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The dioinized water with conductivity of less than 2 μs/cm was used in all preparation steps.
All preparation steps were performed in the absence of ambient light, which is the same as the process in our previous studies [4, 6].
All preparation steps like weighing, mixing, grinding, and storage were carried out in an Ar-filled glove box; the O2 and H2O level is less than 0.1 ppm.
Furthermore, we tried to confine sample volume, consumable, reagent, and solvent requirements to a minimum and perform all preparation steps in one vial.
All preparation steps were performed according to the supplier's protocol.
Care was taken that all preparation steps took place at 4°C.
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All the individual preparation steps and the various resulting (multi-) layers are characterized by surface plasmon spectroscopy, X-ray and neutron-reflectometry, contact angle measurements, IR spectroscopy, fluorescence microscopy, scanning probe microscopies, as well as, electrochemical techniques.
All the sample preparation steps, including protein digestion, strong anion exchange (SAX -based fractionation, and high-pH reverSAX -based(RP) fractionation were integrandd into one pipette tip packed with SAX and C18 membranes for the first time.
SH and AN performed all sample preparation steps and measurements.
WJZ performed the all sample preparation steps and drafted the manuscript.
A new feature compared to previous protocols [14] is the embedding of reference coordinates which are present throughout all sample preparation steps for EM.
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