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. 2020 Dec 18;11:595726. doi: 10.3389/fpls.2020.595726

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Copyright © 2020 Lee, Carroll, Crossett, Connolly, Batarseh and Djordjevic.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Flow chart summarizing the two solubilization and protein cleavage methodologies used in this study. Red box: Negligible insoluble material remained after using the formic acid solubilization, cyanogen bromide, and trypsin Lys-C (FA-CTLC) method (2a, blue arrow), whereas significant insoluble material remained after using the urea solidilization, trypsin Lys-C (UR-TLC) method (2b, red arrow). The remaining insoluble material after UR-TLC was solubilized and digested using the FA-CTLC method. Mass spectrometry (MS) analysis showed the presence of a wide range of proteins in the pellet (Supplementary Table 2). Subsequent centrifugation of this re-solubilized and FA-CTLC-treated material showed that negligible insoluble material remained (2c, green arrow).