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The complexity of a sample can be further reduced prior to mass spectrometric analysis by the addition of extra dimensions of separation.
Although this method was applied to data acquired using mass spectrometry-based detection, it is easily applied to other metabolomics platforms (e.g., NMR), or to additional dimensions of separation combined with MS. We have also indicated the application of the MEDI workflow only on static samples, although the GEDI core software is ideal to determine underlying temporal dynamics.
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While the peaks may appear relatively broad in comparison to C18 reversed-phase chromatography, WCX-HILIC affords a secondary dimension of separation based on the methylation status (Fig. 3b), separating multiple methylated isoforms that contain the same number of acetylations.
Fractions from each of the 8 anion-exchange separation elution steps were automatically transferred onto a reversed-phase column (PorosR2/10, 4.6 mm ID×100 mL, Applied Biosystems) for a second dimension of separation.
The 12 pools were then subjected to a second dimension of separation by reversed-phase chromatography.
Moreover, complementary ESI-MS measurements were carried out, providing another dimension of separation.
The first dimension of separation was carried out on IPGphore (Amersham Biosciences, Uppsala, Sweden) at 20°C.
This is the first dimension of separation in a 2D gel experiment.
The additional dimension of separation based on the size and shape of gas-phase ions allows for the differentiation of isobaric species based on CCS.
For the second dimension of separation, each eluted salt step was desalted with an inline peptide microtrap (Michrom BioResources) with 2% ACN, 0.1% FA at 5 μL/min.
Nano-flow UPLC (300 nl/min) was used for separation of pull-down digests and normal-flow UPLC (400 μl/min) used for the second dimension of separation for lysate digests.
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