Name: an efficient sample preparation method to enhance carbohydrate ion signals in matrix-assisted laser desorption/ionization mass spectrometry
Uploaded: 2018-07-29T13:00:00
Description: Watch this Scientific Journal Video about An Efficient Sample Preparation Method to Enhance Carbohydrate Ion Signals in Matrix-assisted Laser Desorption/Ionization Mass Spectrometry at JoVE.com

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 wate...

<ol>
	<li>Holme, D. J., Peck, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Analytical Biochemistry. , (1998).</li><li>Costello, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time,+life+...+and+mass+spectrometry+-+New+techniques+to+address+biological+questions.">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="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+bacterial+lipopolysaccharides.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix-assisted+laser+desorption/ionization+time-of-flight+mass+spectrometry+in+clinical+chemistry.">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="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix-assisted+laser+desorption/ionization+mass+spectrometry+of+carbohydrates.">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="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+rapid+method+for+the+permethylation+of+carbohydrates.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivatization+of+carbohydrates+for+chromatographic,+electrophoretic+and+mass+spectrometric+structure+analysis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reverse+thin+layer+method+for+enhanced+ion+yield+of+oligosaccharides+in+matrix-assisted+laser+desorption/ionization.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+matrix+crystal+structure+on+ion+abundance+of+carbohydrates+by+matrix-assisted+laser+desorption/ionization+Fourier+transform+ion+cyclotron+resonance+mass+spectrometry.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+the+fast-evaporation+sample+preparation+method+for+improving+quantification+of+angiotensin+II+by+matrix-assisted+laser+desorption/ionization.">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/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> 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+Homogeneous+MALDI+Samples+for+Quantitative+Applications.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancing+carbohydrate+ion+yield+by+controlling+crystalline+structures+in+matrix-assisted+laser+desorption/ionization+mass+spectrometry.">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="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+'near'+homogeneous+samples+for+the+analysis+of+matrix-assisted+laser+desorption/ionisation+processes.">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="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystallite+size+dependence+of+volatilization+in+matrix-assisted+laser+desorption+ionization.">Crystallite size dependence of volatilization in matrix-assisted laser desorption ionization.</a> Applied Surface Science. 127 (Supplement C), 226-234 (1998).</li></ol>

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.

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			<td height="23" style="height:23px;width:332px;">Reagent</td>
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			<td height="23" style="height:23px;width:332px;">Detergent powder</td>
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			<td height="23" style="height:23px;">2,5-dihydroxybenzoic acid (DHB)</td>
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			<td style="width:137px;">A11459</td>
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			<td height="23" style="height:23px;">sialyl-lewis A (SLeA)</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td style="width:137px;">S1782</td>
			<td></td>
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		<tr height="23">
			<td height="23" style="height:23px;">Maltoheptaose</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td style="width:137px;">M7753</td>
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			<td height="23" style="height:23px;width:332px;">Nitrogen gas flowmeter</td>
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			<td height="23" style="height:23px;width:332px;">Stereomicroscope</td>
			<td style="width:157px;">Olympus</td>
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			<td></td>
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			<td height="23" style="height:23px;width:332px;">Temperature controllable drying chamber</td>
			<td style="width:157px;">This lab</td>
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			<td height="23" style="height:23px;width:332px;">Ultraflex II TOF/TOF mass spectrometer</td>
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			<td height="23" style="height:23px;width:332px;">Flexcontrol Version 3.4</td>
			<td style="width:157px;">Bruker Daltonics</td>
			<td style="width:137px;"></td>
			<td>Control software</td>
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			<td height="23" style="height:23px;width:332px;">Fleximaging Version 2.1</td>
			<td style="width:157px;">Bruker Daltonics</td>
			<td style="width:137px;"></td>
			<td>Imaging software</td>
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			<td height="23" style="height:23px;width:332px;">Flexanalysis Version 3.4</td>
			<td style="width:157px;">Bruker Daltonics</td>
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			<td>Analysis software</td>
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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 &#181;m. After recrystallization by MeOH, the sample has a larger area covered evenly with fine flake-like cryst...

Watch this Scientific Journal Video about An Efficient Sample Preparation Method to Enhance Carbohydrate Ion Signals in Matrix-assisted Laser Desorption/Ionization Mass Spectrometry at JoVE.com

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

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) ...

A simple sample preparation procedure was developed to improve the conventional method in matrix-assisted laser desorption ionization mass spectrometry by reforming the simple morphology during the drying process. The advantage of this technique is that the signal intensity of carbohydrate samples prepared by this optimized method can be effectively enhanced. Generally, individuals new to this method will struggle because the methanol depressurization step is very crucial and it needs to be done in one go.Any hesitation will decrease the data quality. To begin, while wearing nitrile gloves, use 100 milliliters of detergent solution to hand wash the sample plate. Use distilled-deionized, or DD, water to hand wash the plate.Then, with 30 milliliters of methanol, rinse its surface. Insert the sample plate into a 600 milliliter beaker and fill it with DD water until the plate is fully immersed. Then place the beaker into an ultrasonic bath and sonicate the plate for 15 minutes.Remove the sample plate from the beaker and use pressurized nitrogen to blow off the water drops. Then deposit 0.2 microliters of methanol on the sample plate to check whether it spreads to other spots. If it does, repeat the wash, sonication and nitrogen treatment as just demonstrated.After preparing the drying chamber according to the text protocol, premix 0.25 microliters of 2, 5-Dihydroxybenzoic acid solution and 0.25 microliters of Sialyl-Lewis A, or maltoheptaose solution in the micro centrifuge tube. Then vortex the tube for three seconds. Spin down the mixed solution in the mini centrifuge at 2000 times G for two seconds.Then immediately pipette 0.1 microliter of the pre-mixed solution on to the sample plate. When the sample has dried, while working quickly to avoid evaporation, pipette 0.2 microliters of methanol directly on to the sample spot. The sample will redissolve and then immediately dry out.The methanol deposition step is the most difficult step that determines data quality and reproducibility. To ensure the best performance, we recommend that the user finish the deposition in three to five seconds to avoid significant evaporation loss of methanol. Wear nitrile gloves and carefully remove the sample plate from the drying chamber.Then, under a microscope, examine the sample. If the crystal morphologies are not as expected, repeat mixing the sample and spotting onto the sample plate. To ensure the sample crystals have the optimal morphology after recrystallization, always use a microscope to exam them.It is difficult to determine the quality of crystal morphology correctly with the naked eye. To analyze a one microliter sample, premix 2.5 microliters of 2, 5-Dihydroxybenzoic acid solution and 2.5 microliters of Sialyl-Lewis A or maltoheptaose solution in a micro centrifuge tube. Vortex the pre-mixed solution for five seconds.Spin down the mixed solution at 2000 times G for two seconds, then immediately pipette one microliter of the pre-mixed solution on to the sample plate. After the sample dries out, pipette 1.5 microliters of methanol directly on to the dried sample spot. The sample will redissolve and immediately dry out.Carefully remove the sample from the drying chamber. Examine the sample under a microscope. If the crystal morphologies are not as expected, repeat the solution preparation as just demonstrated.To carry out mass spectrometry, open the mass spectrometer control software and insert the sample plate into the machine. Select the pre-optimized data acquisition method in the software, then using the imaging software, register the whole sample region for imaging mass spectrometry. Start data acquisition in batch mode of the control software.When data acquisition is complete, use the imaging software to plot the ion images and analyze the data according to the text protocol. Representative SEM images of Sialyl-Lewis A mixed with 2, 5-Dihydroxybenzoic acid prepared using dried droplet and recrystallization methods are shown here. A typical 2, 5-Dihydroxybenzoic acid morphology is large, needle-shaped crystals at the rim and fine crystalline structures in the center of sample spots.After recrystallization by methanol, the sample has a larger area covered evenly with fine, flake-like crystals. This figure depicts imaging mass spectrometry results of Sialyl-Lewis A and maltoheptaose with and without methanol recrystallization. After recrystallization, the distribution of Sialyl-Lewis A and maltohepaose signals match well with the bright-field images of sample spots.The signal intensities of the recrystallized carbohydrate samples are also enhanced compared to conventional dry droplet samples. This graph compares the signal intensity of sodiated or positive ion mode and deprotonated or negative ion mode carbohydrates of recrystallized samples with respect to that of dried droplet samples. On average, recrystallization of Sialyl-Lewis A and maltoheptaose samples increased sodiated signals by factors of 3.9 and 3.3.Deprotonated Sialyl-Lewis A ion signals were enhanced by a factor of 4.7 after recrystallization. Once mastered, samples can be prepared in 10 minutes using this technique if it is performed properly. While attempting this procedure, it's important that the methanol droplet be immediately and precisely deposited on the sample spot.After watching this video, you should have a good understanding of how to effectively control crystal morphology of multi samples to obtain the best ion signals for routine analysis.

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