Name: An Efficient Sample Preparation Method to Enhance Carbohydrate Ion Signals in Matrix-assisted Laser Desorption/Ionization Mass Spectrometry (Video) | JoVE Journal
Uploaded: 2018-07-29T13:00:00.000+00:00
Duration: 7 min 12 s
Description: Learn an optimized sample preparation method for MALDI mass spectrometry that enhances carbohydrate ion signals through controlled crystalline structure reforming. This protocol improves data quality for biomolecular analysis.

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The authors have no acknowledgements.

1. Sample Plate Preconditioning
<ol>
	<li>Cleaning of the sample plate
	<ol>
		<li>Wear nitrile gloves to avoid contamination of the sample plate during cleaning.</li>
		<li>Hand-wash the sample plate with 100.0 mL of detergent solution (1.0 mg/mL).</li>
		<li>Hand-wash the sample plate with distilled-deionized water (DDW).</li>
		<li>Rinse the sample plate surface with 30.0 mL of MeOH.</li>
		<li>Put the sample plate in a 600 mL beaker and fill with DDW until the sample plate is fully immersed in water.</li>
		<li>Put the beaker in an ultrasonic bath (see Table of Materials) and sonicate the sample plate for 15 min (200 W, 40 kHz).</li>
		<li>Take out the sample plate and blow off water drops using pressurized nitrogen.</li>
		<li>Deposit 0.2 &#181;L of MeOH on the sample plate to check whether MeOH spreads to other spots. 
		Note: If MeOH merges with other spots, repeat steps 1.1.3-1.1.5; if not, proceed to the next step.</li>
	</ol>
	</li>
	<li>Regulation of drying chamber temperature
	<ol>
		<li>Use a drying chamber under steady conditions to dry droplets, as described previously11,12,13. In particular, use the detailed procedure described in steps 2.1-2.5 of Ou, Y.-M. et al. 201612. Briefly:
		<ol>
			<li>Purge the drying chamber by room temperature nitrogen at a constant flow rate to maintain a low relative humidity environment.</li>
			<li>Keep the sample plate temperature constant at regular- (25 &#176;C) or fast-drying conditions (50 &#176;C), regulated by a temperature-controlled copper block in the drying chamber.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Preparation of matrix and analyte solutions
	<ol>
		<li>Preparation of matrix solutions
		<ol>
			<li>Dissolve DHB in 50% acetonitrile (ACN):50% DDW to prepare a 0.1 M solution.</li>
		</ol>
		</li>
		<li>Preparation of carbohydrate analytes
		<ol>
			<li>Dissolve SLeA in DDW to prepare a 10-4 M solution.</li>
			<li>Dissolve MH in DDW to prepare a 10-4 M solution.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Sample Deposition and Recrystallization
Note: The optimized procedures for analyzing small and regular amounts of samples are described here. Ensure that the sample plate temperature is stabilized at the desired temperature before depositing the solutions. If the sample spreads over a large area to cover other sample spots during recrystallization, prepare a new sample or repeat step 1.1.
<ol>
	<li>For analysis of a small amount (0.1 &#181;L) of sample 
	Note: The following steps have been developed for minimizing sample and time consumption. It is suitable for quantitative analysis of real samples with a limited amount or quick IMS for quantification analysis.
	<ol>
		<li>Premix 0.25 &#181;L of DHB solution and 0.25 &#181;L of SLeA or MH solution in a microcentrifuge tube.</li>
		<li>Vortex the mixed solution using a vortex mixer for 3 s.</li>
		<li>Spin down the mixed solution in a mini centrifuge for 2 s (2000 x g).</li>
		<li>Use a pipette to draw out 0.1 &#181;L of the premixed solution and immediately deposit it on the sample plate. 
		Note: When depositing a small amount of sample, DO NOT keep the premixed solution in the tip of pipette for over 10 s.</li>
		<li>Wait for the sample to dry out. Typical drying times are listed in Table 1.</li>
		<li>Use a pipette to deposit 0.2 &#181;L of MeOH right onto the dried sample spot. The sample will get wet and dry out immediately. 
		Note: Ensure that the deposition procedure is finished in 3 s to avoid significant evaporation loss of MeOH.</li>
		<li>Examine the sample using a microscope. If the crystal morphologies are not as expected (see Figure 1 for example of desired results), repeat steps 2.1.1-2.1.6 to prepare a new sample.</li>
		<li>Wear nitrile gloves and carefully take out the sample plate from the drying chamber.</li>
	</ol>
	</li>
	<li>For analysis of a regular amount (1 &#181;L) of sample 
	Note: The following steps are developed for maximizing the homogeneity of carbohydrate samples with typically utilized MALDI sample amount. The process is suitable for routine and quantitative analyses. The recrystallization process redistributes samples and matrixes evenly to larger areas.
	<ol>
		<li>Premix 2.5 &#181;L of DHB solution and 2.5 &#181;L SLeA or MH solution in a microcentrifuge tube.</li>
		<li>Vortex the premixed solution with a vortex mixer for 5 s.</li>
		<li>Spin down the mixed solution in a mini centrifuge for 2 s (2000 x g).</li>
		<li>Use a pipette to draw out 1.0 &#181;L of the premixed solution and immediately deposit it on the sample plate. 
		Note: DO NOT use the remaining premixed solution again after depositing the samples.</li>
		<li>Wait for the sample to dry out. Typical drying times are listed in Table 1.</li>
		<li>Use a pipette to deposit 1.5 &#181;L of MeOH right onto the dried sample spot. The sample will get wet and dry out immediately. 
		Note: In cases with high sample plate temperature (50 &#176;C), the recrystallization step should be done within 5 s to minimize evaporation of MeOH in pipette tip.</li>
		<li>Examine the sample using a microscope. If the crystal morphologies are not as expected (see Figure 1 for example of desired results), repeat steps 2.2.1-2.2.6 to prepare a new sample.</li>
		<li>Wear nitrile gloves and carefully take out the sample plate from the drying chamber.</li>
	</ol>
	</li>
</ol>
3. Mass Spectrometry Data Acquisition and Analysis
Note: The analysis is performed using a commercial time-of-flight mass spectrometer (Table of Materials) equipped with a MALDI ion source. The instrument is operated by specific control software (Table of Materials) with pre-optimized extraction delay and laser energy. Spectra are recorded in linear mode with a mass range of m/z = 0 &#8211; 1500. The sample plate potential is &#177;25 keV and every spectrum averages 10 laser shots. Users should conduct instrument optimization and sample analysis using compatible software and follow instrument manufacturer&#39;s instructions.
<ol>
	<li>Open the instrument control software (see Table of Materials).</li>
	<li>Insert the sample plate into the mass spectrometer.</li>
	<li>Select the pre-optimized data acquisition method in the software.</li>
	<li>Register the whole sample region for IMS using the imaging software (see Table of Materials). 
	Note: Skip this step if not doing IMS.</li>
	<li>Start data acquisition in the batch mode of the control software.</li>
	<li>Plot the ion images using the imaging software after data acquisition is complete.</li>
	<li>Analyze mass spectra using analysis software (see Table of Materials) if the data is recorded without an ion image.</li>
</ol>

<ol><li>Holme, D. J., Peck, H. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aHolme%2C+DJ+%22Analytical+Biochemistry%22">Analytical Biochemistry</a>. , 3rd edn, Addison Wesley Longman Limited. (1998).</li>
<li>Costello, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Time%2C+life+...+and+mass+spectrometry+-+New+techniques+to+address+biological+questions%5BTitle%5D)+AND+%22Biophysical+Chemistry%22%5BJournal%5D)">Time, life ... and mass spectrometry - New techniques to address biological questions.</a> Biophysical Chemistry. 68 (1-3), 173-188 (1997).</li>
<li>Caroff, M., Karibian, D. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/14670707">Structure of bacterial lipopolysaccharides.</a> Carbohydrate Research. 338 (23), 2431-2447 (2003).</li>
<li>Marvin, L. F., Roberts, M. A., Fay, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Matrix-assisted+laser+desorption%2Fionization+time-of-flight+mass+spectrometry+in+clinical+chemistry%5BTitle%5D)+AND+%22Clinica+Chimica+Acta%22%5BJournal%5D)">Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in clinical chemistry.</a> Clinica Chimica Acta. 337 (1), 11-21 (2003).</li>
<li>Harvey, D. J. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/10639030">Matrix-assisted laser desorption/ionization mass spectrometry of carbohydrates.</a> Mass Spectrometry Reviews. 18 (6), 349-450 (1999).</li>
<li>Ciucanu, I., Kerek, F. <a target="_blank" href="https://www.sciencedirect.com/science/article/abs/pii/0008621584852428">A simple and rapid method for the permethylation of carbohydrates.</a> Carbohydrate Research. 131 (2), 209-217 (1984).</li>
<li>Lamari, F. N., Kuhn, R., Karamanos, N. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Derivatization+of+carbohydrates+for+chromatographic%2C+electrophoretic+and+mass+spectrometric+structure+analysis%5BTitle%5D)+AND+%22Journal+of+Chromatography+B%22%5BJournal%5D)">Derivatization of carbohydrates for chromatographic, electrophoretic and mass spectrometric structure analysis.</a> Journal of Chromatography B. 793 (1), 15-36 (2003).</li>
<li>Nishikaze, T., Amano, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reverse+thin+layer+method+for+enhanced+ion+yield+of+oligosaccharides+in+matrix-assisted+laser+desorption%2Fionization%5BTitle%5D)+AND+%22Rapid+Communications+in+Mass+Spectrometry%22%5BJournal%5D)">Reverse thin layer method for enhanced ion yield of oligosaccharides in matrix-assisted laser desorption/ionization.</a> Rapid Communications in Mass Spectrometry. 23 (23), 3787-3794 (2009).</li>
<li>Williams, T. I., Saggese, D. A., Wilcox, R. J., Martin, J. D., Muddiman, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+matrix+crystal+structure+on+ion+abundance+of+carbohydrates+by+matrix-assisted+laser+desorption%2Fionization+Fourier+transform+ion+cyclotron+resonance+mass+spectrometry%5BTitle%5D)+AND+%22Rapid+Communications+in+Mass+Spectrometry%22%5BJournal%5D)">Effect of matrix crystal structure on ion abundance of carbohydrates by matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry.</a> Rapid Communications in Mass Spectrometry. 21 (5), 807-811 (2007).</li>
<li>Nicola, A. J., Gusev, A. I., Proctor, A., Jackson, E. K., Hercules, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Application+of+the+fast-evaporation+sample+preparation+method+for+improving+quantification+of+angiotensin+II+by+matrix-assisted+laser+desorption%2Fionization%5BTitle%5D)+AND+%22Rapid+Communications+in+Mass+Spectrometry%22%5BJournal%5D)">Application of the fast-evaporation sample preparation method for improving quantification of angiotensin II by matrix-assisted laser desorption/ionization.</a> Rapid Communications in Mass Spectrometry. 9 (12), 1164-1171 (1995).</li>
<li>Lai, Y. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reducing+Spatial+Heterogeneity+of+MALDI+Samples+with+Marangoni+Flows+During+Sample+Preparation%5BTitle%5D)+AND+%22Journal+of+the+American+Society+for+Mass+Spectrometry%22%5BJournal%5D)">Reducing Spatial Heterogeneity of MALDI Samples with Marangoni Flows During Sample Preparation.</a> Journal of the American Society for Mass Spectrometry. 27 (8), 1314-1321 (2016).</li>
<li>Ou, Y. -M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Preparation+of+Homogeneous+MALDI+Samples+for+Quantitative+Applications%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Preparation of Homogeneous MALDI Samples for Quantitative Applications.</a> Journal of Visualized Experiments. (116), e54409(2016).</li>
<li>Lee, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enhancing+carbohydrate+ion+yield+by+controlling+crystalline+structures+in+matrix-assisted+laser+desorption%2Fionization+mass+spectrometry%5BTitle%5D)+AND+%22Analytica+Chimica+Acta%22%5BJournal%5D)">Enhancing carbohydrate ion yield by controlling crystalline structures in matrix-assisted laser desorption/ionization mass spectrometry.</a> Analytica Chimica Acta. , 49-55 (2017).</li>
<li>Allwood, D. A., Perera, I. K., Perkins, J., Dyer, P. E., Oldershaw, G. A. <a target="_blank" href="https://www.sciencedirect.com/science/article/pii/0169433296005302">Preparation of 'near' homogeneous samples for the analysis of matrix-assisted laser desorption/ionisation processes.</a> Applied Surface Science. 103 (3), 231-244 (1996).</li>
<li>Sadeghi, M., Vertes, A. <a target="_blank" href="https://www.sciencedirect.com/science/article/pii/S0169433297006363">Crystallite size dependence of volatilization in matrix-assisted laser desorption ionization.</a> Applied Surface Science. 127 (Supplement C), 226-234 (1998).</li>
</ol>

The authors have nothing to disclose.

Sample heterogeneity is a crucial problem in MALDI-MS. DD is the most commonly used sample preparation method, but the resultant crystals are highly heterogeneous. Such samples show poor shot-to-shot and sample-to-sample signal reproducibility. Therefore, searching for &#34;sweet spots&#34; in sample areas during data acquisition is a common procedure in MALDI experiments. Such heterogeneous samples are unsuitable for quantification in routine analyses.
In the current study, MALDI sample morph...

Carbohydrate analysis is an important and challenging subject. Carbohydrates and their derivatives play important roles in living organisms1,2,3. These molecules have complicated structures and are prone to decompose. Many of them cannot be clearly characterized due to difficulties in separation and detection. Although matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) has been applied to analysis of a wide range of biomolecules, due to its sensitivity and comprehensible results4, analyzing carbohydrates using MALDI-MS continues to be a major challenge due to the low ionization efficiency of such molecules5. Chemical derivatization is a common way to improve ionization efficiency of carbohydrates6,7, but such procedures are time and sample consuming. Besides, the ionization efficiency of derivatized carbohydrates is still lower than that of proteins. Thus, the development of methods to improve carbohydrate signal in MALDI-MS without complicated procedures is necessary.
The application of MALDI-MS to quantitative analysis is another challenging subject. A major problem of MALDI-MS is that its sensitivity and data reproducibility relies critically on sample preparation protocols and experimental parameters. In many cases, quantitative analysis by MALDI-MS is unreliable due to heterogeneous sample morphologies and analyte distribution. A well-known example is samples prepared with a 2,5-dihydroxybenzoic acid (DHB) MALDI matrix. When DHB is crystallized slowly under ambient environment, the extent of analyte incorporation into matrix crystals is unpredictable, because resultant samples show irregular morphologies. Such samples normally consist of large needle-shaped and fine crystals. When DHB is prepared using a volatile solvent and/or a heated sample plate, a fast drying process results in more homogeneous fine crystals and better quantitative results8,9,10. This technique is known as &#34;recrystallization&#34; of MALDI samples. The improvement is attributed to better incorporation of analytes into fine matrix crystals during the fast crystallization process. We have also demonstrated that adjusting the sample preparation environment reduced the heterogeneity of carbohydrate signal and improved quantitative results11,12. The findings in these works suggest that sample morphology is a critical factor in determining carbohydrate signal quality. To develop a general strategy for daily analysis, an efficient sample reformation method providing improved carbohydrate sensitivity is required.
We have systematically examined the correlation between sample morphology and carbohydrate sensitivity in MALDI-MS in a recent report13. The results obtained using several important carbohydrates and matrixes show that the best signal enhancement is fulfilled by recrystallizing dried MALDI samples. The morphology of samples prepared using the conventional dried droplet (DD) method is reformed by fast recrystallization with methanol (MeOH). The detailed sample preparation protocols are demonstrated here. The protocol consists of three main steps, including sample plate preconditioning, sample deposition and recrystallization, and mass spectrometry analysis. The utilized carbohydrates include sialyl-lewis A (SLeA) and maltoheptaose (MH). DHB is used as a model matrix. The results show that carbohydrate signal intensity and spatial distribution improved markedly after recrystallization. Such a method can be applied to samples with other popular matrixes, including 2,4,6-trihydroxyacetophenone (THAP) and &#945;-cyano-4-hydroxycinnamic acid. This method serves as a general approach that can be easily integrated in the laboratory routine for carbohydrate analysis.

<table><tbody><tr><td>&lt;strong&gt;Reagent&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Detergent powder</td><td>Alconox</td><td>242985</td><td></td></tr><tr><td>Methanol</td><td>Merck</td><td>106009</td><td></td></tr><tr><td>Acetonitrile</td><td>Merck</td><td>100003</td><td></td></tr><tr><td>2,5-dihydroxybenzoic acid (DHB)</td><td>Alfa Aesar</td><td>A11459</td><td></td></tr><tr><td>sialyl-lewis A (SLe&lt;sup&gt;A&lt;/sup&gt;)</td><td>Sigma-Aldrich</td><td>S1782</td><td></td></tr><tr><td>Maltoheptaose</td><td>Sigma-Aldrich</td><td>M7753</td><td></td></tr><tr><td>Pipette tips</td><td>Mettler Toledo</td><td>17005091</td><td></td></tr><tr><td>Microcentrifuge tube</td><td>Axygen</td><td>MCT-150-C</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Milli-Q water purification system</td><td>Millipore</td><td>ZMQS6VFT1</td><td></td></tr><tr><td>Powder-free nitrile gloves</td><td>Microflex</td><td>SU-690</td><td></td></tr><tr><td>600 mL beaker</td><td>Duran</td><td>2110648</td><td></td></tr><tr><td>Ultrasonic cleaner</td><td>Delta</td><td>DC300H</td><td></td></tr><tr><td>Hygrometer</td><td>Wisewind</td><td>5330</td><td></td></tr><tr><td>Nitrogen gas flowmeter</td><td>Dwyer</td><td>RMA-6-SSV</td><td></td></tr><tr><td>K-type thermocouples</td><td>Digitron</td><td>311-1670</td><td></td></tr><tr><td>Vortex mixer</td><td>Scientific Industries</td><td>&amp;nbsp;SI-0236</td><td></td></tr><tr><td>Mini centrifuge</td><td>Select BioProducts</td><td>Force Mini&amp;nbsp;</td><td></td></tr><tr><td>Pipette</td><td>Rainin</td><td>pipet-lite XLS</td><td></td></tr><tr><td>Stereomicroscope</td><td>Olympus</td><td>SZX16</td><td></td></tr><tr><td>Temperature controllable drying chamber</td><td>This lab</td><td></td><td></td></tr><tr><td>Ultraflex II TOF/TOF mass spectrometer</td><td>Bruker Daltonics</td><td></td><td></td></tr><tr><td>MTP 384 target plate polished steel BC</td><td>Bruker Daltonics</td><td>8280781</td><td></td></tr><tr><td>Flexcontrol Version 3.4</td><td>Bruker Daltonics</td><td></td><td>Control software</td></tr><tr><td>Fleximaging Version 2.1</td><td>Bruker Daltonics</td><td></td><td>Imaging software</td></tr><tr><td>Flexanalysis Version 3.4</td><td>Bruker Daltonics</td><td></td><td>Analysis software</td></tr></tbody></table>

an efficient sample preparation method to enhance carbohydrate ion signals in matrix-assisted laser desorption/ionization mass spectrometry

An Efficient Sample Preparation Method to Enhance Carbohydrate Ion Signals in Matrix-assisted Laser Desorption/Ionization Mass Spectrometry

Sample preparation is a critical process in mass spectrometry (MS) analysis of carbohydrates. Although matrix-assisted laser desorption/ionization (MALDI) MS is the method of choice in carbohydrate analysis, poor ion signal and data reproducibility of carbohydrate samples continue to be severe problems. For quantitative analysis of carbohydrates, an effective analytical protocol providing superior data quality is necessary. This video demonstrates sample preparation protocols to improve signal intensity and minimize data variation of carbohydrates in MALDI-MS. After drying and crystallization of sample droplets, the crystal morphology is reformed by methanol before mass spectrometric analysis. The enhancement in carbohydrate signal is examined with MALDI imaging mass spectrometry (IMS). Experimental results show that crystal reformation adjusts crystalline structures and redistributes carbohydrate analytes. In comparison with the dried droplet preparation method in conventional MALDI-MS, reforming carbohydrate crystal morphologies with methanol shows significantly better signal intensity, ion image distribution, and data stability. Since the protocols demonstrated herein do not involve changes in sample composition, they are generally applicable to various carbohydrates and matrixes.

Representative SEM images of SLeA mixed with DHB prepared using DD and recrystallization methods are shown in Figure 1. A typical DHB morphology as prepared by the DD method is large needle-shaped crystals at the rim and fine crystalline structures in the center of sample spots. The typical lengths of such needle-shaped crystals are ~100 µm. After recrystallization by MeOH, the sample has a larger area covered evenly with fine flake-like crystals. ...

A protocol for enhancing carbohydrate ion signals in MALDI mass spectrometry by reforming crystalline structures during sample preparation processes is demonstrated.

Sample preparation is a critical process in mass spectrometry (MS) analysis of carbohydrates. Although matrix-assisted laser desorption/ionization (MALDI) ...

an-efficient-sample-preparation-method-to-enhance-carbohydrate-ion

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Learn an optimized sample preparation method for MALDI mass spectrometry that enhances carbohydrate ion signals through controlled crystalline structure reforming. This protocol improves data quality for biomolecular analysis.

An Efficient Sample Preparation Method to Enhance Carbohydrate Ion Signals in Matrix-assisted Laser Desorption/Ionization Mass Spectrometry (Video) | JoVE Journal

An Efficient Sample Preparation Method to Enhance Carbohydrate Ion Signals in Matrix-assisted Laser Desorption/Ionization Mass Spectrometry

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