A method for protein extraction from different subcellular fractions of laticifer latex in Hevea brasiliensis compatible with 2-DE and MS

Abstract

BackgroundProteomic analysis of laticifer latex in Hevea brasiliensis has been received more significant attentions. However, the sticky and viscous characteristic of rubber latex as cytoplasm of laticifer cells and the complication of laticifer latex membrane systems has made it challenge to isolate high-quality proteins for 2-DE and MS.ResultsBased on the reported Borax/PVPP/Phenol (BPP) protocol, we developed an efficient method for protein preparation from different latex subcellular fractions and constructed high-resolution reference 2-DE maps. The obtained proteins from both total latex and C-serum fraction with this protocol generate more than one thousand protein spots and several hundreds of protein spots from rubber particles as well as lutoid fraction and its membranes on the CBB stained 2-DE gels. The identification of 13 representative proteins on 2-DE gels by MALDI TOF/TOF MS/MS suggested that this method is compatible with MS.ConclusionThe proteins extracted by this method are compatible with 2-DE and MS. This protein preparation protocol is expected to be used in future comparative proteomic analysis for natural rubber latex.

Systematic comparison of technical details in CBB methods and development of a sensitive GAP stain for comparative proteomic analysis

“…Mass spectra were obtained on a Ultraflex MALDI-TOF/TOF mass spectrometer (Bruker Daltonics, Bremen, Germany). The spectra were analyzed by flexAnalysis software (Version 3.2, Bruker-Daltonics, USA) and searched against the taxonomy of mammals for BSA and green plants for other samples in nonredundant NCBI (NCBInr) database using the MASCOT software (Version 2.3) as described in [20][21][22][23]. The identification was considered only with a high MASCOT score (72 as the threshold), maximum peptide coverage and additional experimental confirmation of protein positions on the gels.…”

“…Proteins were identified by MALDI TOF/TOF MS/MS as described in [4,[20][21][22][23]. Before spot/band picking, the preserved gels were transferred into 50 volumes of ddH 2 O and shaken gently overnight.…”

“…Proteins were produced by the Borax/PVPP/Phenol (BPP) protocol [23] and its developed version for rubber latex [21]. The air-dried pellets were dissolved in the lysis buffer (7 M urea, 2 M thiourea, 2% CHAPS, 13 mM DTT) and the Bradford assay was used to determine the protein concentration.…”

“…This method has been proven to generate high-resolution 2-DE gels as well as good MS compatibility [3,[20][21][22]. However, it requires multiple steps and developed technical skills as well as a long time for additional sensitization.…”

Ethrel-stimulated prolongation of latex flow in the rubber tree (Hevea brasiliensisMuell. Arg.): an Hev b 7-like protein acts as a universal antagonist of rubber particle aggregating factors from lutoids and C-serum

“…First, the protein was digested in-gel with bovine trypsin according to a reported method (22). After digestion, the protein peptides were collected and vacuum dried.…”