Reprosil 100 c18 column

Purifications of synthesized products were performed on a Reprosil 100 C18 column (Dr. Maisch GmbH, 40 × 250 mm, 10 µm particle size, flow rate 25 mL/min) or Reprosil 100 C18-XBD column (Dr. Maisch GmbH, 20 × 250 mm, 10 µm particle size, flow rate 10 mL/min) by using a SpotPrep (Armen) system and several gradient methods. The mobile phase consisted of 0.08% TFA in acetonitrile (ACN) (solvent A) and 0.1% TFA in water (solvent B). The column was maintained at ambient temperature. The eluted solution was monitored with an UV detector at 220 and 254 nm. Eluates were fractioned and analyzed by analytical RP-HPLC.
Analytical separations were performed on a Kinetex XB-C18 column (Phenomenex, 4.6 × 150 mm, 5 µm particle size, flow rate was 1 mL/min) using a Shimadzu Prominence system and several gradient methods. The mobile phase, ambient temperature and the parameters of UV monitoring were the same as those mentioned for the Reprosil columns.
The chemical structure of the obtained conjugates are presented in Fig. 1.Figure 1

The chemical structure of the synthesized conjugates in question. The conjugates: (a) Van-PEG₃-TP10 (conjugate I); (b) Van-PEG₄-TP10 (conjugate II); (c) TP10-Ala(PEG₄-Van) (conjugate III); (d) [Lys⁷(PEG₄-Van)]TP10 (conjugate IV); (e) Fl[Lys⁷(PEG₄-Van)]TP10 (conjugate V).

For simultaneous mass spectrometry analysis, each peptide sample was individually labeled with a specific TMTpro 16 plex label reagent (Thermo Fisher Scientific, Rockford, USA) according to the user manual provided with the kit. Afterwards, 10% of each labeled peptide sample was combined into two pools, and salt contaminants from the labeling process were removed by passing this complex peptide mixture across an OASIS HLB 1 cc Flangeless cartridge (Waters GmbH, Eschborn, Germany). The solvent was removed by vacuum evaporation, and the dried peptides were stored at −20 °C until further use. Samples were redissolved in 20 mM ammonium formate (pH 10) and fractionated by reversed-phase chromatography at elevated pH with a Reprosil 100 C18 column (3 µm 125 × 4 mm, Dr. Maisch GmbH, Ammerbuch-Entringen, Germany). Sixty fractions were combined into 6 pools and dried in a vacuum concentrator⁵⁵ . Peptides were purified by solid phase extraction (Oasis HLB cartridges, Waters GmbH, Eschborn, Germany).