High-pH fractions were solubilized in 60 µl of LC-solution (3% ACN (v/v) and 0.2% Formic acid (v/v) in by placing them on a Thermomixer at room temperature for 30 minutes with an agitation at 1800 rpm. 7 µl of each fraction was transferred into LC-vail inserts for mass spectrometry analysis. LC-MS/MS analysis was carried out on a Thermo Lumos ETD Tribrid mass spectrometer inline with 3000 ultimate RSLC nano-liquid chromatography system. Sample was injected into pre-column (C18, 5µm, 100Ao, 100µ, 2cm Nano-viper column # 164564, Thermo Scientific) at 5 µl/min flow rate and subsequently loaded onto the analytical column (C18, 5µm, 50cm, 100Ao Easy nano spray column # ES903, Thermo Scientific) for the separation of peptides using nano-pump operated at 300 nl/min flow rate. 85 min non-linear gradient was applied (5% Solvent B (80 %ACN v/v in 0.1% Formic acid v/v) to 22% B for 70 min and increased to 35% B for another 10 min for a total of 100 min run time. The eluted peptides were electrosprayed into the mass spectrometer using easy nano source. The data was acquired in a data dependent acquisition (DDA) mode in SPS MS3 (FT-IT-HCD-FT-HCD) method and was acquired using top speed for 2 sec for each duty cycle. The Full MS1 scan was acquired at 120,000 resolution at m/z 200 and analyzed using Ultra high filed Orbitrap mass analyzer in the scan range of 375-1500 m/z. The precursor ions for MS2 were isolated using Quadrupole mass filter at 0.7 Da isolation width and fragmented using normalized 35% Higher-energy collisional dissociation (HCD) of in Ion routing multipole analyzed using Ion trap. Top 10 MS2 fragment ions in a subsequent scan were isolated and fragmented using HCD at 65% normalized collision energy and analyzed using Orbitrap mass analyzer at 50,000 resolution in the scan range of 100-500 m/z.