ashman for allowing use of the LTQ.
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