Polar advantage 2 acclaim column

The purified peptides were analyzed by liquid chromatography–mass spectrometry (LC-MS) on a high-resolution electrospray—quadrupole—time of flight (ESI-Q-TOF) instrument, using either a 1290 Infinity II UPLC (chromatography system connected to a hybrid ion mobility Q-TOF instrument (6560, Agilent), for MccJ25, McC and MccB17, or an Ultimate 3000-RSLC system (Thermo Fisher Scientific) connected to a Maxis II ETD ESI-Q-TOF instrument (Bruker Daltonics), for MccE492. For the former LC-MS system, the separation was achieved on a Poroshell 120 EC-C18 column (2.1 × 100 mm, 2.7 μm, Agilent) at a flow rate of 400 μL/min, using an A2/ACN gradient from 10% to 100% ACN over 15 min. For the latter system, the separation was achieved on a Polar Advantage II Acclaim column (2.2 μm, 120 Å, 2.1 × 100 mm, Thermo Fisher Scientific) at a flow rate of 300 μL/min, using an A2/ACN gradient from 10% to 100% ACN over 15 min. The MS detection was performed in positive mode.

Digested protein extracts were analysed independently by ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) using a workflow described previously [34 (link)]. Separation was performed on an Ultimate 3000-RSLC system (Thermo Fisher Scientific) with a RSLC Polar Advantage II Acclaim column (2.1 × 100 mm, 120 Å, 2.2 µm) using a flow rate of 300 µl min^–1 and mobile phase gradient of A: H₂O + formic acid (FA) 0.1% and B: acetonitrile + FA 0.08%. We used a high-resolution ESI-Q-TOF mass spectrometer (Maxis II ETD, Bruker Daltonics) in positive mode and data-dependent auto-MS/MS mode on the m/z range 200–2200. MS/MS spectra were generated using collision-induced dissociation by selection of ions with charge states between 2⁺ and 5⁺ and on m/z range 300–2200. Calibration was carried out for each run with sodium formate clusters and experimental blanks analysed every five samples for each of the runs with modern references and archaeological samples.

The purified peptides were analyzed by liquid chromatography–mass spectrometry (LC-MS) on a high-resolution electrospray—quadrupole—time of flight (ESI-Q-TOF) instrument, using either a 1290 Infinity II UPLC (chromatography system connected to a hybrid ion mobility Q-TOF instrument (6560, Agilent), for MccJ25, McC and MccB17, or an Ultimate 3000-RSLC system (Thermo Fisher Scientific) connected to a Maxis II ETD ESI-Q-TOF instrument (Bruker Daltonics), for MccE492. For the former LC-MS system, the separation was achieved on a Poroshell 120 EC-C18 column (2.1 × 100 mm, 2.7 μm, Agilent) at a flow rate of 400 μL/min, using an A2/ACN gradient from 10% to 100% ACN over 15 min. For the latter system, the separation was achieved on a Polar Advantage II Acclaim column (2.2 μm, 120 Å, 2.1 × 100 mm, Thermo Fisher Scientific) at a flow rate of 300 μL/min, using an A2/ACN gradient from 10% to 100% ACN over 15 min. The MS detection was performed in positive mode.