Fluorescence Labeling Protein Quantification and Identification by High Performance Liquid Chromatog

• 作 者: Xiangmin Zhang Junyan Liu Yang Liu Mingxia Gao Chunhui Deng
• 机 构: DepartmentofChemistryandInstitutesofBiomedicalSciences,FudanUniversity,Shanghai200433,China
• 出 处: 第2届大连国际色谱学术报告会、第37届国际高效液相色谱及相关技术会议、第18届全国色谱学术报告会
• 发表日期: 2011年10期
• 关键词: Quantitative proteomics Fluorescence labeling HPLC Mass spectrometry

　　A new approach to proteomic quantification and identification was developed to make full uses of multi-dimensional HPLC with high-sensitive fluorescence detection in quantification and mass spectrometry (MS) in identification.Requirement should be met for fluorescence labeling in MS identification: Labeling completeness, minimum side-reactions, simple MS spectra and no extra tandem MS fragmentations for easy structure elucidations.Additionally, high reproducibility and sensitivity, wide linearity of quantification in chromatography are also necessary.Quantitative labeling was achieved after optimization of fluorescence types and reacting conditions.A so-call fluorescent tag absolute quantification (FTAQ) method was developed.A fluorescence dye, 5-iodoacetamidofluorescein, was finally chosen to label proteins on all cysteine residues.Model proteins, BSA (35 cysteines), OVA and other proteins with different cysteine-sites, were used for verifying the completeness of labeling.A synthesized pure peptide as internal standard, parts of proteins in human liver proteome were quantified and identified using this method.Accurate absolute quantification results were demonstrated.