Trypsin protease

Proteolysis was performed on Se7942 McdB at 30 µM in buffer containing 150 mM KCl, 50 mM HEPES, pH 7.7, and 2 mM BME. Trypsin protease (Thermo Fisher) was added at a 1:100 ratio of protease:protein. The reaction was incubated at 30°C and samples were quenched at the indicated time points by diluting into 4× Laemmli SDS–PAGE sample buffer containing 8% SDS. Degradation over time was visualized by running time points on a 4–12% Bis-Tris NuPAGE gel (Invitrogen) and staining with InstantBlue Coomassie Stain (Abcam).
Bands that were N-terminally sequenced were separated via SDS–PAGE as above, but transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad) prior to staining. Transfer of bands was performed using a Trans-Blot Turbo Transfer System (Bio-Rad). N-terminal sequences of these bands were then determined using Edman degradation.

The S. cerevisiae strain used in this study is a wild type strain with a BY4742 genetic background (MATalpha, his3Δ1, leu2Δ0, lys2Δ0, ura3Δ0). It is commercially available and was purchased at Horizon Scientific (Cambridge, UK). YPD (yeast extract, peptone, dextrose) media was from Sunrise Science (Knoxville, TN, USA). Tandem Mass Tag (TMTpro) isobaric tagging reagents, BCA (Bicinchoninic acid) protein quantification kit, Pierce protease inhibitor tablets, trypsin protease, and Pierce C18 tips were purchased from ThermoFisher Scientific (Rockford, IL, USA). C18 StageTip—(Empore) material was purchased from CDSanalytical (Oxford, PA, USA). Sep-Pak cartridges (100 mg) were acquired from Waters (Milford, MA, USA). Lys-C protease was from Fujifilm Wako (Richmond, VA, USA). Mass spectrometry grade reagents (i.e., water, formic acid, methanol, and acetonitrile) were purchased from J.T. Baker (Center Valley, PA, USA).

Five microliters of cell lysate per sample were transferred to a tube containing 20 μL of urea denaturing buffer (6 M urea, 2 M thiourea and 10 mM HEPES; pH 8.0). Disulfide bonds from the cell lysate proteins were reduced by adding 1 μL of dithiothreitol (10 mM) and incubating for 30 min at room temperature. The samples were alkylated by adding 1 μL of iodoacetamide (55 mM) solution and incubated at room temperature for another 30 min in the dark. Four volumes of ammonium bicarbonate buffer (40 mM) were added to each sample, and overnight digestion was carried out at room temperature by adding 1 μg of trypsin protease (Thermo Scientific, Waltham, MA, USA). To stop the digestion reactions, acidification of the samples was achieved by adjusting the final concentrations to 5% acetonitrile and 0.03% trifluoroacetic acid (TFA). Next, the samples were desalted using C18 StageTips with Empore™ C18 Extraction Disks (StageTip format) as previously described [50 (link)], and the peptides eluted from the StageTips were dried using vacuum centrifugation.

Following FACS-based separation of viable progenitor-rich salivary single cells from SMG and PG, cells were sorted, and reactions were performed on a 384-well plate using the cellenONE system (Cellenion, France) ^{42 (link)}. Lysis buffer was prepared with the concentration of 0.2% DDM (324355–1GM, Millipore, USA), 100 mM TEAB (T7408–500ML, Sigma-Aldrich, USA) and 2 ng/μl trypsin protease (90057, Thermo Scientific, USA). Prior to single-cell deposition, 1000 droplets (~350 nl) of lysis buffer were dispensed into each well of 384-well plate. Single cells were then isolated and deposited into the wells containing lysis buffer. Plate was incubated on the heating deck inside the cellenONE at 37°C for 1 hour. Digestion was then quenched by adding 100 nl of 5% formic acid. Digested samples from each well were reconstituted in 4 μl of 0.1% formic acid containing 0.05x iRT peptides (Biognosys, Ki-3002–1).

Protein extract was reduced with 10 mM dithiothreitol (BioShop) at 37 °C water bath for 30 min, and carboxymethylated with 55 mM iodoacetamide (Sigma-Aldrich) in the dark at room temperature for 30 min. Alkylated proteins were digested with Lys-C (1:100 w/w) (WAKO, Osaka, Japan) for 3 h and then digested with trypsin protease (1:100 w/w) (Thermo Fisher Scientific) overnight at 37 °C. The trypsin reaction was inactivated by acidified the peptide solution to a pH < 3 using trifluoroacetic acid (Sigma-Aldrich). Then, acidified peptide solution was combined with an equal volume of ethyl acetate (Sigma-Aldrich) and agitated vigorously for 1 min, followed by centrifugation at 15,700×g for 2 min to separate the aqueous and organic phases. The solution from the aqueous phase was dried using a centrifugal evaporator and then subjected to desalting using Styrenedivinylbenzene Empore disk membranes (SDB-XC) StageTips (#2340, 3 M, Nazarethm, Belgium) and eluted in a buffer containing 0.1% (v/v) TFA and 80% (v/v) acetonitrile (ACN) [27 (link)].