TY - JOUR
T1 - Improving the Identification and Coverage of Plant Transmembrane Proteins in Medicago Using Bottom–Up Proteomics
AU - Lee, Han Chung
AU - Carroll, Adam
AU - Crossett, Ben
AU - Connolly, Angela
AU - Batarseh, Amani
AU - Djordjevic, Michael A.
N1 - Publisher Copyright:
© Copyright © 2020 Lee, Carroll, Crossett, Connolly, Batarseh and Djordjevic.
PY - 2020/12/18
Y1 - 2020/12/18
N2 - 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 solubilization and protein cleavage methods to identify shoot-derived TMPs from the legume Medicago. We compared a urea solubilization, trypsin Lys-C (UR-TLC) cleavage method to a formic acid solubilization, 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 hydrophobic TMPs than the UR-TLC method. FA-CTLC identified more TMPs, particularly transporters, whereas UR-TLC preferentially identified TMPs with one TMD, particularly signaling proteins. 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.
AB - 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 solubilization and protein cleavage methods to identify shoot-derived TMPs from the legume Medicago. We compared a urea solubilization, trypsin Lys-C (UR-TLC) cleavage method to a formic acid solubilization, 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 hydrophobic TMPs than the UR-TLC method. FA-CTLC identified more TMPs, particularly transporters, whereas UR-TLC preferentially identified TMPs with one TMD, particularly signaling proteins. 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.
KW - Medicago truncatula
KW - TRAMDOMI algorithm
KW - detergent-free purification
KW - liquid chromatography
KW - mass spectrometry
KW - transmembrane domain
KW - transmembrane protein
UR - http://www.scopus.com/inward/record.url?scp=85098696377&partnerID=8YFLogxK
U2 - 10.3389/fpls.2020.595726
DO - 10.3389/fpls.2020.595726
M3 - Article
SN - 1664-462X
VL - 11
JO - Frontiers in Plant Science
JF - Frontiers in Plant Science
M1 - 595726
ER -