定量应用同质MALDI样品的制备
概要
一种通过调节过程中样品干燥过程中衬底温度降低MALDI质谱离子信号的空间分异协议证明。
Abstract
This protocol demonstrates a simple sample preparation to reduce spatial heterogeneity in ion signals during matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The heterogeneity of ion signals is a severe problem in MALDI, which results in poor data reproducibility and makes MALDI unsuitable for quantitative analysis. By regulating sample plate temperature during sample preparation, thermal-induced hydrodynamic flows inside droplets of sample solution are able to reduce the heterogeneity problem. A room-temperature sample preparation chamber equipped with a temperature-regulated copper base block that holds MALDI sample plates facilitates precise control of the sample drying condition. After drying of sample droplets, the temperature of sample plates is returned to room temperature and removed from the chamber for subsequent mass spectrometric analysis. The areas of samples are examined with MALDI-imaging mass spectrometry to obtain the spatial distribution of all components in the sample. In comparison with the conventional dried-droplet method that prepares samples under ambient conditions without temperature control, the samples prepared with the method demonstrated herein show significantly better spatial distribution and signal intensity. According to observations using carbohydrate and peptide samples, decreasing substrate temperature while maintaining the surroundings at ambient temperature during the drying process can effectively reduce the heterogeneity of ion signals. This method is generally applicable to various combinations of samples and matrices.
Introduction
Mass spectrometry (MS) is one of the most important analytical techniques for analyzing the molecular compositions of complex samples. Among all the ionization methods used in MS, matrix-assisted laser desorption/ionization (MALDI) is the most sensitive and widely used method in bioanalytical applications.1 In comparison to other ionization techniques, MALDI has the highest sensitivity and high tolerance to salt contaminants. Such analytical properties make MALDI the first choice for carbohydrate analysis and many proteomics applications. However, sample preparation is a crucial step for obtaining high quality data in MALDI-MS.
The most commonly used sample preparation method for MALDI-MS is the dried-droplet method, in which sample droplets are deposited on a surface and dried under ambient conditions. This drying method is simple and generally effective.2-5 However, a common problem in the dried-droplet method is that the resultant analyte/matrix crystals normally distribute irregularly. In many cases, the crystals aggregate at the periphery of sample areas, resulting in the so-called ring-stain formation.6-8 The heterogeneous crystal morphologies affect the spatial distribution of analyte molecules, which results in severe fluctuation in ion signal over sample areas. Such severe signal fluctuations and poor data reproducibility are known as the “sweet spot” problem in MALDI-MS.9 Thus, there is a great need for reducing spatial heterogeneities in MALDI-MS dried droplet applications.
Hydrodynamic flows in the sample droplet play an important role in determining the spatial distribution of samples prepared with the dried-droplet method.10-12 It was found that the evaporation of solvent induces outward capillary flows within droplets, which are responsible for the ring-stain formation.7,10 In contrast, recirculation flows induced by tangential surface-tension gradients may counterbalance the outward capillary flows.13 If the recirculation flow speeds are higher than that of the outward capillary flows, the samples can be efficiently redistributed to reduce the heterogeneity problem.14
In this work, we demonstrate a detailed protocol for preparing samples with a simple drying chamber to induce efficient recirculation flows during droplet drying processes. Droplet drying conditions are precisely controlled, including the temperatures of the sample plate and its surroundings, and the relative humidity within the chamber. The model analytes include maltotriose and bradykinin chain (1-7). The matrix used for the demonstration is 2,4,6-trihydroxyacetophenone (THAP). The samples are examined with time-of-flight (TOF) MS, and the data are analyzed quantitatively to show the reduction of heterogeneity.
Protocol
Representative Results
Discussion
根据以往的理论预测，液滴内温度引起的水动力流可以向外克服溶剂挥发诱导毛细血管流动。当温度液滴增加内梯度分子的这种内部再循环的效率提高。根据预测结果，保持样品板温度时低于5℃，同时保持其周围环境温度，再循环流动的液滴内的平均流速大于向外毛细管流动的速度的4倍左右。如果样品板温度是相同的环境中，再循环流的平均速度比向外毛细流慢1800倍。这一计算的结果表明，样?…
開示
The authors have nothing to disclose.
Acknowledgements
This work is supported by the Genomics Research Center, Academia Sinica and the Ministry of Science and Technology of Taiwan, the Republic of China (Contract No. 104-2119-M-001-014).
Materials
| Name of Reagent | |||
| Detergent powder | Alconox | 242985 | |
| Methanol | Merck | 106009 | |
| Acetonitrile | Merck | 100003 | |
| 2,4,6-trihydroxyacetophenone (THAP) | Sigma-Aldrich | T64602 | |
| Bradykinin fragment (1-7) | Sigma-Aldrich | B1651 | |
| Maltotriose | Sigma-Aldrich | 47884 | |
| Pipette tips | Mettler Toledo | 17005091 | |
| Microcentrifuge tube | Axygen | MCT-150-C | |
| Name of Equipment | |||
| Milli-Q water purification system | Millipore | ZMQS6VFT1 | |
| Powder-free nitrile gloves | Microflex | SU-690 | |
| 600 mL beaker | Duran | 2110648 | |
| Ultrasonic cleaner | Delta | DC300H | |
| Hygrometer | Wisewind | 5330 | |
| Nitrogen gas flowmeter | Dwyer | RMA-6-SSV | |
| K-type thermocouples | Digitron | 311-1670 | |
| Centrifuge | Select BioProducts | Force Mini | |
| Pipette | Rainin | pipet-lite XLS | |
| Stereomicroscope | Olympus | SZX16 | |
| Temperature controllable drying chamber | this lab | ||
| Synchronized dual-polarity time-of-flight imaging mass spectrometer (DP-TOF IMS) | this lab | ||
| MALDI-TOF stainless steel sample target | this lab |
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