title: Thermal denaturation produced degenerative proteins and interfered with MS for proteins dissolved in lysis buffer in proteomic analysis.
abstract: In 1-DE, proteins were traditionally mixed with the standard Laemmli buffer and boiled for several minutes. Recently, proteins dissolved in lysis buffer were used to produce better-resolved 2-DE gels, but thermal denaturation procedure still remained in some proteomic analysis. To determine the detailed effects of thermal denaturation on SDS-PAGE and MS, both 1-DE and 2-DE were performed using proteins heated at 100°C for different periods of time, and 17 protein bands/spots were positively identified by MALDI TOF/TOF MS/MS. Protein profiles on both 1-DE and 2-DE gels changed obviously and more polydisperse bands/spots were observed with increased heating time for over-heated samples. Based on these observations, an alternative protein marker-producing method was designed by directly dissolving protein standards without BSA into lysis buffer. This new kind of protein marker could be stored at room temperature for a long time, thus was more convenient for using and shipping. The identification of 17 proteins via MS and comparison of their identities revealed MASCOT-searched scores, number of both matched peptides, total searched peptides and sequence coverage became progressively lower with increasing denaturation intensity, probably due to the interference of thermal denaturation on trypsin cleavage efficiency and produced redundant modified peptides. Therefore, it was concluded that thermal denaturation not only changed the protein profiles and produced more polydisperse protein bands/spots, but also heavily interfered with the subsequent MS analysis, hence not recommended in future proteomic analysis for proteins dissolved in lysis buffer.
authors.affiliation: Key Laboratory of Tropical Crop Biotechnology, Ministry of Agriculture, Institute of Tropical Biosciences and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou Hainan, PR China. xchwanghainan@163.com
authors.forename: Xuchu
authors.initials: X
authors.lastname: Wang
authors.forename: Haiyan
authors.initials: H
authors.lastname: Wang
authors.forename: Dan
authors.initials: D
authors.lastname: Wang
authors.forename: Dongyang
authors.initials: D
authors.lastname: Wang
authors.forename: Bing
authors.initials: B
authors.lastname: Han
authors.forename: Weimin
authors.initials: W
authors.lastname: Tian
authors.forename: Anping
authors.initials: A
authors.lastname: Guo
mesh_terms: D000595:Amino Acid Sequence; D001798:Blood Proteins / Q000032:analysis*; D002460:Cell Line; D015180:Electrophoresis, Gel, Two-Dimensional / Q000295:instrumentation / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004926:Escherichia coli / Q000378:metabolism; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis; D010940:Plant Proteins / Q000032:analysis*; D011489:Protein Denaturation; D012710:Serum Albumin, Bovine / Q000032:analysis* / Q000378:metabolism; D012967:Sodium Dodecyl Sulfate / Q000592:standards; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods; D014325:Tromethamine / Q000592:standards
chemical_list: D001798:Blood Proteins; C088816:Laemmli buffer; D010455:Peptides; D010940:Plant Proteins; D014325:Tromethamine; D012710:Serum Albumin, Bovine; D012967:Sodium Dodecyl Sulfate