High-throughput flow injection analysis of labeled peptides in cellular samples – ICP-MS analysis versus fluorescence based detection

摘要

A high throughput method based on flow injection analysis was developed and validated for the quantification of the peptide Bβ15-42 in cellular samples comparing different labeling strategies and detection methods. The used labels were 1,4,7,10-tetraazacyclododecane-N, N′, N′′, N′′′-tetraaceticacid (In-DOTA) and 2-(4-isothiocyanatobenzyl) - 1,4,7,10-tetraazacyclododecane-N, N′, N′′, N′′′-tetraacetic acid (In-DOTA-Bn) for elemental labeling. 6-Hydroxy-9-(2-carboxyphenyl)- (3H)-xanthen-3-on (fluorescein) was employed as fluorescence label. The explored peptide (mass = 3 kD) is a novel candidate drug, which shows an anti-inflammatory effect after an event of myocardial infarction. The analysed samples were fractioned cell compartments of human umbilical cord vein endothelial cells (HUVEC) maintained via lysis with Triton X buffer. In order to enhance sensitivity and selectivity of peptide quantification via flow injection the peptide was labeled prior to incubation using elemental and fluorescence labels. Quantification of the elemental and fluorescence labeled peptide was performed via flow injection analysis combined with inductive coupled plasma sector field mass spectrometry (FIA-ICP-SFMS) or fluorescence detection (FIA-FLD), respectively. The employed quantification strategies were external calibration in the case of fluorescence detection and external calibration with and without internal standardization and on-line IDMS in the case of ICP-MS detectionThe limit of detection (LOD) for FIA-ICP-MS was 9 pM In-DOTA-Bβ15-42 (0.05 fmol absolute) whereas FIA-FLD showed a LOD of 100 pM (3 fmol absolute) for the fluorescein labeled peptide. Short term precision of FIA-ICP-MS was superior for all ICP-MS based quantification strategies compared to FIA-FLD (FIA-ICP-SFMS: 0.3-3.3%; FIA-FLD: 6.5%). Concerning long term precision FIA-ICP-SFMS with on-line IDMS and internal standardization showed the best results (3.1 and 4.6%, respectively) whereas the external calibration of both applied methodological approaches was only in the range of 10 %.The concentrations in the Triton X soluble fraction relative to the applied amount of Indium in the cell culture were in the range of 0.75-1.8% for In-DOTA or 0.30-0.79% for the 2-(4-isothiocyanatobenzyl) - 1,4,7,10-tetraazacyclododecane-N, N′, N′′, N′′′-tetraacetic acid (In-DOTA-Bn) labeled peptide Bβ15-42. In the Triton X insoluble fraction the relative concentrations of Indium were 0.03-0.18% for the In-DOTA labeled peptide and 0.03-0.13% for Bβ15-42-In-DOTA-Bn.