Protein extraction from plant samples is usually challenging due to the low protein content and high level of contaminants.Therefore,the 2-DE pattern resolution is strongly influenced by the procedure of sample preparation.To understand mechanisms in cold resistance of alpine plants,a proteomic analysis of callus from a Tibet alpine plant Saussurea involucrata grown at 0℃ was performed.Three improved protein extraction methods,i.e.TCA/aceton extraction,urea/thiourea extraction and phenol extraction,were adopted to extract proteins from samples.The prepared protein samples were separated by twodimensional electrophoresis,followed by colloidal Coomassie Blue G-250 staining.The three methods were compared in protein yield,protein purity,and electrophoregram.The results showed that:(1)Most proteins can be extracted by the phenol protein extraction.In addition,the background is more clear,and less longitudinal and transverse stripes exist.(2)About 500 μg protein sample was the most appropriate for 17 cm IPG strip.(3)2D-MALDI-TOF analysis of the callus samples resulted in the identification of 38 proteins that were differentially accumulated during cold acclimation (0℃ for 12 h).33 proteins were up-regulated by 1.5 fold and 5 proteins were downregulated by 2 fold.(4)NADP-dependent isocitrate dehydrogenase,adenosylhomocysteinase,resistance protein RPP8-like protein,hypothetical protein gi|13129470,and alpha-tubulin were induced in coldacclimated callus.The proteins are related to metabolism,plant defense,energy metabolism,and cell structure,respectively.