Complete and accurate characterization of intact proteins by mass spectrometry is possible today thanks to the latest technological development. We developed a standard mixture for LCMS quality control and method development. Here we present the top-down analysis of this mixture using various fragmentation techniques and MSn capabilities available on a modified Orbitrap Fusion Lumos Tribrid.
We performed direct infusion experiments and LC-MS experiments using a Pierce intact protein standard mix ranging from ~9 kDa to ~70kDa MW on an Orbitrap Fusion Lumos Tribrid MS modified with a 213 nm UVPD source and coupled with a Vanquish UHPLC system. Intact proteins were separated using 2.1 mm by 50 mm Acclaim columns with a 4 µm particle size. We performed CID, HCD, ETD, EThcD and UVPD fragmentation MSn experiments for structural investigation. Intact Protein spectra were deconvoluted with ReSpect or Xtract in Biopharma Finder 2.0. MS/MS spectra were analyzed with ProSightPC 4.1 and the ProSightPD node in Proteome Discoverer 2.1
Results and Conclusions
Optimal dissociation parameters were determined as a function of ProSight P score. It was determined, for example, that UVPD reaction times spanned from 15 to 50 ms for proteins ranging from 9 – 68 kD. ETD reaction times ranged from 20 to 10 ms for proteins ranging 9 – 68 kD To test consistency between infusion and LCMS, these optimized settings were translated to LCMS methods. These data not only confirms the translatability of these results and the value of these standards to optimize conditions during MS method development, but also intact protein LC method development and quality control. We will present here the range of optimized values in each fragmentation mode for proteins across the MW range, and discuss theoretical reasoning behind these experimentally determined settings.