Masslynx maxent1

Purified protein was exchanged into LC-MS grade water or 50 mM triethyl-ammonium bicarbonate with PD-10 columns, diluted to 50 μM and analyzed with the Waters Synapt G2 Mass Spectrometer at the Mass Spectrometry Facility at Oregon State University. The deconvoluted masses were obtained by using Waters MassLynx MaxEnt1 software.

Denatured protein samples (~10μM) were subjected to LC-MS analysis. Briefly, proteins were separated on a C4 BEH 1.7μm, 1.0 x 100mm UPLC column (Waters, UK) using a modified nanoAcquity (Waters, UK) to deliver a flow of approximately 50 μl/min. The column was developed over 20 minutes with a gradient of acetonitrile (2% v/v to 80% v/v) in 0.1% v/v formic acid. The analytical column outlet was directly interfaced via an electrospray ionisation source, with a hybrid quadrupole time-of-flight mass spectrometer (Xevo G2, Waters, UK). Data was acquired over a m/z range of 300–2000, in positive ion mode with a cone voltage of 30V. Scans were summed together manually and deconvoluted using MaxEnt1 (Masslynx, Waters, UK). The theoretical molecular weights of proteins with ncAAs was calculated by first computing the theoretical molecular weight of wild-type protein using an online tool (http://web.expasy.org/protparam/) and then manually correcting for the theoretical molecular weight of ncAAs.

Denatured protein samples (~10 µM) were subjected to liquid chromatography-mass spectrometry analysis. Briefly, proteins were separated on a BEH C4 UPLC (1.7µm; 1.0 x 100mm; Watewrs) column using a modified nanoAcquity (Waters) to deliver a flow of approximately 50 µl min^-1. The column was developed over 20 min with a gradient of acetonitrile (2% vol/vol to 80% vol/vol) in 0.1% vol/volformic acid. The analytical column outlet was directly interfaced via an electrospray ionization source, with a hybrid quadrupole time-of-flight mass spectrometer (Xevo G2, Waters, UK). Data were acquired over an m/z range of 300–2000, in positive ion mode with a cone voltage of 30 V. Scans were summed together manually and deconvoluted using MaxEnt1 (Masslynx; Waters). The theoretical molecular weights of proteins with ncAAs were calculated by first computing the theoretical molecular weight of wild-type protein using an online tool (http://web.expasy.org/protparam/) and then manually correcting for the theoretical molecular weight of ncAAs.

Denatured protein samples (~10 μM) were subjected to liquid chromatography-mass spectrometry analysis. Briefly, proteins were separated on a C4 BEH 1.7 μm, 1.0 × 100 mm ultraperformance liquid chromatography column (Waters) using a modified nanoAcquity (Waters) to deliver a flow of approximately 50 μl min^-1. The column was developed over 20 min with a gradient of acetonitrile (2–80% v/v) in 0.1% v/v formic acid. The analytic column outlet was directly interfaced via an electrospray ionization source, with a hybrid quadrupole time-of-flight mass spectrometer (Xevo G2, Waters). Data were acquired over a m/z range of 300-2,000, in positive-ion mode with a cone voltage of 30 V. Scans were summed together manually and deconvoluted using MaxEnt1 (Masslynx, Waters). The theoretical molecular weights of proteins with ncAAs was calculated by first computing the theoretical molecular weight of wild-type protein using an online tool (http://web.expasy.org/protparam/) and then manually correcting for the theoretical molecular weight of ncAAs.