Tandem mass spectrometry identifies sites of three post-translational modifications of spinach light-harvesting chlorophyll protein II. Proteolytic cleavage, acetylation, and phosphorylation.

摘要: The photosynthetic membranes of spinach (Spinacia oleracea L.) chloroplasts were incubated with [gamma-32P] ATP. When the thylakoid membrane kinase was activated light, 25- and 27-kDa forms light-harvesting chlorophyll a/b protein (LHC II) phosphorylated on their amino termini. Treatment proteinase K or thermolysin released phosphopeptides which purified by ferric ion affinity chromatography reverse phase high performance liquid chromatography. Sequencing performed tandem quadrupole mass spectrometry. Three different Ac-RKTAGKPKT, Ac-RKTAGKPKN, Ac-RKSAGKPKN originating from class I LHC II examined after release thermolysin. One phosphopeptide, Ac-RRTVKSAPQ, K. Each four derived terminus a distinct protein. Peptides acetylated at amino-terminal arginine either threonine serine in third position. We conclude that proteolytic processing pre-LHC occurs conserved methionyl-arginyl bond is followed acetylation nearby phosphorylation mature II. Eight peptides synthesized nonacetylated as substrates for kinase. From comparison kinetics phosphate incorporation into peptides, we basic residues both sides site are important enzyme recognition. Acetylation not required phosphorylation.