title: Sequential enrichment of singly- and multiply-phosphorylated peptides with zwitterionic hydrophilic interaction chromatography material.
abstract: An interesting and novel method for the selective and sequential enrichment of singly- and multiply-phosphorylated peptides with a zwitterionic material "Click TE-Cys" is presented. Retention mechanisms between phosphopeptides and Click TE-Cys are systematically investigated by checking the influence of acetonitrile content, pH value, and buffer concentration on the retention of phosphopeptides. Both hydrophilic interaction and electrostatic interaction are involved in retention between phosphopeptides and Click TE-Cys. Based on these results, an optimized method is established for selective enrichment of phosphopeptides using Click TE-Cys. This method not only exhibits high selectivity for phosphopeptides, but also fractionates singly- and multiply-phosphorylated peptides into two fractions. This method was evaluated using relatively complex samples, including peptide mixtures of α-casein and bovine serum albumin (BSA) at a molar ratio of 1:10 and skim milk. This efficient and optimized protocol has great potential for enriching multiply-phosphorylated peptides and could be a valuable tool for specific enrichment of phosphopeptides in phosphoproteome analysis.
authors: Shanghai Key Laboratory of Functional Materials Chemistry, East China University of Science and Technology, Shanghai 200237, China.
authors: Qianying
authors: Q
authors: Sheng
authors: Shanghai Key Laboratory of Functional Materials Chemistry, East China University of Science and Technology, Shanghai 200237, China.
authors: Kaiya
authors: K
authors: Yang
authors: Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
authors: Xingya
authors: X
authors: Xue
authors: Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China; Co-innovation Center of Neuroregeneration, Nantong University, Nantong, 226019, China. Electronic address: lixiuling@dicp.ac.cn.
authors: Xiuling
authors: X
authors: Li
authors: Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
authors: Zhimou
authors: Z
authors: Guo
authors: Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
authors: Aijin
authors: A
authors: Shen
authors: Engineering Research Center of Pharmaceutical Process Chemistry, East China University of Science and Technology, Shanghai 200237, China.
authors: Yanxiong
authors: Y
authors: Ke
authors: Shanghai Key Laboratory of Functional Materials Chemistry, East China University of Science and Technology, Shanghai 200237, China; State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China. Electronic address: minbolan@ecust.edu.cn.
authors: Minbo
authors: M
authors: Lan
authors: Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
authors: Xinmiao
authors: X
authors: Liang
mesh_terms: D000097:Acetonitriles; D002364:Caseins / Q000737:chemistry; D002845:Chromatography / Q000295:instrumentation / Q000379:methods; D057930:Click Chemistry; D003545:Cysteine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D010748:Phosphopeptides / Q000737:chemistry*; D010766:Phosphorylation; D012710:Serum Albumin, Bovine / Q000737:chemistry; D012822:Silicon Dioxide / Q000737:chemistry; D014753:Vinyl Compounds / Q000737:chemistry
chemical_list: D000097:Acetonitriles; D002364:Caseins; D010748:Phosphopeptides; D014753:Vinyl Compounds; D012710:Serum Albumin, Bovine; D012822:Silicon Dioxide; D003545:Cysteine; C032159:acetonitrile
keywords: Mass spectrometry; Mixed-mode retention mechanisms; Multiply-phosphorylated peptides; Sequential enrichment; Zwitterionic material