MALDI-TOF MS detection of dilute, volume-limited peptide samples with physiological salt levels.
MALDI-TOF MS detection of dilute, volume-limited peptide samples with physiological salt levels.
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DOI：
10.1039/b407322c
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发表时间：
2004-09
期刊：
影响因子：
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通讯作者：
Xiaoyan Zhao;Jennifer Barber-Singh;S. Shippy
中科院分区：
文献类型：
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作者：
Xiaoyan Zhao;Jennifer Barber-Singh;S. Shippy
提出了一种高效的基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）样品前处理技术。该研究的目的是使用常规的MALDI支持物直接和方便地检测具有生理盐水平的体积有限样品中的亚nM水平的肽。在这种新方法中，通过将毛细管尖端直接接触不锈钢MALDI板，可以方便地产生直径为500 μ m的高度均匀的基质-硝酸纤维素斑点。一个阵列的50个微点可以从1微升基质硝化纤维素溶液在1分钟内印迹。有人发现，除了高浓度的硝化纤维素的α-氰基-4-羟基肉桂酸（CHCA）基质溶液是形成微点的关键。将样品沉积在这些微点的顶部并孵育3 min。CHCA-硝酸纤维素表面显示出显著的肽结合能力，可结合亚nM水平的肽。将基质斑点直径限制为500微米，因为肽被限制在小的固相表面积上，所以得到分析物富集效果。即使在>0.15 M盐水平下也观察到选择性肽结合。加载具有多个应用的小等分试样的样品允许低至100 pM的低水平肽检测。用微斑点法成功地分析了大鼠纹状体的推拉灌流液。
This paper presents a highly efficient sample preparation technique for matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The purpose of the research is to use a conventional MALDI support to directly and conveniently detect sub-nM levels of peptides from volume-limited samples with physiological salt levels. In this new method, highly uniform matrix-nitrocellulose spots with a 500 microm diameter were conveniently generated by direct contact of a capillary tip to a stainless steel MALDI plate. An array of 50 microspots can be blotted from 1 microL matrix-nitrocellulose solution within 1 min. It was found that the addition of high concentration nitrocellulose to the alpha-cyano-4-hydroxycinnamic acid (CHCA) matrix solution is critical for the formation of microspots. Samples are deposited on top of those microspots and incubated for 3 min. The CHCA-nitrocellulose surface shows a significant peptide binding capability for sub-nM levels of peptide. Restricting the matrix spot diameter to 500 microm gives an analyte enrichment effect because the peptides are confined to a small solid-phase surface area. Selective peptide binding is seen even with >0.15 M salt levels. Loading small aliquots of samples with multiple applications allows low level peptide detection down to 100 pM. Push-pull perfusates collected from the rat striatum were successfully analyzed with the microspot method.