Proteomics of Medicago sativa cell walls.
A method for the sequential extraction and profiling by two-dimensional gel electrophoresis (2-DE) of Medicago sativa (alfalfa) stem cell wall proteins is described. Protein extraction included freezing, grinding in a sodium acetate buffer, separation by filtration of cell walls from cytosolic contents, and extensive washing. Cell wall proteins were then extracted sequentially with a solution containing 200 mM CaCl2 and 50 mM sodium acetate, followed by extraction with 3.0 M LiCl and 50 mM sodium acetate. Cell wall proteins from both the CaCl2 and LiCl fractions were profiled by 2-DE. Approximately 150 protein spots were extracted from these two gels, digested with trypsin, and analyzed using nanoscale HPLC coupled to a hybrid quadrupole time-of-flight (Q-tof) tandem mass spectrometer (LC/MS/MS). More than 100 proteins were identified and used in conjunction with the 2-DE profiles to generate proteomic reference maps for cell walls of this important legume. Identified proteins include classical cell wall proteins as well as proteins traditionally considered as non-secreted. Two unique extracellular proteins were also identified.
Plant Biology Division, The Samuel Roberts Noble Foundation, PO Box 2180, Ardmore, OK 73402, USA.
Bonnie S
BS
Watson
Zhentian
Z
Lei
Richard A
RA
Dixon
Lloyd W
LW
Sumner
D002473:Cell Wall; D015180:Electrophoresis, Gel, Two-Dimensional; D013058:Mass Spectrometry; D000455:Medicago sativa / Q000378:metabolism*; D010940:Plant Proteins / Q000032:analysis* / Q000302:isolation & purification; D020543:Proteome / Q000032:analysis; D040901:Proteomics / Q000379:methods*
D010940:Plant Proteins; D020543:Proteome