title: Directed analysis of cyanobacterial membrane phosphoproteome using stained phosphoproteins and titanium-enriched phosphopeptides.
abstract: Gel-free shotgun phosphoproteomics of unicellular cyanobacterium Synechocystis sp. PCC 6803 has not been reported up to now. The purpose of this study is to develop directed membrane phosphoproteomic method in Synechocystis sp. Total Synechocystis membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and phosphoprotein-stained gel bands were selectively subjected to in-gel trypsin digestion. The phosphorylation sites of the resulting peptides were determined by assigning the neutral loss of [M-H(3)PO(4)] to Ser, Thr, and Tyr residues using nano-liquid chromatography 7 Tesla Fourier transform mass spectrometry. As an initial application, 111 proteins and 33 phosphoproteins were identified containing 11 integral membrane proteins. Identified four unknown phosphoproteins with transmembrane helices were suggested to be involved in membrane migration or transporters based on BLASTP search annotations. The overall distribution of hydrophobic amino acids in pTyr was lower in frequency than that of pSer or pThr. Positively charged amino acids were abundantly revealed in the surrounding amino acids centered on pTyr. A directed shotgun membrane phosphoproteomic strategy provided insight into understanding the fundamental regulatory processes underlying Ser, Thr, and Tyr phosphorylation in multi-layered membranous cyanobacteria.
authors.affiliation: Biological Disaster Analysis Group, Korea Basic Science Institute, Daejeon, 305-806, Republic of Korea.
authors.forename: Dong-Gi
authors.initials: DG
authors.lastname: Lee
authors.forename: Joseph
authors.initials: J
authors.lastname: Kwon
authors.forename: Chi-Yong
authors.initials: CY
authors.lastname: Eom
authors.forename: Young-Moon
authors.initials: YM
authors.lastname: Kang
authors.forename: Seong Woon
authors.initials: SW
authors.lastname: Roh
authors.forename: Kyung-Bok
authors.initials: KB
authors.lastname: Lee
authors.forename: Jong-Soon
authors.initials: JS
authors.lastname: Choi
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D010748:Phosphopeptides / Q000737:chemistry* / Q000302:isolation & purification; D010750:Phosphoproteins / Q000737:chemistry* / Q000302:isolation & purification; D010766:Phosphorylation; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D046939:Synechocystis / Q000737:chemistry*; D053719:Tandem Mass Spectrometry; D014025:Titanium; D014443:Tyrosine / Q000737:chemistry
chemical_list: D000596:Amino Acids; D001426:Bacterial Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D010748:Phosphopeptides; D010750:Phosphoproteins; D020543:Proteome; D014443:Tyrosine; D014025:Titanium
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