Mass spectrometry analysis of protein purity

Sample purity and integrity was analyzed by on-line buffer exchange MS using an UltiMate™ 3000 RSLC (Thermo Fisher Scientific) coupled to an Exactive Plus EMR Orbitrap instrument (Thermo Fisher Scientific) modified to incorporate a quadrupole mass filter and allow for surface-induced dissociation^{43 (link)}. 40 pmole protein (5 μL of 8 μM protein in TBS) were injected and on-line buffer exchanged to 200 mM ammonium acetate, pH 6.8 (AmAc) by a self-packed buffer exchange column⁴⁴ (P6 polyacrylamide gel, BioRad) at a flow-rate of 100 μL per min. Mass spectra were recorded for 1000 – 12000 m/z at 8750 resolution as defined at 200 m/z. The injection time was set to 200 ms. Voltages applied to the transfer optics were optimized to allow ion transmission while minimizing unintentional ion activation. Mass spectra were deconvoluted with UniDec version 2.6.5^{45 (link)} using the following processing parameters: sample mass every 1 Da; peak FWHM 1 Thompson, Gaussian peak shape function. Organic source corrected average masses calculated with NIST Mass and Fragment Calculator v1.32^{46 (link)} from the His-tag-cleaved sequences were listed as the expected masses.