Signal amplification using "spot on-a-chip" technology for the identification of proteins via MALDI-TOF MS
Signal amplification using "spot on-a-chip" technology for the identification of proteins via MALDI-TOF MS
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DOI：
10.1021/ac000734u
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发表时间：
2001-01-15
影响因子：
7.4
通讯作者：
Laurell, T
中科院分区：
文献类型：
--
作者：
Ekström, S;Ericsson, D;Laurell, T
所提出的“单片点”技术可以方便地富集低纳摩尔（1-5 nM）范围内的样品，并为进行基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF MS）分析提供了一种快速可靠的自动化样品制备方法，具有高灵敏度和高通量。通过微点胶，可以精确沉积60 pl液滴，稀释样品通过在纳米瓶中进行多个液滴沉积来富集。样品被限制在一个确定的斑点区域（300 x 300 mum），多次沉积增加了纳米瓶中分析物的表面密度，从而提供了低原子水平的检测。研究了纳米瓶的几何形状对沉积在微制硅瓶中的多肽的MALDI-TOF MS分辨率的影响，并确定了最佳的几何形状和尺寸。点上芯片技术，即微点配、微机械硅纳米瓶和现场富集的结合，与普通样品制备相比，提供了至少10-50倍的信号放大。在5 nM水平上对多肽混合物进行分析，表明富集效果呈线性。芯片上的点富集所提供的信号放大可以通过相关生物样品的分析、尖刺细胞上清中的白细胞介素-8以及高灵敏度银染色二维电泳凝胶分离中切除点的成功蛋白质鉴定来证明。
The presented "spot-on-a-chip" technology enables easy enrichment of samples in the low nanomolar (1-5 nM) range and provides a fast and reliable automated sample preparation method for performing matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis with high sensitivity and throughput. Through microdispensing, which allows accurate deposition of 60-pL droplets, dilute samples were enriched by making multiple droplet depositions in nanovials. The sample was confined to a defined spot area (300 x 300 mum), and multiple depositions increase the surface density of analyte in the nanovial, thereby providing detection of low attomole levels. The impact of the nanovial geometry with respect to the MALDI-TOF MS resolution for peptides deposited in the microfabricated silicon vials was investigated and the optimal geometry and size were determined. The spot-on-a-chip technology, that is, the combination of microdispensing, micromachined silicon nanovials and on-spot enrichment provides a signal amplification of at least 10-50 times as compared to an ordinary sample preparation. The linearity of the enrichment effect is shown by the analysis of a peptide mixture at the 5 nM level. The signal amplification provided by the spot-on-a-chip enrichment is demonstrated by the analysis of relevant biological samples, interleukin-8 from a spiked cell supernatant, and by successful protein identification of an excised spot from a high-sensitivity silver-stained two-dimensional electrophoresis gel separation.