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Preparation of Homogeneous MALDI Samples for Quantitative Applications

Published: October 28th, 2016



1Genomic Research Center, Academia Sinica, 2Department of Chemistry, National Taiwan University, 3Department of Chemical Engineering and Biotechnology, National Taipei University of Technology

A protocol for reducing spatial heterogeneities of ion signals in MALDI mass spectrometry by regulating substrate temperature during sample drying processes is demonstrated.

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.

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.


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NOTE: This protocol is developed for reducing the spatial heterogeneity of maltotriose and bradykinin fragment (1-7) prepared with the dried-droplet method. The protocol consists of three main steps, including preparation and preconditioning, sample deposition and drying, and mass spectrometry data analysis. The procedures are outlined and described in more detail below:

1. Preparation and Preconditioning

  1. Cleaning the Sample Plate
    1. Wear nitrile gloves and hand-wash the sample plate gently wi.......

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The bright-field images as well as the MS images of maltotriose and bradykinin fragment (1-7) prepared with sample plate temperature of 5 and 25 °C are shown in Figure 1. In the case of sodiated maltotriose, the ion signal mainly populates at the periphery of the sample area when it is prepared with a sample plate temperature of 25 °C. By decreasing the sample plate temperature to 5 °C, the signal populates homogeneously over the entire sample area. The onl.......

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Based on previous theoretical predictions, temperature-induced hydrodynamic flows within droplets can overcome outward capillary flows induced by solvent evaporation. The efficiency of such internal recirculation of molecules is enhanced when the temperature gradients within a droplet increase. According to the predicted results, when keeping the sample plate temperature under 5 °C while maintaining its surroundings at ambient temperature, the average velocity of recirculation flows within the droplet is about 4 tim.......

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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).


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Name Company Catalog Number Comments
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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