Oasis hlb 1 cc cartridges

Human KG1a cells (ATCC^® CCL-246.1™) were maintained and propagated in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C in a 5% CO₂ atmosphere, and cells were lysed in buffer containing 4% SDS. Lysates were sonicated (Ningbo Scientz) and centrifuged at 15,000 × g for 15 min. The protein concentration of the supernatant was determined by using the BCA assay kit. Proteins (200 µg) were digested overnight with Lys-C (Wako Chemicals) and trypsin (Promega) using the filter-aided sample preparation protocol^{33 (link)}. Peptides were recovered and desalted using Oasis HLB 1-cc cartridges (Waters Corp.). In brief, peptides were loaded onto a HLB cartridge, washed with 0.1% trifluoroacetic acid (TFA), and eluted with 30% ACN and 60% ACN in 0.1% TFA. Flow through from the Oasis HLB was then loaded onto a Sep Pak C18 cartridge (Waters), washed with 0.1% TFA, and then eluted with 30% ACN and 60% ACN in 0.1% TFA. Eluates from HLB and C18 cartridges were combined and lyophilized in a vacuum centrifuge for LC-MS/MS proteome analysis as described below. E. coli cells were incubated at 37 °C with 200 rpm shaking and harvested at mid-log phase. Cells were pelleted at 10,000 × g, and then washed three times with phosphate-buffered saline pH 7.5. Harvested cell pellets were digested using the methods mentioned above.

Samples were resuspended in 2% phosphoric acid and desalted using Oasis HLB 1CC cartridges (Waters) according to the manufacturer’s instructions. Vacuum-dried samples were resuspended in HPLC grade water with 20 mM ammonium formate for high-pH reversed phase fractionation on an Agilent 1100 series HPLC with a Poroshell 300 Extended C18 column (Agilent). Fractions were collected from 1-37 min at 1.2 min intervals at a flow rate of 200 μL/min using a gradient of 100% acetonitrile + 20 mM ammonium formate. Collected fractions were vacuum dried, resuspended in water and vacuum dried again.