Lc ms grade acetone

Cell lysis of the purified T. pallidum extract was performed by conducting three consecutive freeze-thaw cycles, followed by ultrasonication on ice with an amplitude of 50% and a pulser frequency of 2 seconds for 2 minutes (Sonics, Vibra cell; Newton USA). Protein concentration was determined by loading a small fraction of the lysed sample on a high performance liquid chromatographic (HPLC) reversed phase C4 system that was calibrated using a serial dilution of a protein standard mixture. Protein concentrations were determined based on the area under the curve (AUC at 214 nm). Approximately 400–500 μg of protein was extracted from each biological replicate; a large proportion of this amount was host protein in the form of albumin. Samples were acetone precipitated by adding 6 volumes of LC-MS grade acetone (Biosolve, Valkenswaard, Netherlands) and incubated overnight at -20°C. In all cases, lo-bind Eppendorf tubes (Eppendorf, Hamburg, Germany) were used to ensure high recovery rates of proteins/peptides.

Carboxymethylcellulose (CMC), norharmane, red phosphorus, ammonium acetate (NH₄OAc 7.5 M solution) and formic acid (FA) were obtained from Sigma (Merck Life Science B.V., Overijse, Belgium, molecular grade). LC-MS-grade acetone, isopropanol (IPA), methanol (MeOH) and water were obtained from Biosolve B.V. (Valkenswaard, The Netherlands), while n-hexane was obtained from VWR International BVBA (Leuven, Belgium).