Proteins of Mycoplasma bovis biotinylation in vitro

For the DB approach, M. bovis was cultured in 10 mL PPLO medium for 36 h and precipitated by centrifugation at 12,000 × g for 5 min. The pellet of M. bovis was suspended in 2 mL of phosphate-buffered saline (PBS) with 1× Halt Protease Inhibitor Cocktail (100×) (Thermo Fisher Scientific, Rockford, USA) and lysed by sonication at 200 W on ice for 15 min for a total of 60 cycles. Each sonication cycle was as follows: sonication 5 s, resting period 10 s. Whole cell lysate proteins of M. bovis were isolated in the supernatant after centrifugation at 12,000 × g for 10 min. Proteins concentration was measured with a BCA kit (Beyotime, China). Proteins were then labeled with biotin using the EZ-Link™ Sulfo-NHS-LC-Biotinylation Kit (Thermo Scientific, Rockford, USA). Biotin is a small naturally occurring vitamin that binds with high affinity to avidin and streptavidin proteins. Because of its size, biotin can be conjugated to many proteins without altering their biological activities. N-Hydroxysuccinimide (NHS) ester-activated biotins are the most popular type of biotinylation reagent. NHS esters react efficiently with primary amino groups (-NH2) in pH 7–9 buffers to form stable amide bonds. Because proteins generally contain multiple lysine (K) residues in addition to the N-terminus of each polypeptide, they have multiple primary amines available as targets for labeling with NHS-activated reagents. Generally, 10 mg whole cell proteins in 0.5 mL PBS mixed with 135 μL 10 mM Sulfo-NHS-LC-Biotin solution was used. The reaction was incubated on ice for 2 h and excess biotin reagent was removed using a desalting column.

BoMac cells at 80% confluency in a 10 cm dish were treated with 10 mg biotin- labeled proteins for 24 h. Cytoplasmic and nuclear proteins of BoMac cells were extracted using a Minute™ Cytoplasmic and Nuclear Fraction Kit (Invent, Beijing, China) according to the manufacturer’s instructions. Western blotting was used to assess contamination the nuclear proteins with cytoplasmic proteins. Proteins were resolved by SDS-PAGE and transferred to a PVDF membrane. Immunodetection was achieved with antibodies against α-tubulin as the internal reference for cytoplasmic proteins (Abcam, Cambridge, UK) or PARP (Abcam, Cambridge, UK) for nuclear proteins. Proteins were visualized with a WesternBright™ ECL western blotting detection kit (Advansta, San Jose, CA, USA). Nuclear proteins were incubated with 100 μL Dynabeads™ M-280 Streptavidin (Invitrogen, Carlsbad, CA, USA) for 2 h. After five washes with PBS, proteins were subjected to LC–MS/MS analysis (Applied Protein Technology Co. Ltd., Shanghai, China) for sequencing.