Kretschy Daniela, Gröger Marion, Zinkl Daniela, Petzelbauer Peter, Koellensperger Gunda, Hann Stephan
University of Natural Resources and Life Sciences, BOKU Vienna, Department of Chemistry, Division of Analytical Chemistry, Muthgasse 18, A-1190 Vienna, Austria.
Int J Mass Spectrom. 2011 Oct 1;307(1-3):105-111. doi: 10.1016/j.ijms.2011.02.008.
A high throughput method based on flow injection analysis was developed and validated for the quantification of the peptide Bβ(15-42) 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-Bβ(15-42) (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 Bβ(15-42). 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 Bβ(15-42)-In-DOTA-Bn.
开发了一种基于流动注射分析的高通量方法，并对其进行了验证，用于比较不同标记策略和检测方法时对细胞样品中肽Bβ(15 - 42)的定量分析。所用的标记物为用于元素标记的1,4,7,10 - 四氮杂环十二烷 - N,N',N'',N'''- 四乙酸（铟 - DOTA）和2 - (4 - 异硫氰酸苄基)-1,4,7,10 - 四氮杂环十二烷 - N,N',N'',N'''- 四乙酸（铟 - DOTA - Bn）。6 - 羟基 - 9 - (2 - 羧基苯基)-(3H)-呫吨 - 3 - 酮（荧光素）用作荧光标记物。所研究的肽（质量 = 3 kD）是一种新型候选药物，在心肌梗死事件后具有抗炎作用。分析的样品是通过用Triton X缓冲液裂解来维持的人脐静脉内皮细胞（HUVEC）的细胞区室。为了通过流动注射提高肽定量的灵敏度和选择性，在孵育前使用元素和荧光标记物对肽进行标记。分别通过流动注射分析结合电感耦合等离子体扇形场质谱（FIA - ICP - SFMS）或荧光检测（FIA - FLD）对元素标记和荧光标记的肽进行定量。所采用的定量策略在荧光检测情况下为外标法，在ICP - MS检测情况下为有和没有内标法的外标法以及在线同位素稀释质谱法（IDMS）。FIA - ICP - MS的检测限（LOD）为9 pM铟 - DOTA - Bβ(15 - 42)（绝对量0.05 fmol），而FIA - FLD对荧光素标记的肽显示的LOD为100 pM（绝对量3 fmol）。与FIA - FLD相比，FIA - ICP - MS的短期精密度在所有基于ICP - MS的定量策略中更优（FIA - ICP - SFMS：0.3 - 3.3%；FIA - FLD：6.5%）。关于长期精密度，采用在线IDMS和内标的FIA - ICP - SFMS显示出最佳结果（分别为3.1%和4.6%），而两种应用方法的外标法仅在10%的范围内。相对于细胞培养中铟的应用量，Triton X可溶部分中铟 - DOTA标记的肽Bβ(15 - 42)的浓度范围为0.75 - 1.8%，2 - (4 - 异硫氰酸苄基)-1,4,7,10 - 四氮杂环十二烷 - N,N',N'',N'''- 四乙酸（铟 - DOTA - Bn）标记的肽Bβ(15 - 42)的浓度范围为0.30 - 0.79%。在Triton X不溶部分中，铟 - DOTA标记的肽中铟的相对浓度为0.03 - 0.18%，Bβ(15 - 42)-铟 - DOTA - Bn中铟的相对浓度为0.03 - 0.13%。
