AUTHOR=Lee Han Chung , Carroll Adam , Crossett Ben , Connolly Angela , Batarseh Amani , Djordjevic Michael A. 

TITLE=Improving the Identification and Coverage of Plant Transmembrane Proteins in Medicago Using Bottom–Up Proteomics

JOURNAL=Frontiers in Plant Science

VOLUME=Volume 11 - 2020

YEAR=2020

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2020.595726

DOI=10.3389/fpls.2020.595726

ISSN=1664-462X

ABSTRACT=Plant transmembrane proteins (TMPs) are essential for normal cellular homeostasis, nutrient exchange and responses to environmental cues. Commonly used bottom-up proteomic approaches fail to identify a broad coverage of peptide fragments derived from TMPs. Here, we used mass spectrometry (MS) to compare the effectiveness of two solubilisation and protein cleavage methods to identify shoot-derived TMPs from the legume Medicago. We compared a urea solubilisation, trypsin Lys-C (UR-TLC) cleavage method to a formic acid solubilisation, cyanogen bromide and trypsin Lys-C (FA-CTLC) cleavage method. We assessed the effectiveness of these methods by (i) comparing total protein identifications, (ii) determining how many TMPs were identified, and (iii) defining how many peptides incorporate all, or part, of transmembrane domains (TMD) sequences. The results show that the FA-CTLC method identified nine-fold more TMDs, and enriched more TMPs than the UR-TLC method. FA-CTLC identified more TMPs, particularly transporters, whereas, UR-TLC preferentially identified TMPs with one TMD, particularly signalling proteins. We identified 8993 protein groups including 3289 TMPs in young shoot tissues, which accounts for around 36 % of the predicted Medicago TMPs. The results suggest that combining plant membrane purification techniques with both the FA-CTLC and UR-TLC methods will achieve a more complete identification and coverage of TMPs.