smegmatis cells in the “”trypsin shaving”" incubation buffer without trypsin for 2 hours. The Cell Cycle inhibitor digestion mixtures were centrifuged at 3,500 × g for 10 min at 4°C,

and the supernatants (Fresh trypsin was added) were incubated at 37°C for around 12~14 hrs for full digestion after being filtered using 0.22 μm pore-size filters (Millipore, Etobicoke, ON, Canada). Protease reactions were stopped with formic acid at 0.1% final concentration. Peptide fractions were concentrated with a Speed-vac centrifuge (Savant), and kept at -20°C until further analysis. Sample digestion Protein sample was separated by 12.5% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE), run for 1 h at 30 W, then for 4.5 h at 180 W. The gels were Coomassie Brilliant Blue stained and the lane corresponding to the cell wall proteins was cut into 6 equal pieces. The gel pieces were individually check details in-gel digested as described previously with some modifications [50]. Briefly, after in-gel digestion using trypsin, the digested solution was transferred into a clean 0.6 ml tube. Fifty microliters of 50% acetonitrile (ACN)/5% formic acid (FA) was added to the gel pieces and sonicated

for 30 min. This extraction procedure was repeated three times, and a total of 150 μl of extracts was collected. All extracts were pooled and concentrated to less than 10 μl using an SPD 2010 SpeedVac system (Thermo Electron, Waltham, MA). Thereafter, the sample was diluted with 0.1% FA in HPLC water to 100 μL for direct LC-MS/MS AZD1390 manufacturer Protein kinase N1 analysis or reconstituted with trifluoroacetic acid (TFA) to a final concentration of 0.1% and subjected to sample cleanup steps using C18 ZipTips (Millipore) prior to LC-MS/MS analysis. The C18 ZipTips were conditioned with 100% ACN and then equilibrated three times with 0.1% TFA. The peptides

were bound to the ZipTip pipet tip by aspirating and dispensing the sample for at least 15 cycles, washed with 0.1% TFA, and eluted by 20 μL of elution buffer (75% ACN, 0.1% TFA). Protein identification by LC-MS/MS Digests were analyzed using an integrated Agilent 1100 LC-ion-Trap-XCT-Ultra system fitted with an Agilent ChipCube source sprayer. Injected samples were first trapped and desalted on a Zorbax 300 SB-C18 Precolumn (5 μm, 5 × 300-μm inside diameter; Agilent) for 5 min with 0.2% formic acid delivered by the auxiliary pump at 0.3 μl/min. The peptides were then reverse eluted from the trapping column and separated on an analytical Zorbax 15 cm-long 300SB-C18 HPLC-Chip 0.3 μl/min. Peptides were eluted with a 5-45% acetonitrile gradient in 0.2% formic acid over a 50 min interval. Data-dependent acquisition of collision-induced dissociation MS/MS was utilized, and parent ion scans were run over the mass range m/z 400-2,000 at 8,100. For analysis of LC-MS/MS data, Mascot searches used the following parameters: 1.4 Da MS error, 0.8 Da MS/MS error, 1 potential missed cleavage, and variable oxidation (Methionine) [51].