Ammonium bicarbonate, 2,5-dihydroxybenzoic acid (DHB), α-cyano-4-hydroxycinnamic acid (α-cyano), and formic acid (FA) were purchased from Sigma-Aldrich Chemical Company (St. Louis, MO). Acetonitrile (ACN) was purchased from Caledon (Georgetown, Ontario, Canada), trifluoroacetic acid (TFA) was acquired from Pierce (Rockford, IL), and phosphoric acid (PA) was from EMD Chemicals (Gibbstown, NJ). All solvents were HPLC grade. Sequencing-grade modified porcine trypsin was purchased from Promega Corporation (Madison, WI). All solutions and buffers were prepared using deionized water (18 MΩ) from an in-house Hydro Services (Durham, NC) Picopure 2 water system. Compact reaction columns (CRC) and filters (10 μm pore size) were obtained from USB Corporation (Cleveland, OH). Titansphere® TiO2 resin was a gift from GL Sciences (Torrance, CA).
2.2 Cloning and Expression of the Intracellular Domain of Mouse CD34
The intracellular domain (amino acids 309 to 382) of mouse CD34 (AAH06607) was amplified from mouse keratinocytes isolated from CD-1 mice as described previously [3
] then expressed using the Gateway system from Invitrogen (Carlsbad, CA). The fragment was PCR cloned in 2 steps. The first round of PCR introduced a stop codon and the attB2 recombination site with the following primers: 5’
. The product from the first round of PCR was gel purified and used as a template for the second round. The second round of PCR introduced the attB1 site using the forward primer 5’
and the same reverse primer from round 1. The PCR product from the second round was gel purified and recombined into pDONR221. The gene fragment was then migrated to pDEST17 for expression with an N-terminal His tag. The His-CD34 fragment was expressed using the E. coli
strain Rosetta(DE3)pLacI (Novagen, EMD Chemicals, Inc., Gibbstown, NJ). The cells were grown in LB media and induced with 0.2 mM IPTG at 30 °C. Four hours after induction the cells were harvested and stored at −80 °C until used.
2.3 Purification of the His-CD34 domain fusion
The cell pellet was suspended in B-PER (Pierce, Rockford, IL) containing EDTA free complete protease inhibitor cocktail (Roche, Indianapolis, IN). After a 10 minute incubation at room temperature, the sample was cooled on ice and sonicated 3 times for 30 seconds using continuous bursts at max power with a Branson Sonifier 450 (Danbury, CT). Insoluble material was removed by centrifugation at 27,000 × g for 30 min. The soluble protein was then flow loaded onto a Ni-NTA (QIAGEN, Inc, Valencia, CA) column pre-equilibrated with 50 mM HEPES (pH. 7.5), 200 mM NaCl, and 10 mM imidazole. Bound protein was eluted with a linear gradient from 10 mM to 500 mM imidazole. The His-CD34 fusion protein eluted as a single peak centered at ~175 mM imidazole. The peak fractions were pooled and dialyzed overnight at 4° C in 20 mM MOPS (pH 7.2) prior to use in the PKC assay to ensure buffer compatibility.
2.4 Protein Kinase C Assays
In vitro PKC phosphorylation of the purified CD34 fusion protein (20 mM MOPS, pH 7.2) was performed using a PKC Isoform Panel Miniature Set (Millipore/Upstate Biotechnology, Inc., Lake Placid, NY) per the manufacture’s recommendations. Briefly, just prior to each assay, the isoforms were diluted to a concentration of 10–20 ng/μl in 20 mM HEPES (pH 7.4), 2mM EDTA, 2 mM EDTA, 5 mM DTT, 100 mM NaCl, 0.05% Triton X-100, and 50% glycerol. Depending upon the isoform, CD34 was diluted in the following buffers (supplied in the kit): 20 mM MOPS (pH 7.2), 25 mM β-glycerophosphate, 1 mM sodium orthovanadate, 1 mM DTT, and 1 mM CaCl2 for PKC α , βI, βII, and γ , and 20 mM MOPS (pH 7.2), 25 mM β-glycerophosphate, 1 mM sodium orthovanadate, and 1 mM DTT for PKC δ , ε, ζ , and η. Each individual kinase reaction consisted of 500 ng CD34 substrate, 25 ng of each PKC isoform, 1 μCi of [γ-32P]-ATP (3000 Ci/mmol) (DuPont-NEN), 5 μg phosphatidylserine, and 0.5 μg diglycerides. The kinase reactions were incubated at 30 ºC for 15 minutes. To verify the specificity of PKC, 1 μM of inhibitor residues 19–31 pseudosubstrate was added 1 hr prior to reaction. Following the kinase reactions, SDS-PAGE was performed using 4–12% Bis-Tris NuPAGE SDS gradient gels (Invitrogen, Carlsbad, CA). Following electrophoresis, the gels were stained with Coomassie brilliant blue R250 (CBB) R-250 and subjected to autoradiography using Typhoon 8600 phosphorimager (GE Healthcare Biosciences).
2.5 Other Kinase Assays
The CD34 fusion protein (0.5 μg) was mixed together with active AKT2, JNK2, or IKKβ kinase (Millipore/Upstate Biotechnology, Inc., Lake Placid, NY), 1 μCi of [γ-32P]-ATP (3000 Ci/mmol) and 1 X kinase buffer. Kinase reactions were performed according to the manufacturer's protocol using the supplied reagents with the assay. The kinase reaction mixtures were incubated at 30 ºC for 15 min followed by separation using a 15% NuPAGE gel (Invitrogen, Carlsbad, CA) and exposed to X-ray film for autoradiography.
2.6 Solution Digestion Conditions
Following the protein kinase assay, the protein samples were subjected to tryptic digestion by adding sequencing-grade modified porcine trypsin at a protein:enzyme ratio of 20:1. The reactions were allowed to proceed overnight at 37 °C.
2.7 Cloning, Expression, and Purification of Full-Length Mouse CD34
Using mouse integrin α 6+CD34+ keratinocyte stem cells as a source of total RNA, the full-length CD34 was amplified from cDNA (by RT-PCR using a pair of gene specific primers containing a Kozak sequence) and was cloned directionally into the vector pcDNA3.1D/V5-His (Invitrogen, Carlsbad, CA). The mouse CD34 full-length cDNAs were then subcloned and ligated into the protein expression vector pFLAG-CMV-5c (Sigma-Aldrich, St. Louis, MO) using Hind III/Not I restriction enzyme sites. All DNA sequences were verified by the NIEHS DNA Sequencing Core Facility using a BigDye Terminator kit and automated sequencer (Applied Biosystems, Foster City, CA).
For protein expression studies, the HEK293F cells were transiently transfected either with empty vector (pFLAG-CMV-5c) or plasmid containing wild-type CD34 cDNA using Lipofectamine PLUSTM reagents (Invitrogen, Carlsbad, CA). After 48 hours of incubation, total protein lysates were obtained using the radioimmunoprecipitation assay (RIPA) buffer including protease inhibitors (aprotinin, leupeptin, pepstatin and PMSF) and phosphatase inhibitors (sodium orthovanadate and NaF), followed by centrifugation at 14,000xg for 15 min at 4 ºC. Protein concentration was determined by BCA protein assay (Pierce, Rockford, IL).
CD34 protein was immunoprecipitated at 4ºC overnight with EZ View Red resin-bound anti-FLAG M2 monoclonal antibody (Sigma-Aldrich). Immunoprecipitates were centrifuged at 2500 rpm for 3 min at 4 ºC and washed four times with RIPA buffer. The CD34-FLAG bound fractions were eluted with an excess of FLAG3 peptide (Sigma-Aldrich) (100 μg/mL), concentrated with acetone (1/5 = v/v), subjected to 4–12% Bis-Tris NuPAGE SDS gradient gel (Invitrogen, Carlsbad, CA), and stained with CBB R250.
2.8 In-gel Digestion
The protein band corresponding to full-length CD34 was manually excised from the gel, cut into small pieces, and transferred into a 96-well microtiter plate. Gel pieces were subjected to automatic tryptic digestion using an Investigator Progest protein digestion station (Genomic Solutions, Ann Arbor, MI). Briefly, gel bands were sequentially washed twice with 25 mM ammonium bicarbonate buffer (pH 7) and acetonitrile, dehydrated, rehydrated with 25 μL of the enzyme solution, and digested at 37 ºC for 8 hrs. The enzyme solution used was sequencing grade modified trypsin at a concentration of 0.01 mg/mL in 25 mM ammonium bicarbonate buffer (pH 7). Resulting tryptic peptides were extracted from the gel, lyophilized, and stored at −80 ºC. Prior to mass spectrometric analysis, the peptides were reconstituted in 40 μL of a 97:3 solution of water:acetonitrile (0.1% formic acid).
2.9 Titanium Dioxide Enrichment
The titanium dioxide (TiO2
) phosphopeptide enrichment procedure was adopted from Larsen, et al. [21
]. Briefly, 1 mg of Titansphere TiO2
resin was added to 10 μL bead buffer (80% ACN, 19.9% H2
O, 0.1% TFA) and applied to a CRC tube. Binding of phosphopeptides to the TiO2
column was achieved by first draining the bead buffer, then loading the protein digestion mixture with 20 μL protein buffer (100 mg/mL DHB in 80% ACN, 19.9% H2
O, 0.1% TFA). The mixture was allowed to incubate at room temperature for 10 minutes, after which, the column was drained and washed 1 × 30 μL protein buffer and 1 × 30 μL bead buffer. Elution of the bound peptides was accomplished by adding 5 μL of 200 mM ammonium bicarbonate buffer (pH 10.5) to the column and incubating for 5 minutes at room temperature. The 5 μL elution solution was then drained and diluted with 5 μL water. 0.5 μL of the eluent solution was spotted on a MALDI target with 0.5 μL of DHB matrix (20 mg/mL DHB in 50% ACN, 49% H2
O, 1% PA).
2.10 MALDI Mass Spectrometry
MALDI analyses were performed using either an ABI Voyager Super DE-STR or an ABI 4700 Proteomics Analyzer (Applied Biosystems, Inc., Framingham, MA) in the positive ion reflector mode. For sample analyses not involving titanium dioxide enrichment, the MALDI matrix was prepared initially as a saturated solution of α-cyano-4-hydroxycinnamic acid in 50:50 acetonitrile:water containing 0.1% FA (v/v). This saturated solution of α-cyano was then diluted 1:2 (v/v) with 50:50 acetonitrile:water containing 0.1% FA, of which 0.3 μL is mixed with 0.3 μL of the peptide digestion solution on a 100-well MALDI sample target. All MALDI spectra were obtained over the mass range of 700–4000 Da with 1000 laser shots per spectrum. For each sample analysis on the ABI 4700 Proteomics Analyzer, data dependent acquisitions were acquired in a fully automated mode such that a MALDI mass spectrum is acquired followed by MS/MS of the five most abundant ions in the spectrum (excluding ions from matrix and trypsin autolysis products). Additionally, ions that matched in nominal mass to putatively phosphorylated tryptic peptides of CD34 were also further interrogated manually by MS/MS. In the MS/MS mode, 1kV was used for the fragmentation energy. Ions corresponding in mass to trypsin autolysis products were used to internally calibrate in the MS mode; thereby, allowing a routine mass accuracy of 10 ppm or less, while in the MS/MS mode an external calibration was employed using the fragment ions of Angiotensin 1 (m/z 1296.6853). Following the analyses, mass spectra were processed manually or analyzed using a Global Proteome Server Explorer workstation and software (Applied Biosystems, Framingham, MA).
2.11 Electrospray Mass Spectrometry
For the intact protein analyses, a Waters Micromass Q-Tof Ultima Global (Milford, MA) hybrid tandem mass spectrometer was used. This instrument is equipped with a nanoflow electrospray source and consists of a quadrupole mass filter and an orthogonal acceleration time-of-flight mass spectrometer. The needle voltage was ~3500 V, collision energy was 10 eV, the scan range was 300–3000, and the scan time for the MS analyses was 1.9 sec/scan. For the LC analyses, a Waters CapLC HPLC system (Milford, MA) consisting of binary pumps (solvent A=water, 0.1% formic acid and solvent B=acetonitrile, 0.1% formic acid) and a micro autosampler was used to deliver the gradients. Injections of 5 picomoles of CD34 were made onto the column. For the chromatographic separations, the gradients were as follows: 5% B for 30 minutes, then a linear gradient of 5–95% B over 60 min followed by ten minutes at 95% B. The column used was a 5 cm × 75 μm C4 PepMap300 column (LC Packings, San Francisco, CA) at a flow rate of 500 nL/min.
For the LC/MS/MS analyses, an Agilent Technologies XCT Ultra ion trap (Santa Clara, CA) was used. The Agilent XCT Ultra ion trap is equipped with an HPLC-Chip Cube MS interface and an Agilent 1100 nanoLC system. Injections of 30 μL from a 1:10 dilution of the peptide digests were made onto a 40 nL enrichment column followed by a 43 mm × 75 μm analytical column packed with ZORBAX 300SB C18 particles. Peptides were separated and eluted using a linear gradient of 3–50% acetonitrile (0.1% formic acid) over 40 min, followed by a linear gradient of 50–95% acetonitrile over 7 min at a flow rate of 500 nL/min. The ion trap mass spectrometer was operated in the positive ion mode, standard enhanced mode using the following settings: capillary voltage, −2150 V; mass range, 300–1500; ICC smart target (number of ions in the trap prior to scan out), 100000 or 200 milliseconds of accumulation; and MS/MS fragmentation amplitude, 1.0 V. During the LC/MS/MS analyses, automated data dependent acquisition software was employed with the six most abundant ions (threshold requirement of 10000 counts) from each spectrum selected for MS/MS analysis. Following the analyses, the MS/MS data were extracted and analyzed using Spectrum Mill MS Proteomics software (Agilent Technologies, Inc). To generate peak lists, the raw data files were processed using the Data Extractor function with the following parameters: deconvoluted ions of 300–6000 Da and a retention time of 10 to 60 min. MS scans with the same precursor m/z were merged based on a +/− 1.4 m/z window and a +/− 15 sec retention time window. Using the extracted data, searches were performed against an in-house database using the MS/MS search function. All sites of phosphorylation were manually validated.