Microflex lrf maldi tof

All peptides (Table 1) were manufactured by solid-phase peptide synthesis using 9-fluorenylmethoxycarbonyl (Fmoc)-chemistry as described previously (Bolscher et al., 2011 (link); Van Dijk et al., 2015 (link)). The peptides were purified by High-Performance Liquid Chromatography (RF-HPLC, Dionex Ultimate 3000, Thermo Scientific, Breda, Netherlands) to a purity of at least 95%. The authenticity was confirmed by mass spectrometry with a Microflex LRF MALDI-TOF (Bruker Daltonik GmbH, Bremen, Germany) as previously described (Bolscher et al., 2011 (link); Van Dijk et al., 2015 (link)). During synthesis, part of Hst1 was labeled with the fluorescent dye ATTO-647N (ATTO-TEC GmbH, Siegen, Germany). An equimolar amount of the dye was coupled to the ε-amino group of the side chain of lysine residue number 17 (lys17, K of Hst1 after removal of the specific protective (ivDde)-OH group by hydrazine (2% hydrazine hydrate).

Different variants of glutamate-urea-lysine-based PSMA-targeting ligands, varying in their linker moieties, were synthesized using solid phase chemistry, coupled to IRDye700DX, and labeled with ¹¹¹In. A detailed description of the synthetic procedures and chemical analyses (HPLC, ESI-ion trap, MALDI-ToF) is provided in the supplemental information (Supplementary Materials and Methods and Results, Figure S1, Figure S2, Figure S8).All amino acids were purchased from Iris Biotech GmbH or Fluorochem unless stated otherwise. DOTA-NHS and DOTAGA anhydride were purchased from CheMatech SAS. IRDye700DX was purchased from LI-COR Biosciences. Other reagents were purchased from Fisher Scientific unless stated otherwise and were reagent grade. All water used was highly pure (18 mΩ) and all HPLC-grade solvents were purchased from Biosolve B.V. Identity of the peptides was confirmed using high-resolution mass spectra recorded on a JEOL AccuTOF (ESI-MS), and the dye-conjugated peptides with MALDI-ToF Mass spectrometry on a Bruker Microflex LRF MALDI-ToF.