Universal sample preparation method integrating trichloroacetic acid/acetone precipitation with phenol extraction for crop proteomic analysis

Abstract

Crop plants contain large amounts of secondary compounds that interfere with protein extraction and gel-based proteomic analysis. Thus, a protein extraction protocol that can be easily applied to various crop materials with minimal optimization is essential. Here we describe a universal protocol for total protein extraction involving trichloroacetic acid (TCA)/acetone precipitation followed by SDS and phenol extraction. Through SDS extraction, the proteins precipitated by the TCA/acetone treatment can be fully resolubilized and then further purified by phenol extraction. This protocol combines TCA/acetone precipitation, which aggressively removes nonprotein compounds, and phenol extraction, which selectively dissolves proteins, resulting in effective purification of proteins from crop tissues. This protocol can also produce high-quality protein preparations from various recalcitrant tissues, and therefore it has a wide range of applications in crop proteomic analysis. Designed to run on a small scale, this protocol can be completed within 5 h.

Bioorthogonal Noncanonical Amino Acid Tagging (BONCAT) Enables Time-Resolved Analysis of Protein Synthesis in Native Plant Tissue

“…Samples were prepared with protocols adapted from Wang et al (2006) and Wu et al (2014). Frozen aerial tissues were macerated in liquid nitrogen with a mortar and pestle.…”

“…Specifically, we pulsed Aha into Arabidopsis seedlings and allowed nascent proteins to be labeled for 3 h at room temperature. To remove nonprotein contaminants and obtain better quality samples for mass spectrometry, we isolated total protein from seedlings via trichloroacetic acid-acetone precipitation followed by phenol extraction (Wang et al, 2006;Wu et al, 2014).…”

Neogenin-1 Promotes Cell Proliferation, Motility, and Adhesion by Up-Regulation of Zinc Finger E-Box Binding Homeobox 1 Via Activating the Rac1/PI3K/AKT Pathway in Gastric Cancer Cells

“…In addition, ZEB1 was identified as an independent prognostic factor of GC, and there is a significant correlation between ZEB1 expression and diffuse phenotype in GC [38]. Previous studies suggested that a variety of factors such as microRNAs (miRNAs) and ubiquitin ligase CUL4A can regulate the EMT process by modulating ZEB1 expression [40][41][42][43][44]. Our study provided new evidence that Neo1 promoted the EMT process in GC by regulating the expression of ZEB1.…”

Identification of proteins from wild cardoon flowers (Cynara cardunculus L.) by a proteomic approach

“…The aqueous phase dissolves cell debris, nucleic acids, and carbohydrates, while the phenolic phase carries proteins and lipids [29]. Proteins from the phenolic phase are then precipitated with methanol and ammonium acetate [30].…”

“…Furthermore, three spots were found at low molecular levels, with an approximate molecular mass in the range of 10 and 20 kDa. In fact, according to several studies of cardoon's protein profile [14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31], proteins with low Mw, represented the β-subunits of cardosins (16.5 and 13.5 kDa), since there were a positive correlation between band intensities and milk-clotting activities.…”