Summary

Kantitatif Uygulamalar için Homojen MALDI Örneklerin Hazırlanması

Published: October 28, 2016
doi:

Summary

Örnek Kurutma işlemleri esnasında alt-tabaka sıcaklığının ayarlanması sayesinde yöntemde MALDI kütle spektrometresi iyon sinyallerin uzaysal farklılıklarını azaltmak için bir kuralı gösterilmektedir.

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

NOT: Bu protokol, kurutuldu-damlacık yöntemiyle hazırlanan maltotrioz ve bradikinin fragmanı (1-7) mekansal heterojen azaltmak için geliştirilmiştir. Protokol hazırlanması ve ön koşullandırma, örnek bırakma ve kurutma ve kütle spektrometrisi veri analizi de dahil olmak üzere, üç ana adımdan oluşur. prosedürleri tarif ve aşağıda daha ayrıntılı olarak tarif edilmiştir: 1. Hazırlık ve Önkoşullanma Örnek Plate Temizleme nitril eldiven giyin ve deterjan ve distile-…

Representative Results

Parlak alan görüntüleri ve örnek plakası 5 sıcaklığı ve 25 ° C ile hazırlanan maltotrioz ve bradikinin fragmanı (1-7) MS Fotoğraflar, Şekil 1 'de gösterilmiştir. Sodiated maltotrioz, iyon sinyali esas olarak doldurulur halinde örnek alanın çevresine 25 ° C lik bir örnek plakası sıcaklığı hazırlandığında. 5 ° C'ye örnek plaka sıcaklığı düşürerek, sinyal homojen tüm numune alanı üzerinde doldurur. 5 ° C'nin altında ö…

Discussion

önceki teorik tahminlere dayanarak, damlacıklar içindeki sıcaklık kaynaklı hidrodinamik akışları dışa çözücü buharlaştırma yoluyla uyarılan kılcal akışları üstesinden gelebilir. sıcaklık damlacık artışı içinde gradyanlar zaman moleküllerin bu iç sirkülasyonu verimliliği artırılmıştır. çevre sıcaklığında çevresi muhafaza edilirken, 5 ° C'nin altında Örnek plaka sıcaklığı tutmak zaman tahmin edilen sonuçlara göre, damlacık içindeki sirkülasyon akımlarının ort…

Disclosures

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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Cite This Article
Ou, Y., Tsao, C., Lai, Y., Lee, H., Chang, H., Wang, Y. Preparation of Homogeneous MALDI Samples for Quantitative Applications. J. Vis. Exp. (116), e54409, doi:10.3791/54409 (2016).

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