Identification of proteins expressed at extremely low level in Arabidopsis leaves.
2-DE related techniques have been broadly used for protein separation and identification in recent years. However, many important cellular components often escape detection by 2-DE due to its inherent low resolution. Here we use a polyU-affinity column to enrich proteins with nucleotide-binding motifs. As a result, 26 enriched protein spots, including three new Arabidopsis RNA-binding proteins cp31A, cp31B, and CSP41, are identified by MALDI-TOF-MS. In the sum, 15 protein spots encoded by 12 individual genes are observed on the 2-DE only after binding to the polyU-affinity column. Two of these genes are expressed at lower than 0.1% the level of TUB4 that were considered mainly as non-expressed genes in several ATH1 microarray analysis of Arabidopsis leaf samples. Our results suggest that polyU or similar media may be combined with 2-DE techniques to identify cellular proteins with extremely low transcription levels to fulfill specific research needs.
Xu
Yu
Y
The National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871, China.
Wang
Bai-Chen
BC
Zhu
Yu-Xian
YX
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics*; D015180:Electrophoresis, Gel, Two-Dimensional; D018506:Gene Expression Regulation, Plant; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis; D018515:Plant Leaves / Q000378:metabolism*; D011072:Poly U / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D011072:Poly U; D012313:RNA