Purified proteins were de-salted by dialysis and freeze-dried, or de-salted and concentrated using Microsep TM centrifugal concentrators (VivaScience AG, Hannover, Germany).
It is also necessary to de-salt the eluent prior to detection, and in this work, ion chromatography suppressors were used for this step.
It was demonstrated that suppressed ion-exchange separation using a complex KOH elution profile could be coupled with ELSD, with the suppressor effectively de-salting the eluent, producing a stable baseline.
All samples were de-salted by C18 ZipTip before mass spectrometry analysis.
Protein samples were de-salted in 10 K microcon, diluted with 100 ml of ammonium bicarbonate buffer (100 mM).
The samples were de-salted on the trap column for 5 minutes using solvent A [0.1% formic acid (aq)] at 30 μL/min.
The samples were de-salted through Econo-Pac 10DG desalting columns (Bio-Rad, Hercules, CA, USA) and concentrated with Amicon Ultra (EMD Millipore).
The samples were immunodepleted by multiple affinity removal system (MARS HPLC column, 4.6 × 100 mm; Agilent technologies, UK) and de-salted using 5 K molecular weight cut off spin filters (Agilent technologies, UK).
The peptides were then mixed, dried in a SpeedVac concentrator and subsequently de-salted using a 50 mg, 1 cc Sep-Pak SPE C18 cartridge and dried again in a SpeedVac concentrator.
De-salting was performed using UltraMicro spin columns (Nest group).
NHS-fluorescein and Zeba de-salting column were purchased from Pierce Biotechnology (Rockford, IL).
