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

ABSTRACT: Detailed energy dependence investigations were carried out on AGP digest using 2.2 pmol glycoprotein per run and on the mixture of AGP, fetuin and transferrin digests injecting 2.2 pmol from all glycoproteins in each run. Stepped CE settings were applied with 80% of the time allocated to the higher energy component and the low CE/high CE ratio was set to 0.5. The CE was systematically varied from 6.25% to 175% of the Hinneburg et al.'s setting in steps of 6.25% resulting in 27 different nano-LC-MS/MS runs. Experiments were performed with the use of three inclusion lists, based on DDA measurements taken with Hinneburg et al.'s CE method. Two lists for the mixture (3GP - SPLv5b, SPLv6) and one list for AGP (AGP - SPLv7) were created. The higher component of the collision energy is marked by a 3,4,5 digit number after the SPL designation, containing 0,1,2 decimal digits, respectively (eg "100"->100%, "08125"-->81.25%).

INSTRUMENT(S): maXis II

ORGANISM(S): Bos Taurus (ncbitaxon:9913) Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Agnes Revesz

PROVIDER: MSV000089657 | MassIVE | Wed Jun 15 12:51:00 BST 2022

REPOSITORIES: MassIVE

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Project description: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.

Project description:The industrially important fungus Aspergillus niger feeds naturally on decomposing plant material, for which it is equipped with a range of enzyme systems. A significant proportion of plant material are lipids that might be available either as for energy storage or as membrane building blocks. With 63 potential lipase-encoding genes in its genome, A. niger has the tools to degrade these extracellular lipids. In contrast to polysaccharide-degrading enzyme networks not much is known about the signalling and regulatory processes that control lipase expression and activity in fungi both under laboratory and natural occurring conditions. A pulse of 1 mM of various oils was applied to four bioreactor-grown A. niger cultures to examine (i) whether A. niger responds at the level of gene transcription, (ii) at what time point this effect is detected most accurately, and (iii) whether differences between the response towards oils are observed. The triglyceride olive oil induces genes encoding peroxins and enzymes of fatty acid metabolism. A complex oil mixture extracted from wheat gluten, which is enriched for digalactosyl-diglycerides, induces genes encoding peroxins as well as enzymes of fatty acid metabolism, but with different expression profile when compared to olive oil. Pure digalactosyldiglyceride, a proxy for plant membrane lipids, does not trigger a transcriptional response. Keywords: time course; induction experiment In one week, 4 fermentor cultures were run in 2.2-liter batch fermentors in which A. niger was grown on 100 mM sorbitol. At 14 hours after oxygen supply had switched from headspace to sparger-inlet each fermentor was induced with 22 mL medium which contained 100 mM (final concentration in fermentor: 1 mM) of either olive oil, a complex oil mixture extracted from wheat gluten, pure wheat digalactosyldiglycerides, or was induced with a solution of minimal medium containing only 0.2% (in fermentor, final concentration 0.002%) triton X-100 which served as emulsifier agent. For each fermentor vessel, a sample of 10 mL was taken prior to induction (T=0), or 30 minutes, 1 hour, or 2 hours after induction. Every sample was hybridized onto a single microarray, yielding in total 16 DNA microarrays.

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

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