MassIVE MSV000090218

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Diversity matters: Optimal collision energies for tandem mass spectrometric analysis of a large set of N-glycopeptides - part 2

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a) Ratio of the lower and higher energy component. Tests were done on the mixture of AGP, fetuin and transferrin digests using 2.2 pmol from all glycoproteins in each run. Five different high CE setting was used which were 50%, 75%, 100%, 125% and 150% of the original method of Hinneburg et al. These were combined with three different low CE/high CE ratio, 0.3, 0.5 and 0.7, resulting in 15 different MS/MS settings. b) Time distribution. Tests were performed on the mixture of AGP, fetuin and transferrin digests using 2.2 pmol from all glycoproteins in each run. The fraction of the MS/MS acquisition time allocated to the higher energy condition was system-atically varied from 40% to 90% in steps of 5% resulting in 10 different MS/MS methods. d) Performance check. The above energy-dependence studies allowed optimum energies to be determined for each individual N-glycopeptide, but obviously, we cannot directly apply these in practice since the identity of the glycopeptides is not known at the time of the measurement. The results on individual glycopeptides showed reasonably good m/z-dependent linear trends for the optimum energies for both Byonic and pGlyco search engines and these formed the basis for CE choice in a practical DDA measurement run. We explored the potential gain via CE optimization by comparing the number of hits using the Hinneburg et al.'s literature CE setting and our optimized MS/MS method in actual measure-ments. HeLa digest and blood plasma digest were used. The pellet fraction of acetone precipitation enriched in glycopep-tides were investigated with injection amounts of 750 ng and 1.5 ug in the case of HeLa and blood plasma, respectively. Three repetitions were carried out with each CE setting and data were evaluated using both Byonic and pGlyco search engines. e) Measurements on mAb sample. Nano-LC-MS/MS ex-periments were performed on a mAb sample using 2 pmol tryptic digest in each run. First, the energy dependence study was carried out analogously to the mixture of the 3 glycopro-tein digests and AGP tryptic digests as described above in-volving 27 nano-LC-MS/MS measurements. Then, based on the energy dependence, optimal CE method was designed for the mAb N-glycopeptides. The CE method of Hinneburg et al., the CE setting optimized for all the N-glycopeptides of the glycoprotein mixture and AGP samples and CE optimized for the mAb N-glycopeptides were tested and compared in 5-5 repetitions (15 runs overall). f) Three-stepped methods. We tested the impact of adding a third CE step at the mid-point between the high and low energy levels. [doi:10.25345/C5CN6Z38J] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: N-glycosylation ; collision energy ; optimization ; transfer ; monoclonal antibody

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Principal Investigators:
(in alphabetical order) 		Agnes Revesz, Research Centre for Natural Sciences, Budapest, Hungary
Submitting User: reveszagnes
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