Sub-microlitre dialysis system to enable trace level peptide detection from volume-limited biological samples using MALDI-TOF-MS.
Sub-microlitre dialysis system to enable trace level peptide detection from volume-limited biological samples using MALDI-TOF-MS.
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DOI：
10.1039/b707783a
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发表时间：
2007-09
期刊：
影响因子：
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通讯作者：
Kyaw ThetMaw Myasein;J. Pulido;R. M. Hatfield;C. McCannel;R. F. Dundervill;S. Shippy
中科院分区：
文献类型：
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作者：
Kyaw ThetMaw Myasein;J. Pulido;R. M. Hatfield;C. McCannel;R. F. Dundervill;S. Shippy
由于样品体积小、多肽浓度范围广、内源性蛋白质和盐分含量高，利用质谱法从有限的生物样品中检测多肽是一项具有挑战性的任务。以前，用MALDI-MS从生物盐水平的亚微升样品中检测痕量多肽的微斑点方法被提出。然而，在蛋白质存在的情况下，肽信号显著减少。本文提出了一种新型的透析器，用于去除亚微升样品中的蛋白质，使用半透中空纤维膜来增强多肽的检测。构建了一种亚微升透析装置，用于从复杂样品中去除蛋白质。以血管紧张素-I为模型肽，在生理盐水中制备的350mgL(-1)牛血清白蛋白模拟生物样品。在没有牛血清白蛋白的情况下，血管紧张素I在250 PM出现明显的峰值，而在牛血清白蛋白存在的情况下，几乎没有检测到10 NM的血管紧张素I。透析后，峰值检测提高到500 PM水平。用CE-LIF测定蛋白质的去除量和多肽的回收率(约66%)。低至200nL的临床玻璃体样本在30分钟内成功透析，检测到的多肽峰增加了3倍，信号大大改善。该方法简单，可用于从体积有限的生物样品中检测痕量水平的多肽。
The detection of peptides with mass spectrometry from volume-limited biological samples is a challenging task due to low sample volume, a broad range of peptide concentrations down to trace levels, endogenous high proteins and salt levels. Previously, a microspotting method was presented for trace-level peptide detection with MALDI-MS from sub-microlitre samples with biological salt levels. However, in the presence of proteins, peptide signals are significantly reduced. This paper presents a novel dialysis device for removal of proteins from sub-microlitre samples using a semipermeable hollow fiber membrane to enhance peptide detection. A dialysis device was constructed to perform sub-microlitre dialysis to remove proteins from complex samples. Angiotensin I was used as a model peptide in the presence of 350 mg L(-1) BSA prepared in physiological saline to mimic biological samples. In the absence of BSA, clear angiotensin I peaks were seen at 250 pM, yet in the presence of the BSA, 10 nM angiotensin I was barely detected. After dialysis, peak detection was improved to a 500 pM level. Protein removal and peptide recovery (approximately 66%) were determined using CE-LIF. Clinical vitreous samples as low as 200 nL were successfully dialyzed in 30 min and a 3-fold increase in peptide peaks were detected with greatly improved signals. This method is simple and can be a useful technique for trace level peptide detection from volume-limited biological samples.