Lcq deca xp iontrap esi mass spectrometer

The antigenic peptides hgp100_(25–33) (KVPRNQDWL and KVPRNQDWLC-6-ahx-lysine (biotin)) [81 (link)], mgp100_25–33 (EGSRNQDWL), human gp100_280–288 (YLEPGPVTA and YLEPGPVTAC-6-ahx-lysine (biotin)), Trp1_222–229 (TAYRYHLL and TAYRYHLLC-6-ahx-lysine (biotin)), Trp2_180–188 (SVYDFFVWL and SVYDFFVWLC-6-ahx-lysine (biotin)) and MART1_(26–35) (ELAGIGILTV and ELAGIGILTVC-6-ahx-lysine (biotin)) were synthesized at the GlycO2peptide unit at our lab by solid phase peptide synthesis using Fmoc chemistry on a Symphony peptide synthesizer (Protein Technologies Inc., Tucson, AZ, USA). The linker 6-ahx and the special amino acid lysine(biotin) were purchased at Iris Biotech GmbH (Marktredwitz, Germany) The peptides were purified on a preparative Ultimate 3000 HPLC system (Thermo Fisher Scientific, Breda, the Netherlands) over a Vydac 218MS1022 C18 25 × 250mm column (Grace Davidson, Worms, Germany). Quality control was performed by UPLC-MS on a Ultimate 3000 UHPLC system (Thermo Fisher Scientific) hyphenated with a LCQ-Deca XP Iontrap ESI mass spectrometer (Thermo Finnigan, Waltham, MA, USA) using a RSLC 120 C18 Acclaim 2.2um particle 2.1 × 250 mm column and ionizing the sample in positive mode.

The antigenic peptides gp100 (VTHTYLEPGPVTANRQLYPEWTEAQRLDC), MART-1 (YTTAEELAGIGILTV) containing the immune dominant epitope MART-1_26−35L and MART-1_26−35L (ELAGIGILTV) were synthesized by solid phase peptide synthesis using Fmoc chemistry on a Symphony peptide synthesizer (Protein Technologies Inc., Tucson, U.S.). In the last step of the synthesis, prior to the cleavage of the peptide from the resin, a palmitic tail was added at the N-terminus of the gp100 and MART-1 peptide through a reaction with palmitic anhydride in dichloromethane (DCM). The resulting lipopeptides were cleaved and purified on a preparative Ultimate 3000 HPLC system (Thermo Fisher) over a VYDAC 214MS1022 C4 25 × 250 mm column (Grace Davidson). Mass and purity were confirmed by UPLC-MS on a Ultimate 3000 UHPLC system (Thermo Fisher, Waltham, U.S.) hyphenated with a LCQ-Deca XP Iontrap ESI mass spectrometer (Thermo Finnigan, Waltham, U.S.) using a Prosphere HP C4 5 μm 150 × 4.6 mm column and ionizing the sample in positive mode.

Thz-VTHTYLEPGPVTANRQLYPEWTEAQRLD-(Abu)₃-C peptide was synthesized at the GlycO2pep unit at our lab by microwave assisted solid phase peptide synthesis using Fmoc chemistry on a peptide synthesizer (Liberty blue peptide synthesizer, CEM). The peptide was deprotected with 92.5% TFA, 2.5% MilliQ, 2.5% TIS and 2,5% EDT cleavage solution. After collection, the peptide was lyophilized and purified on a preparative Ultimate 3000 HPLC system (Thermo Fisher) over a Vydac 218MS1022 C18 25x250mm column (Grace Vydac). Mass and purity were confirmed by UHPLC-MS on a Ultimate 3000 UHPLC system (Thermo Fisher) hyphenated with a LCQ-Deca XP Iontrap ESI mass spectrometer (Thermo Finnigan) using a RSLC 120 C18 Acclaim 2.2um particle 2.1 x 250 mm column and ionizing the sample in positive mode.