EV Protein Extraction and In-Gel Digestion

EVs in PBS (15 μg protein) were concentrated in a SpeedVac (Thermo Fisher Scientific) to ∼20 μl and prepared in Laemmli sample buffer (BioRad 1610747) containing 10 mM DTT. Samples were loaded into a 4%–12% NuPAGE Bis‐Tris gel (Invitrogen) and were run until samples travelled 2 cm in the gel. In‐gel digestion was performed as previously described (Yang et al., 2015 (link)). Peptides were extracted with 50% acetonitrile/0.1% formic acid, dried in a SpeedVac, resuspended in 0.1% formic acid, and quantified with a NanoDrop One spectrophotometer (Thermo Fisher Scientific, 205 nm).

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Correll V.L., Otto J.J., Risi C.M., Main B.P., Boutros P.C., Kislinger T., Galkin V.E., Nyalwidhe J.O., Semmes O.J, & Yang L. (2022). Optimization of small extracellular vesicle isolation from expressed prostatic secretions in urine for in‐depth proteomic analysis. Journal of Extracellular Vesicles, 11(2), e12184.

Publication 2022

SpeedVac Laemmli sample buffer NuPAGE Bis‐Tris gel NanoDrop One spectrophotometer

Acetonitrile Bis tris Digestion Formic acid Laemmli buffer Peptides Protein

Corresponding Organization : Eastern Virginia Medical School

Other organizations : University of Toronto, University of California, Los Angeles, University Health Network, Princess Margaret Cancer Centre

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Peptide samples extracted and quantified

control variables

Protein amount (15 μg) used for EV preparation
Laemmli sample buffer with 10 mM DTT used for sample preparation
4%-12% NuPAGE Bis-Tris gel used for protein separation
In-gel digestion protocol followed as previously described (Yang et al., 2015)
Peptides extracted with 50% acetonitrile/0.1% formic acid and dried in a SpeedVac
Peptides resuspended in 0.1% formic acid and quantified using a NanoDrop One spectrophotometer

controls

No positive or negative controls explicitly mentioned

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