Voyager de pro mass spectrometer

For peptide assay, mTOR complex purified from 293T cells (∼1000 nM) were incubated with 2 μg IRF3 peptide in the presence of 0.5 μM okadaic acid and ATP buffer (50 mM Tris -HCl, pH 7.5, 2 mM ATP, 5 mM MgCl2, 0.5 mM DTT) and incubated at 30°C for 60 minutes. The reaction mixture supernatant were then desalted and enriched using a μ-C18 Zip Tip (Millipore, ZTC18S096) and eluted directly onto an MALDI plate containing 2 mL CHCA (a-cyano-4-hydroxycinnamic acid) saturated solution in 50% acetonitrile (ACN) and 0.1% trifluoroacetic acid (TFA). MALDI-TOF spectra were acquired on a Voyager DE Promass spectrometer from Applied Biosystems.
For protein assay, mTOR complex purified from 293T cells (∼1000 nM) were incubated with 2 μg GST-IRF3 in the presence of 0.5 μM okadaic acid and ATP buffer (50 mM Tris -HCl, pH 7.5, 2 mM ATP, 5 mM MgCl2, 0.5 mM DTT) and incubated at 30°C for 60 minutes. The reaction mixtures were analyzed by western blot with indicated antibodies.

With the purpose of confirming chromatographic results, DP of synthesized OsLu was also determined by matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry (MALDI–TOF–MS). Analysis were carried out on a Voyager DE-PRO mass spectrometer (Applied Biosystems, Foster City, CA, USA) equipped with a pulsed nitrogen laser (λ = 337 nm, 3 ns pulse width, and 3 Hz frequency) and a delayed extraction ion source. Ions generated by laser desorption were introduced into a time-of-flight analyzer (1.3 m flight path) with an acceleration voltage of 25 kV, 94% grid voltage, 0.025% ion guide wire voltage, and a delay time of 200 ns in the reflector positive ion mode. Mass spectra were obtained over the m/z range of 500–5000. External mass calibration was applied using the monoisotopic [M + H]⁺ values of des-Arg1 bradykinin and angiotensin I of calibration mixture 1 of the Sequazyme Peptide Mass Standards Kit (Applied Biosystems). 2,5-Dihydroxybenzoic acid (>98%, Fluka) at 10 mg/mL in water was used as the matrix. The sample was diluted 100 times in water and mixed with the matrix at a ratio of 1:4 (v/v). One microliter of this solution was spotted onto a flat stainless steel sample plate and dried in air before analysis.

CMPs were synthesized on glycine‐preloaded Wang or polyethylene glycol‐based resins using Fmoc‐protected monomers from Chem‐Impex, as described previously.^[19 (link)
^] Condensation of Fmoc‐ProHypGly‐OH,^[11b (link)
^] Fmoc‐GlyProHyp‐OH (Bachem), or Fmoc‐GlyPro‐OH (Chem‐Impex) segments was employed wherever applicable. Isolated crude peptides were purified, as described previously,^[16 (link)
^] to >95% purity according to analytical HPLC and MALDI–TOF mass spectrometry (MS). MS analyses were carried out on an Applied Biosystems Voyager DE‐Pro mass spectrometer at the University of Wisconsin–Madison Biophysics Instrumentation Facility (UW–BIF; www.biochem.wisc.edu/bif). m/z calcd for 11/3sb variants [M + H]⁺, 3057.2; found, 3057.1 for OKD; found, 3057.4 for KDO; found, 3057.0 for DOK; found, 3057.3 for DOKctrl. Analytical HPLC results for purified CMPs are shown in Figure S1 (Supporting Information). m/z calcd for 8/2sb‐OKD [M + H]⁺, 2222.3; found, 2222.4. Analytical HPLC chromatograms of purified peptides are presented in Figures S1 and S2A (Supporting Information).

8701-BC cells were grown for 48 h, medium was collected, and proteins quantified with Bradford reagent. Amounts of 2.5, 5 and 10 µg of total proteins were loaded in SDS PAGE. After the incubation of the gel with a Blue Coomassie solution, the most evident band at 55 kDa was excised from the gel and analyzed by MALDI-TOF mass spectrometry [74 (link)].
Briefly, the spot from the gel was destained, reduced, alkylated, and submitted to hydrolysis with endoproteinase Glu-C (SIGMA-Aldrich, Milan, Italy). Peptide mixtures were analyzed by MALDI-TOF mass spectrometry using a Voyager DE-PRO mass spectrometer (Applied Biosystems, Monza, Italy). Samples were freeze-dried and then dissolved in 10 μL of 0.2% trifluoroacetic acid; 1 μL was mixed with 1 μL of a solution of alpha-cyano-4-hydroxycinammic acid, 10 mg/mL in acetonitrile, 0.2% trifluoroacetic acid 7:3 (v:v), and the mixture was applied onto the metallic sample plate and air dried. Mass calibration was performed using a peptide standard mixture provided by the manufacturer. All mass values are reported as monoisotopic masses, and raw data were analyzed using software provided by the manufacturer.