title: Sequential on-line C-terminal sequencing of peptides based on carboxypeptidase Y digestion and optically gated capillary electrophoresis with laser-induced fluorescence detection.
abstract: We report a novel method for sequential on-line C-terminal sequencing of peptides, which combines carboxypeptidase Y (CPY) digestion with on-line derivatization and optically gated capillary electrophoresis with laser-induced fluorescence detection (OGCE-LIF). Various factors that may affect the C-terminal sequencing were investigated and optimized. High repeatability of on-line derivatization and the sequential OGCE-LIF assay of amino acids (AAs) was achieved with relative standard deviation (RSD) (n=20) less than 1.5% and 3.2% for migration time and peak height, respectively. A total of 13 AAs was efficiently separated in the present study, indicating that the method can be used for sequencing of peptides consisting of the 13 AAs studied. Using two synthesized N-terminally blocked peptides as test examples, we show that the present method can on-line monitor the released AAs with a temporal resolution of 50s during the entire CPY digestion process. The rates of AA release as a function of digestion time were easily measured; thus, the AA sequence of the peptide was determined with just one OGCE assay. Our study indicates the present approach is an effective, reliable, and convenient method for rapid analysis of the C-terminal sequence of peptides, with potential application in peptide analysis and proteome research.
authors.affiliation: Key Laboratory of Nanobiosensing and Nanobioanalysis at Universities of Jilin Province, Department of Chemistry, Northeast Normal University, 5268 Renmin Street, Changchun, Jilin Province 130024, PR China.
authors.forename: Miaomiao
authors.initials: M
authors.lastname: Tian
authors.affiliation: Key Laboratory of Nanobiosensing and Nanobioanalysis at Universities of Jilin Province, Department of Chemistry, Northeast Normal University, 5268 Renmin Street, Changchun, Jilin Province 130024, PR China.
authors.forename: Ning
authors.initials: N
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Nanobiosensing and Nanobioanalysis at Universities of Jilin Province, Department of Chemistry, Northeast Normal University, 5268 Renmin Street, Changchun, Jilin Province 130024, PR China.
authors.forename: Xiaoxia
authors.initials: X
authors.lastname: Liu
authors.affiliation: Key Laboratory of Nanobiosensing and Nanobioanalysis at Universities of Jilin Province, Department of Chemistry, Northeast Normal University, 5268 Renmin Street, Changchun, Jilin Province 130024, PR China.
authors.forename: Liping
authors.initials: L
authors.lastname: Guo
authors.affiliation: Key Laboratory of Nanobiosensing and Nanobioanalysis at Universities of Jilin Province, Department of Chemistry, Northeast Normal University, 5268 Renmin Street, Changchun, Jilin Province 130024, PR China. Electronic address: yangl330@nenu.edu.cn.
authors.forename: Li
authors.initials: L
authors.lastname: Yang
mesh_terms: D000595:Amino Acid Sequence; D043402:Cathepsin A / Q000378:metabolism*; D019075:Electrophoresis, Capillary; D007834:Lasers; D010455:Peptides / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D020539:Sequence Analysis, Protein; D013050:Spectrometry, Fluorescence
chemical_list: D010455:Peptides; D043402:Cathepsin A
keywords: C-terminal sequencing; Capillary electrophoresis; Carboxypeptidase Y; Peptide