Trypsin

Protein spots were excised manually and destained with 50% acetonitrile in 25 mM ammonium bicarbonate (NH₄HCO₃) until transparent, then washed with 200 mM NH₄HCO₃ to remove the dye. The gel plugs were vacuum-dried, then incubated overnight at 37 °C with 25 µL of 25 mM NH₄HCO₃ containing 15 ng/µL trypsin (Agilent Technologies, Santa Clara, CA, USA). The resulting peptides were extracted using 50% and 100% acetonitrile, and subsequently vacuum-dried for MS analysis. Prior to MS analysis, dried peptides were reconstituted in 0.1% formic acid, desalted using ZipTip C18™ pipette tips (Millipore, Burlington, MA, USA) and cocrystallized by adding one volume of the same to one volume of α-hydroxy cinnamic acid (10 mg/mL) after which, 0.7 µL was spotted directly on Opti-Tof 384 well (Applied Biosystem/Thermo Fisher Scientific, Bedford, MA, USA). The peptides were analyzed using the 4800 Plus MALDI TOF/TOF analyzer (Applied Biosystems/Thermo Fisher Scientific, Bedford, MA, USA) located at the Medical Biotechnology Laboratory of the Faculty of Medicine, University of Malaya, with the mass standard kit (Applied Biosystems/Thermo Fisher Scientific, Bedford, MA, USA) as the calibrator for the resulting MS and MS/MS mass spectra scales.

Three exosome samples from different individuals were digested with trypsin (Agilent, USA) or Glu-C (Promega) in solution and purified by C18 column (GL Sciences, Tokyo, Japan) as reported previously [12 (link)], and then each sample was analyzed in triplicate by LC–MS/MS (Bruker nanoElute UHPLC—Bruker timsTOF pro, 115 min gradient method). The proteins were identified by Mascot search engine (v2.3.1, Matrix Science) with search parameters; variable modifications: phosphorylation (ST), peptide mass tolerance: 50 ppm, fragment mass tolerance: 0.05 Da, max missed cleavages: 2, false discovery rate: < 1%, protein database: Uniprot-Swissprot (n = 20,386).

SILAC-labeling of porcine kidney cells (IBRS-2) was performed in lysine- and arginine-free DMEM (Gibco) supplemented with 10% (v/v) dialyzed FBS together with unlabeled lysine and arginine [“light” label (L), mock-infection] or lysine 13C6 and arginine 13C6, 15N4 [“heavy” label (H), FMDV infection, MOI = 5]. At 6 h post-infection (hpi), IBRS-2 cells were washed three times with ice-cold phosphate-buffered saline (PBS) and harvested on ice. Cell lysis was performed similarly to what has been previously described. Briefly, the harvested “heavy” and “light” labeled cells were lysed with lysis buffer supplemented with Phosphatase Inhibitor Cocktail Set II and Protease Inhibitor Cocktail Set IV on ice using a high intensity ultrasonic processor (Scientz) for 30 min, respectively. After mixing heavy and light labeled proteins (1:1), trypsin (Promega) was added into protein solution with ratio of trypsin to protein at 1:50 (w/w) for digestion at 37°C for 16 h. The sample was then fractionated into fractions by high pH reverse-phase high-performance liquid chromatography (HPLC) using Agilent 300Extend C18 column. Peptide mixtures were first incubated with Ti4 + IMAC microspheres suspension with vibration. The supernatant containing phosphopeptides was collected and lyophilized for LC-MS/MS analysis.

Myc-tagged GKRP was immunoprecipitated from HeLa cells using an anti-Myc antibody. After immunoblot, protein bands were excised from stained one-dimensional electrophoresis gels and destained with 25 mM ammonium bicarbonate and 50% acetonitrile. In-gel digestion of dried gel pieces was performed using sequencing grade trypsin (Promega, Madison, WI, USA) in 25 mM ammonium bicarbonate buffer overnight at 37 °C. The tryptic peptides were desalted using a GELoader tip (Eppendorf, Hamburg, Germany) packed with 1.5 μg of POROS® 20 R2 resin (PerSpective Biosystems, Ramsey, MN, USA) and applied to a C18 RP-HPLC column (75 m × 150 mm). An Agilent 1100 Series LC system was then used to separate the trypsin-digested peptides, which were eluted with a 0–40% acetonitrile gradient for 60 min. followed by analysis using a Finnigan LCQ Deca (ThermoQuest, San Jose, CA, USA) equipped with a nanoelectrospray ion source. Spray and tube lens voltages were 1.9 kV and 40 V, respectively. The capillary temperature was maintained at 250 °C at 5 V. The individual LC-MS/MS spectra were processed using TurboSEQUEST software (ThermoQuest) and the sequences were searched in NCBI databases using MASCOT software (Matrix Science Ltd., London, UK).

Total urine protein amount used in trypsin digestion was adjusted to less than 20 µg according to the protein concentration calculated by protein assay. A substantial amount of protein containing solution was taken from the precipitated sample tube, and the sample was adjusted to a volume up to 100 µL with 8 M urea/50 mM Tris-HCl (pH 8.0) buffer. The sample was treated with 2 µL of 1 M dithiothreitol (DTT) and 8 µL of 500 mM Iodoacetamide (IAA) at RT for 1 h. Then, the sample was re-treated with 1 µL of 1 M DTT for neutralizing the remaining IAA. After the treatments, the sample was diluted with 700 µL of 50 mM Tris-HCl (pH 8.0). Then, the proteins were incubated with 1 µg of trypsin (Agilent, Santa Clara, California, USA) and activated with 50 mM Ammonium bicarbonate (ABC) (pH 7.8) at 37 °C for 16 h with shaking. After the protein digestion, trypsin reaction was terminated with 1 µL of 50% trifluoroacetic acid (TFA).

Precipitated proteins were dissolved in 100 µL of 8 M urea/50 mM Tris-HCl (pH 8.0) buffer. The sample was treated by 1 µL of 1 M dithiothreitol at RT for 1 h and 8 µL of 500 mM iodoacetamide at RT for 1 h with shading. The alkylation was stopped by 1 µL of 1 M dithiothreitol and then diluted eight times by 50 mM Tris-HCl (pH 8.0). For the digestion, 1 µg of trypsin (Agilent, Santa Clara, CA, USA) was added to the sample and incubated at 37 °C for 16 h with shaking. The digestion was stopped by 50% of trifluoro acetic acid (TFA).
The digested sample was purified by C18 spin column (GL Science, Tokyo, Japan) according to the manual. Briefly, a C18 spin column was activated by 100% and 50% acetonitrile sequentially and then equilibrated by 0.2% formic acid with centrifuging at 3000 g for 30 s. After conditioning, the sample was loaded into the spin column and centrifuged at 3000 g for 90 s. Then, trapped peptides were washed with 0.2% TFA twice and eluted by 95% acetonitrile with 5% formic acid. The eluted sample was dried up by VEC-260 vacuum dryer (Iwaki, Tokyo, Japan). The sample was re-suspended by 0.1% formic acid and the peptide concentration was measured by Nano drop 1000 (Thermo Fisher Scientific, Bremen, Germany). The sample was stored at −80 °C until use.