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://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>

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

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) ...

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

Research

JoVE Journal

Chemistry

7.0K Views.  Academia Sinica. A protocol for enhancing carbohydrate ion signals in MALDI mass spectrometry by reforming crystalline structures during sample preparation processes is demonstrated.

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

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.

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 ...

Biochemistry

Matrix-assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) Mass Spectrometric Analysis of Intact Proteins Larger than 100 kDa

Effectively determining masses of proteins is critical to many biological studies (e.g. for structural biology investigations). Accurate mass determination allows one to evaluate the correctness of protein primary sequences, the presence of mutations and/or post-translational modifications, the possible protein degradation, the sample homogeneity, and the degree of isotope incorporation in case of labelling (e.g. 13C labelling).
Electrospray ionization (ESI) mass spectrometry (MS) is widely used for mass determination of denatured proteins, but its efficiency is affected by the composition of the sample buffer. In particular, the presence of salts, detergents, and contaminants severely undermines the effectiveness of protein analysis by ESI-MS. Matrix-assisted laser desorption/ionization (MALDI) MS is an attractive alternative, due to its salt tolerance and the simplicity of data acquisition and interpretation. Moreover, the mass determination of large heterogeneous proteins (bigger than 100 kDa) is easier by MALDI-MS due to the absence of overlapping high charge state distributions which are present in ESI spectra.
Here we present an accessible approach for analyzing proteins larger than 100 kDa by MALDI-time of flight (TOF). We illustrate the advantages of using a mixture of two matrices (i.e. 2,5-dihydroxybenzoic acid and &#945;-cyano-4-hydroxycinnamic acid) and the utility of the thin layer method as approach for sample deposition. We also discuss the critical role of the matrix and solvent purity, of the standards used for calibration, of the laser energy, and of the acquisition time. Overall, we provide information necessary to a novice for analyzing intact proteins larger than 100 kDa by MALDI-MS.

Matrix-assisted Laser Desorption/Ionization Time of Flight MALDI-TOF Mass Spectrometric Analysis of Intact Proteins Larger than 100 kDa

Accurate mass measurement represents an important step during the investigation of proteins. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) can be used for such determination and its key advantage is the tolerance to salts, detergents and contaminants. Here we illustrate an accessible approach for the analysis of proteins larger than 100 kDa by MALDI-MS.

Effectively determining masses of proteins is critical to many biological studies (e.g. for structural biology investigations). Accurate mass ...

Sheathless Capillary Electrophoresis&#8211;Mass Spectrometry for Metabolic Profiling of Biological Samples

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a protocol is presented for the analysis of anionic and cationic metabolites in biological samples by capillary electrophoresis&#8211;mass spectrometry (CE-MS). CE is well-suited for the analysis of highly polar and charged metabolites as compounds are separated on the basis of their charge-to-size ratio. A recently developed sheathless interfacing design, i.e., a porous tip interface, is used for coupling CE to electrospray ionization (ESI) MS. This interfacing approach allows the effective use of the intrinsically low-flow property of CE in combination with MS, resulting in nanomolar detection limits for a broad range of polar metabolite classes. The protocol presented here is based on employing a bare fused-silica capillary with a porous tip emitter at low-pH separation conditions for the analysis of a broad array of metabolite classes in biological samples. It is demonstrated that the same sheathless CE-MS method can be used for the profiling of cationic metabolites, including amino acids, nucleosides and small peptides, or anionic metabolites, including sugar phosphates, nucleotides and organic acids, by only switching the MS detection and separation voltage polarity. Highly information-rich metabolic profiles in various biological samples, such as urine, cerebrospinal fluid and extracts of the glioblastoma cell line, can be obtained by this protocol in less than 1 hr of CE-MS analysis.

A protocol for metabolic profiling of biological samples by capillary electrophoresis&#8211;mass spectrometry using a sheathless porous tip interface design is presented.

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a ...

MALDI Sample Preparation: the Ultra Thin Layer Method

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

Biology

Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the monitoring of therapeutic efficiency.1-3 The circulating low molecular weight proteome (LMWP) composed of small proteins shed from tissues and cells or peptide fragments derived from the proteolytic degradation of larger proteins, has been associated with the pathological condition in patients and likely reflects the state of disease.4,5 Despite these potential clinical applications, the use of Mass Spectrometry (MS) to profile the LMWP from biological fluids has proven to be very challenging due to the large dynamic range of protein and peptide concentrations in serum.6 Without sample pre-treatment, some of the more highly abundant proteins obscure the detection of low-abundance species in serum/plasma. Current proteomic-based approaches, such as two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE) and shotgun proteomics methods are labor-intensive, low throughput and offer limited suitability for clinical applications.7-9 Therefore, a more effective strategy is needed to isolate LMWP from blood and allow the high throughput screening of clinical samples. 
Here, we present a fast, efficient and reliable multi-fractionation system based on mesoporous silica chips to specifically target and enrich LMWP.10,11 Mesoporous silica (MPS) thin films with tunable features at the nanoscale were fabricated using the triblock copolymer template pathway. Using different polymer templates and polymer concentrations in the precursor solution, various pore size distributions, pore structures, connectivity and surface properties were determined and applied for selective recovery of low mass proteins. The selective parsing of the enriched peptides into different subclasses according to their physicochemical properties will enhance the efficiency of recovery and detection of low abundance species. In combination with mass spectrometry and statistic analysis, we demonstrated the correlation between the nanophase characteristics of the mesoporous silica thin films and the specificity and efficacy of low mass proteome harvesting. The results presented herein reveal the potential of the nanotechnology-based technology to provide a powerful alternative to conventional methods for LMWP harvesting from complex biological fluids. Because of the ability to tune the material properties, the capability for low-cost production, the simplicity and rapidity of sample collection, and the greatly reduced sample requirements for analysis, this novel nanotechnology will substantially impact the field of proteomic biomarker research and clinical proteomic assessment.

We developed a technology based on mesoporous silica thin film for the selective recovery of low molecular weight proteins and peptides from human serum. The physico-chemical properties of our mesoporous chips were finely tuned to provide substantial control in peptide enrichment and consequently profile the serum proteome for diagnostic purposes.

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the ...

Bioengineering

Optimal Preparation of Formalin Fixed Samples for Peptide Based Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Workflows

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host of biomolecules from drugs and lipids to N-glycans. Although various sample preparation techniques exist, detecting peptides from formaldehyde preserved tissues remains one of the most difficult challenges for this type of mass spectrometric analysis. For this reason, we have created and optimized a robust methodology that preserves the spatial information contained within the sample, while eliciting the greatest number of ionizable peptides. We have also aimed to achieve this in a cost effective and simple way, thereby eliminating potential bias or preparation error, which can occur when using automated instrumentation. The end result is a reproducible and inexpensive protocol.

This protocol describes a reproducible and reliable method for the sublimation-based preparation of formalin fixed tissue destined for imaging mass spectrometry.

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host ...

Characterization of Synthetic Polymers via Matrix Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) Mass Spectrometry

There are many techniques that can be employed in the characterization of synthetic homopolymers, but few provide as useful of information for end group analysis as matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). This tutorial demonstrates methods for optimization of the sample preparation, spectral acquisition, and data analysis of synthetic polymers using MALDI-TOF MS. Critical parameters during sample preparation include the selection of the matrix, identification of an appropriate cationization salt, and tuning the relative proportions of the matrix, cation, and analyte. The acquisition parameters, such as mode (linear or reflector), polarization (positive or negative), acceleration voltage, and delay time, are also important. Given some knowledge of the chemistry involved to synthesize the polymer and optimizing both the data acquisition parameters and the sample preparation conditions, spectra should be obtained with sufficient resolution and mass accuracy to enable the unambiguous determination of the end groups of most homopolymers (masses below 10,000) in addition to the repeat unit mass and the overall molecular weight distribution. Though demonstrated on a limited set of polymers, these general techniques are applicable to a much wider range of synthetic polymers for determining mass distributions, though end group determination is only possible for homopolymers with narrow dispersity.

Characterization of Synthetic Polymers via Matrix Assisted Laser Desorption Ionization Time of Flight MALDI-TOF Mass Spectrometry

A protocol for the matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) characterization of synthetic polymers is described including the optimization of sample preparation, spectral acquisition, and data analysis.

There are many techniques that can be employed in the characterization of synthetic homopolymers, but few provide as useful of information for end group ...

Using a Cyclic Ion Mobility Spectrometer for Tandem Ion Mobility Experiments

Accurate characterization of chemical structures is important to understand their underlying biological mechanisms and functional properties. Mass spectrometry (MS) is a popular tool but is not always sufficient to completely unveil all structural features. For example, although carbohydrates are biologically relevant, their characterization is complicated by numerous levels of isomerism. Ion mobility spectrometry (IMS) is an interesting complement because it is sensitive to ion conformations and, thus, to isomerism. 
Furthermore, recent advances have significantly improved the technique: the last generation of Cyclic IMS instruments offers additional capabilities compared to linear IMS instruments, such as an increased resolving power or the possibility to perform tandem ion mobility (IMS/IMS) experiments. During IMS/IMS, an ion is selected based on its ion mobility, fragmented, and reanalyzed to obtain ion mobility information about its fragments. Recent work showed that the mobility profiles of the fragments contained in such IMS/IMS data can act as a fingerprint of a particular glycan and can be used in a molecular networking strategy to organize glycomics datasets in a structurally relevant way. 
The goal of this protocol is thus to describe how to generate IMS/IMS data, from sample preparation to the final Collision Cross Section (CCS) calibration of the ion mobility dimension that yields reproducible spectra. Taking the example of one representative glycan, this protocol will show how to build an IMS/IMS control sequence on a Cyclic IMS instrument, how to account for this control sequence to translate IMS arrival time into drift time (i.e., the effective separation time applied to the ions), and how to extract the relevant mobility information from the raw data. This protocol is designed to clearly explain the critical points of an IMS/IMS experiment and thus help new Cyclic IMS users perform straightforward and reproducible acquisitions.

Ion mobility spectrometry (IMS) is an interesting complement to mass spectrometry for the characterization of biomolecules, notably because it is sensitive to isomerism. This protocol describes a tandem IMS (IMS/IMS) experiment, which allows the isolation of a molecule and the generation of the mobility profiles of its fragments.

Accurate characterization of chemical structures is important to understand their underlying biological mechanisms and functional properties. Mass ...

OLIgo Mass Profiling (OLIMP) of Extracellular Polysaccharides

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is that they contain complex sugar moieties in form of glycoproteins, proteoglycans, and/or polysaccharides. Examples include the extracellular matrix of humans and animal cells consisting mainly of fibrillar proteins and proteoglycans or the polysaccharide based cell walls of plants and fungi, and the proteoglycan/glycolipid based cell walls of bacteria. All these glycostructures play vital roles in cell-to-cell and cell-to-environment communication and signalling.
An extraordinary complex example of an extracellular matrix is present in the walls of higher plant cells. Their wall is made almost entirely of sugars, up to 75% dry weight, and consists of the most abundant biopolymers present on this planet. Therefore, research is conducted how to utilize these materials best as a carbon-neutral renewable resource to replace petrochemicals derived from fossil fuel. The main challenge for fuel conversion remains the recalcitrance of walls to enzymatic or chemical degradation due to the unique glycostructures present in this unique biocomposite.
Here, we present a method for the rapid and sensitive analysis of plant cell wall glycostructures. This method OLIgo Mass Profiling (OLIMP) is based the enzymatic release of oligosaccharides from wall materials facilitating specific glycosylhydrolases and subsequent analysis of the solubilized oligosaccharide mixtures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS)1 (Figure 1). OLIMP requires walls of only 5000 cells for a complete analysis, can be performed on the tissue itself2, and is amenable to high-throughput analyses3. While the absolute amount of the solubilized oligosaccharides cannot be determined by OLIMP the relative abundance of the various oligosaccharide ions can be delineated from the mass spectra giving insights about the substitution-pattern of the native polysaccharide present in the wall.
OLIMP can be used to analyze a wide variety of wall polymers, limited only by the availability of specific enzymes4. For example, for the analysis of polymers present in the plant cell wall enzymes are available to analyse the hemicelluloses xyloglucan using a xyloglucanase5, 11, 12, 13, xylan using an endo-&beta;-(1-4)-xylanase 6,7, or for pectic polysaccharides using a combination of a polygalacturonase and a methylesterase 8. Furthermore, using the same principles of OLIMP glycosylhydrolase and even glycosyltransferase activities can be monitored and determined 9.

OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides

A rapid way is described to gain insights into the structure of polysaccharides in an extracellular matrix. The method takes advantage of the specificity of glycosylhydrolases and the sensitivity of mass spectrometry allowing minute amounts of materials to be analyzed. This technique is adaptable to be used directly on tissue itself.

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is ...

Dithranol as a Matrix for Matrix Assisted Laser Desorption/Ionization Imaging on a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer

Mass spectrometry imaging (MSI) determines the spatial localization and distribution patterns of compounds on the surface of a tissue section, mainly using MALDI (matrix&#160;assisted laser desorption/ionization)-based analytical techniques. New matrices for small-molecule MSI, which can improve the analysis of low-molecular weight (MW) compounds, are needed. These matrices should provide increased analyte signals while decreasing MALDI background signals. In addition, the use of ultrahigh-resolution instruments, such as Fourier transform ion cyclotron resonance (FTICR) mass spectrometers, has&#160;the ability to resolve analyte signals from matrix signals, and this can partially overcome many problems associated with the background originating from the MALDI matrix. The reduction in the intensities of the metastable matrix clusters by FTICR MS can also help to overcome some of the interferences associated with matrix peaks on other instruments. High-resolution instruments such as the FTICR mass spectrometers are advantageous as they can produce distribution patterns of many compounds simultaneously while still providing confidence in chemical identifications. Dithranol (DT; 1,8-dihydroxy-9,10-dihydroanthracen-9-one) has previously been reported as a MALDI matrix for tissue imaging. In this work, a protocol for the use of DT for MALDI imaging of endogenous lipids from the surfaces of mammalian tissue sections, by positive-ion MALDI-MS, on an ultrahigh-resolution hybrid quadrupole FTICR instrument has been provided.

Dithranol (DT; 1,8-dihydroxy-9,10-dihydroanthracen-9-one) has previously been reported as a MALDI matrix for tissue imaging of small molecules; protocols for the use of DT for the MALDI imaging of endogenous lipids on the surface of tissue sections&#160;by positive-ion MALDI-MS on an ultrahigh-resolution quadrupole-FTICR instrument are provided here.

Mass spectrometry imaging (MSI) determines the spatial localization and distribution patterns of compounds on the surface of a tissue section, mainly ...