Sequencing grade modi ed trypsin

Total protein was extracted using the chilled acetone precipitation method according to Wei et al. (2020) with some changes. Brie y, samples were rstly ground into powder in liquid nitrogen, then dissolved in 2 mL lysis buffer containing 8M Urea, 2% SDS, 1× Protease Inhibitor Cocktail, followed by sonication on ice for 20 min and centrifugation at 12 000 rpm for 10 min at 4 ℃. The supernatant was pipetted into a new centrifuge tube. Protein was precipitated with chilled acetone for 16 h at -20 ℃, cleaned with acetone two times, re-dissolved in 8M Urea, and homogenized in ice for 3 min using an ultrasonic homogenizer. The homogenate was centrifuged at 12000 rpm for 15 min at 4 ℃, then the supernatant was preserved and the protein concentration was gauged by the BCA Protein Assay Kit (Thermo Fisher, Waltham, MA). A total of 50 µg protein were suspended in 50 µL 50 mM ammonium bicarbonate, reduced by 10 mM dithiothreitol for 1 h at 55 o C and alkylated by 20 mM iodoacetamide for 1 h at 37 o C in the dark. Thereafter, each sample was precipitated with 500 ul chilled acetone for 16 h at -20 ℃. The precipitates were washed three times with chilled acetone and resuspended in 50 mM ammonium bicarbonate. Finally, all samples were digested with sequencing-grade modi ed trypsin (Promega, Madison, WI) at a substrate/enzyme ratio of 50:1 (w/w) at 37°C for 16 h.

NuPAGE gels (4~12%) were acquired from Invitrogen and PIERCE (Rockford, IL, USA). Sequencing-grade modi ed trypsin was purchased from Promega (Madison, WI, USA). Acetonitrile (ACN; MS grade), water (MS grade), and formic acid (FA; ACS regent grade) were acquired from Aldrich (Milwaukee, WI, USA). Seven isotope-labeled peptides were synthesized as internal standards for MRM assay (AnyGen Co., Gwangju, Korea): TEDTIFL*R, VANYVDWI*NDR, ITLPDFTGDL*R, ELLESYI*DGR, VSLATV*DK, NALALFVLP*K, and AADDTWEP*FASGK for alpha-1-acid glycoprotein 1 (AGP1), hepatocyte growth factor activator (HGFA), lipopolysaccharide-binding protein (LBP), prothrombin, selenoprotein P (SeP), thyroxine-binding globulin (TBG), and transthyretin, respectively. * represents amino acid labeled with 13 C 15 N heavy isotope.

The pelleted of different stages mt-nucleoid were washed with25mM NH 4 HCO 3 solution for 20 min, and then dried in a vacuum centrifuge. Resuspended the protein 50μl 25 mM NH 4 HCO 3 solution with 12.5 ng/μl trypsin (Sequencing Grade Modi ed Trypsin, Promega), and incubated for 12 to 16 h at 37℃. An 50μl solution containing 5% (w/v) formic acid and 50% (w/v) acetonitrile was added to the pelleted, agitated for 1 h, and moved into a new tube. Then repeated with solution containing 2.5% (w/v) formic acid and 50% (w/v) acetonitrile for 1 h to to recover the tryptic peptides.
The tryptic peptides were puri ed with a PepClean C-18 spin column (Pierce). Pellets of tryptic peptides were dissolved in 10 to 20 μl of 0.1% (w/v) formic acid for LC-MS/MS analysis. Data were analyzed with an in-house Mascot search program (v. 2.2.06; Matrix Science) against the UniProtKB/Swiss-Prot Viridiplantae database (green plants, 28,773 protein entries). The database search for protein identi cation was performed as described (Lo et al. 2011) . All stages of data are from duplicate studies.