Cross species applicability of abundant protein depletion columns for ribulose-1,5-bisphosphate carboxylase/oxygenase.
In plants, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is an important enzyme in the Calvin cycle, catalyzing the first step of carbon fixation. Because of its critical role in photosynthesis, RuBisCO comprises 30-60% of the total protein content in green leaf tissue and represents a major protein which can interfere with determination of lower abundance proteins in plant proteomics. A potential solution to aid in the determination of low level proteins in plant proteomics are RuBisCO immunodepletion columns. Two formats, spin and LC, of Seppro IgY RuBisCO depletion columns were evaluated for cross species applicability. The spin and LC columns were found to deplete arabidopsis RuBisCO by greater than 90 and 98%, respectively, and automation could be achieved with the LC format. Canola RuBisCO was depleted to a similar extent, and there was evidence suggesting that corn and tobacco RuBisCO were also highly depleted in flow through fractions. Model proteins were spiked into samples to provide insight into the degree of non-specific binding. Finally, improved detection and identification of lower abundance proteins was demonstrated after depletion.
The Dow Chemical Company, Analytical Sciences, Building 1897, Midland, MI 48667, United States. ncellar@dow.com
Nicholas A
NA
Cellar
Krishnamoorthy
K
Kuppannan
Marsha L
ML
Langhorst
Weiting
W
Ni
Ping
P
Xu
Scott A
SA
Young
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D008969:Molecular Sequence Data; D010449:Peptide Mapping / Q000379:methods; D018515:Plant Leaves / Q000201:enzymology; D010940:Plant Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D012273:Ribulose-Bisphosphate Carboxylase / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014026:Nicotiana / Q000201:enzymology; D003313:Zea mays / Q000201:enzymology
D010940:Plant Proteins; D012273:Ribulose-Bisphosphate Carboxylase