Liquid-chromatography-mass spectrometry of thylakoid membrane proteins.
The present chapter describes methods for the separation and identification of photosynthetic proteins of thylakoid membranes present in chloroplasts by using different detergents, high-performance liquid chromatography and mass spectrometry. Thylakoid membranes represent a good model for setting up analytical methods suitable for membrane protein characterization.The first step in the procedure is the preparation of purified membrane fractions from plant tissues, followed by the fractionation of membrane proteins by differential solubilization using different detergents. Thus, several protein complexes can be isolated, collected, separated by ion-pair reversed-phase chromatography and detected online by UV-absorption and/or mass spectrometry. Finally, identification of the eluting proteins is accomplished by comparing the molecular mass determined in silico with the molecular mass measured by mass spectrometry.
Huber
Christian G
CG
Instrumental Analysis and Bioanalysis, Saarland University, Saarbrücken, Germany.
Timperio
Anna-Maria
AM
Toll
Hansjörg
H
Zolla
Lello
L
D005591:Chemical Fractionation; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003902:Detergents / Q000737:chemistry; D003911:Dextrans / Q000737:chemistry; D004072:Digitonin / Q000737:chemistry; D018527:Hydroponics; D013058:Mass Spectrometry / Q000379:methods*; D045331:Photosystem I Protein Complex / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D045332:Photosystem II Protein Complex / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D010940:Plant Proteins / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D011092:Polyethylene Glycols / Q000737:chemistry; D018724:Spinacia oleracea / Q000737:chemistry / Q000166:cytology / Q000254:growth & development; D020524:Thylakoids / Q000737:chemistry*
D003902:Detergents; D003911:Dextrans; D045331:Photosystem I Protein Complex; D045332:Photosystem II Protein Complex; D010940:Plant Proteins; D011092:Polyethylene Glycols; D004072:Digitonin