Name: preparation of homogeneous maldi samples for quantitative applications
Uploaded: 2016-10-28T13:00:00
Description: Watch this Scientific Journal Video about Preparation of Homogeneous MALDI Samples for Quantitative Applications at JoVE.com

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).

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
<ol>
	<li>Cleaning the Sample Plate
	<ol>
		<li>Wear nitrile gloves and hand-wash the sample plate gently wi...

<ol>
	<li>Karas, M., Hillenkamp, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+Desorption+Ionization+of+Proteins+with+Molecular+Masses+Exceeding+10000+Daltons.">Laser Desorption Ionization of Proteins with Molecular Masses Exceeding 10000 Daltons.</a> Anal. Chem. 60, 2299-2301 (1988).</li><li>Beavis, R. C., Chait, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Velocity+Distributions+of+Intact+High+Mass+Polypeptide+Molecule+Ions+Produced+by+Matrix+Assisted+Laser+Desorption.">Velocity Distributions of Intact High Mass Polypeptide Molecule Ions Produced by Matrix Assisted Laser Desorption.</a> Chem. Phys. Lett. 181, 479-484 (1991).</li><li>Beavis, R. C., Chaudhary, T., Chait, B. 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Mass Spectrom. 27, 472-480 (1992).</li><li>Strupat, K., Karas, M., Hillenkamp, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2,5-Dihydroxybenzoic+Acid+-+a+New+Matrix+for+Laser+Desorption+Ionization+Mass-Spectrometry.">2,5-Dihydroxybenzoic Acid - a New Matrix for Laser Desorption Ionization Mass-Spectrometry.</a> Int. J. Mass Spectrom. Ion Process. 111, 89-102 (1991).</li><li>Hu, H., Larson, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaporation+of+a+Sessile+Droplet+on+a+Substrate.">Evaporation of a Sessile Droplet on a Substrate.</a> J. Phys. Chem. B. 106, 1334-1344 (2002).</li><li>Deegan, R. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capillary+Flow+as+the+Cause+of+Ring+Stains+from+Dried+Liquid+Drops.">Capillary Flow as the Cause of Ring Stains from Dried Liquid Drops.</a> Nature. 389, 827-829 (1997).</li><li>Hu, J. -. B., Chen, Y. -. C., Urban, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coffee-Ring+Effects+in+Laser+Desorption/Ionization+Mass+Spectrometry.">Coffee-Ring Effects in Laser Desorption/Ionization Mass Spectrometry.</a> Anal. Chim. Acta. 766, 77-82 (2013).</li><li>Schwartz, S. A., Reyzer, M. L., Caprioli, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+Tissue+Analysis+Using+Matrix-Assisted+Laser+Desorption/Ionization+Mass+Spectrometry:+Practical+Aspects+of+Sample+Preparation.">Direct Tissue Analysis Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry: Practical Aspects of Sample Preparation.</a> J. Mass Spectrom. 38, 699-708 (2003).</li><li>Hu, H., Larson, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marangoni+Effect+Reverses+Coffee-Ring+Depositions.">Marangoni Effect Reverses Coffee-Ring Depositions.</a> J. Phys. Chem. B. 110, 7090-7094 (2006).</li><li>Bhardwaj, R., Fang, X., Attinger, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pattern+Formation+During+the+Evaporation+of+a+Colloidal+Nanoliter+Drop:+A+Numerical+and+Experimental+Study.">Pattern Formation During the Evaporation of a Colloidal Nanoliter Drop: A Numerical and Experimental Study.</a> New J. Phys. 11, 075020 (2009).</li><li>Savino, R., Paterna, D., Favaloro, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Buoyancy+and+Marangoni+Effects+in+an+Evaporating+Drop.">Buoyancy and Marangoni Effects in an Evaporating Drop.</a> J Thermophys Heat Tr. 16, 562-574 (2002).</li><li>Probstein, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Surface Tension. in Physicochemical Hydrodynamics : An Introduction. , 305-361 (1994).</li><li>Lai, Y. -. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reducing+Spatial+Heterogeneity+of+MALDI+Samples+with+Marangoni+Flows+During+Sample+Preparation.">Reducing Spatial Heterogeneity of MALDI Samples with Marangoni Flows During Sample Preparation.</a> J. Am. Soc. Mass Spectrom. 27, 1314-1321 (2016).</li><li>Hsiao, C. -. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+Molecular+Imaging+of+Photolabile+Surface+Samples+with+Synchronized+Dual-Polarity+Time-of-Flight+Mass+Spectrometry.">Comprehensive Molecular Imaging of Photolabile Surface Samples with Synchronized Dual-Polarity Time-of-Flight Mass Spectrometry.</a> Rapid Commun. Mass Spectrom. 25, 834-842 (2011).</li><li>Vorm, O., Roepstorff, P., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+Resolution+and+Very+High-Sensitivity+in+MALDI+TOF+of+Matrix+Surfaces+Made+by+Fast+Evaporation.">Improved Resolution and Very High-Sensitivity in MALDI TOF of Matrix Surfaces Made by Fast Evaporation.</a> Anal. Chem. 66, 3281-3287 (1994).</li><li>Gabriel, S. J., Schwarzinger, C., Schwarzinger, B., Panne, U., Weidner, S. 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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 &#176;C while maintaining its surroundings at ambient temperature, the average velocity of recirculation flows within the droplet is about 4 tim...

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.

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			<td height="21" style="height:21px;width:303px;">Name of Reagent</td>
			<td style="width:157px;"></td>
			<td style="width:139px;"></td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Detergent powder</td>
			<td style="width:157px;">Alconox</td>
			<td style="width:139px;">242985</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Methanol</td>
			<td style="width:157px;">Merck</td>
			<td style="width:139px;">106009</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Acetonitrile</td>
			<td style="width:157px;">Merck</td>
			<td style="width:139px;">100003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">2,4,6-trihydroxyacetophenone (THAP)</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td style="width:139px;">T64602&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Bradykinin fragment (1-7)</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td style="width:139px;">B1651</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Maltotriose</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td style="width:139px;">47884</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Pipette tips</td>
			<td style="width:157px;">Mettler Toledo</td>
			<td style="width:139px;">17005091</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Microcentrifuge tube</td>
			<td style="width:157px;">Axygen</td>
			<td style="width:139px;">MCT-150-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;"></td>
			<td style="width:157px;"></td>
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			<td height="21" style="height:21px;width:303px;">Name of Equipment</td>
			<td style="width:157px;"></td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Milli-Q water purification system</td>
			<td style="width:157px;">Millipore</td>
			<td style="width:139px;">ZMQS6VFT1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Powder-free nitrile gloves</td>
			<td style="width:157px;">Microflex</td>
			<td style="width:139px;">SU-690</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">600 mL beaker</td>
			<td style="width:157px;">Duran</td>
			<td style="width:139px;">2110648</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Ultrasonic cleaner</td>
			<td style="width:157px;">Delta</td>
			<td style="width:139px;">DC300H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Hygrometer</td>
			<td style="width:157px;">Wisewind</td>
			<td style="width:139px;">5330</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Nitrogen gas flowmeter</td>
			<td style="width:157px;">Dwyer</td>
			<td style="width:139px;">RMA-6-SSV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">K-type thermocouples</td>
			<td style="width:157px;">Digitron</td>
			<td style="width:139px;">311-1670</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Centrifuge</td>
			<td style="width:157px;">Select BioProducts</td>
			<td style="width:139px;">Force Mini&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Pipette</td>
			<td style="width:157px;">Rainin</td>
			<td style="width:139px;">pipet-lite XLS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Stereomicroscope</td>
			<td style="width:157px;">Olympus</td>
			<td style="width:139px;">SZX16</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Temperature controllable drying chamber</td>
			<td style="width:157px;">this lab</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">Synchronized dual-polarity time-of-flight imaging mass spectrometer (DP-TOF IMS)</td>
			<td style="width:157px;">this lab</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">MALDI-TOF stainless steel sample target</td>
			<td style="width:157px;">this lab</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of homogeneous maldi samples for quantitative applications

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.

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 &#176;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 &#176;C. By decreasing the sample plate temperature to 5 &#176;C, the signal populates homogeneously over the entire sample area. The onl...

Watch this Scientific Journal Video about Preparation of Homogeneous MALDI Samples for Quantitative Applications at JoVE.com

Preparation of Homogeneous MALDI Samples for Quantitative Applications

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 ...

The overall goal of this sample preparation method from MALDI MS is to reduce the spatial heterogeneity in ion signals during MALDI MS.A well controlled drying environment was built to optimize the conventional dried-droplet method, by manipulating the hydrodynamic flows inside the sample droplet during the drying process. The main advantage of this technique is that the spatial heterogeneity of the ion signals for the sample prepared by the optimized dried-droplet method can be effectively reduced. Though this method can provide homogenous samples for the THAP Metric System.It can also be applied to other metric systems, such as alpha-Cyano-4-hydroxycinnamic acid. Generally, individuals new to this method were struggled because controlling the drying condition is not easy. To begin this procedure, open the door of a drying chamber and immediately place a previously cleaned sample plate on the copper-based block.Then, close the door. Manually adjust the gas-flow meter to set the nitrogen flow rate to 10 standard cubic feet per hour. Following this, fine-tune the gas-flow meter to ensure the relative humidity is always below 25%in the drying chamber, as measured by a hygrometer.Using K Type Thermocouples to monitor the temperature, adjust the water circulator temperature manually, until the sample plate reaches 5 degrees Celsius for the experiment. Ensure the required temperatures and the relative humidity are reached before sample deposition. Mix 0.25 microliters of 0.1 Molar THAP solution and 0.25 microliters of the desired analyte in a microcentrifuge tube.Vortex the sample for three seconds. Then, centrifuge the sample for two seconds to collect the solution at the bottom of the microcentrifuge tube. Following centrifugation, open the door of the drying chamber and carefully deposit 0.1 microliters of the solution on the sample plate with a pipette.Close the door immediately and wait for the sample droplet to dry out. When keeping the sample plate temperature lower than its surroundings, the average velocity of recirculation flows within the droplet is significantly increased to redistribute molecules. Sample deposition is the most critical step that determines data quality and reproducibility.The premixed sample solution needs to be deposit immediately on the sample plate to ensure the sample is crystallized under controlled condition. After drying, open the door. of the drying chamber.Then, set the water circulator temperature to 25 degrees Celsius. Once the sample plate temperature returns to 25 degrees Celsius, remove the sample plate from the drying chamber. Examine the sample morphology under a five times magnified Stereo microscope, and take a snapshot brightfield image.At this point, insert the sample plate into a MALDI Mass Spectrometer. Then, perform imaging mass spectrometry analysis. Select a characteristic mass peak from the mass list shown in the result window and click 2D to plot a two-dimensional ion image.Next, click the adjustment buttons in the pop-up window to determine the upper and lower limits of the signal intensity, to increase the image resolution, and click save a picture. Observe and compare the ion image with the previously acquired brightfield image to obtain the essential sample region. Now, click the null spots and the cracked regions in the ion image shown in the result window to fine-adjust the region to be analyzed.Click the find edge button to find the outer-most layer of the ion image. Following this, click deduct to save the ion abundance information of the outmost layer in a database, and remove this layer from the ion image simultaneously. After repeating the previous steps until the center of the ion image is defined, click and select all the checkboxes in the output data list and click export to export the data.Finally, open the exported data using spreadsheet software to calculate the average ion abundance of every layer, to obtain the spatial distribution information of ions. In the brightfield and MALDI images of maltotriose, the ion signal of the sodiated form mainly populates at the periphery of the sample area, when it is prepared with a sample plate temperature of 25 degrees Celsius. By decreasing the temperature to five degrees Celsius, the signal populates homogeneously over the entire sample area, suggesting that preparing samples under a lower sample plate temperature can significantly redistribute the molecules and reduce heterogeneity.The protonated bradykinin fragment ion image shows a similar trend as the sodiated maltotriose. Statistical analyses show that the heterogeneity of ion signals obtained under a sample plate temperature of 5 degrees Celsius reduces by 60 to 80 percent, with respect to that under 25 degrees Celsius. In the case of sodiated maltotriose, with a sample plate temperature of 25 degrees Celsius, the signal intensities at the centers are much lower than those with the temperature of 5 degrees Celsius.The protonated bradykinin fragment shows less variation when decreasing the sample plate temperature from 25 to 5 degrees Celsius. While attempting this drying method, the temperatures and relative humidity in the drying chamber need to be carefully controlled and monitored. Once mastered, a sample prepared by this technique can be done in 30 minutes if it is performed properly.After it's development, this technique paved the way for researchers in the field of MALDI MS to explore quantitative analysis for dried-droplet samples. After watching this video, you should have a good understanding of how to prepare homogeneous dried-droplet samples for MALDI MS analysis.

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