Membrane proteomic analysis of Arabidopsis thaliana using alternative solubilization techniques.
This study presents a comparative proteomic analysis of the membrane subproteome of whole Arabidopsis seedlings using 2% Brij-58 or 60% methanol to enrich and solubilize membrane proteins for strong cation exchange fractionation and reversed-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 441 proteins were identified by our Brij-58 method, and 300 proteins were detected by our methanol-based solubilization approach. Although the total number of proteins obtained using the nonionic detergent was higher than the total obtained by organic solvent, the ratio of predicted membrane proteins to total proteins identified indicates up to an 18.6% greater enrichment efficiency using methanol. Using two different bioinformatics approaches, between 31.0 and 40.0% of the total proteins identified by the methanol-based method were classified as containing at least one putative transmembrane domain as compared to 22.0-23.4% for Brij-58. In terms of protein hydrophobicity as determined by the GRAVY index, it was revealed that methanol was more effective than Brij-58 for solubilizing membrane proteins ranging from -0.4 (hydrophilic) to +0.4 (hydrophobic). Methanol was also approximately 3-fold more effective than Brij-58 in identifying leucine-rich repeat receptor-like kinases. The ability of methanol to effectively solubilize and denature both hydrophobic and hydrophilic proteins was demonstrated using bacteriorhodopsin and cytochrome c, respectively, where both proteins were identified with at least 82% sequence coverage from a single reversed-phase LC-MS/MS analysis. Overall, our data show that methanol is a better alternative for identifying a wider range of membrane proteins than the nonionic detergent Brij-58.
Mitra
Srijeet K
SK
Department of Horticultural Science, North Carolina State University, Raleigh, North Carolina 27695-7609, USA.
Gantt
John A
JA
Ruby
James F
JF
Clouse
Steven D
SD
Goshe
Michael B
MB
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000737:chemistry* / Q000166:cytology; D029681:Arabidopsis Proteins / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification*; D001436:Bacteriorhodopsins / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification; D002592:Cetomacrogol / Q000737:chemistry; D002853:Chromatography, Liquid / Q000379:methods; D045304:Cytochromes c / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification; D008565:Membrane Proteins / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification*; D000432:Methanol / Q000737:chemistry; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D020543:Proteome / Q000032:analysis*; D012995:Solubility; D013501:Surface-Active Agents / Q000737:chemistry; D053719:Tandem Mass Spectrometry / Q000379:methods
D029681:Arabidopsis Proteins; D008565:Membrane Proteins; D010455:Peptides; D020543:Proteome; D013501:Surface-Active Agents; D001436:Bacteriorhodopsins; D002592:Cetomacrogol; D045304:Cytochromes c; D000432:Methanol