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).

BEMÆRK: Denne protokol er udviklet til at reducere den rumlige heterogenitet maltotriose og bradykinin fragment (1-7) udarbejdet med tørrede-dråbe metoden. Protokollen består af tre trin, herunder forberedelse og forkonditionering prøve deposition og tørring, og massespektrometri dataanalyse. Procedurerne er beskrevet og beskrevet mere detaljeret nedenfor: 1. Forberedelse og Forkonditionering <ol><li> Rengøring af Sample Plate <ol><li> Bær nitrilhandsker og hånd-vaske prøven pladen forsigtigt med ren...

<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. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha-Cyano-4-Hydroxycinnamic+Acid+as+a+Matrix+for+Matrix-Assisted+Laser+Desorption+Mass-Spectrometry.">Alpha-Cyano-4-Hydroxycinnamic Acid as a Matrix for Matrix-Assisted Laser Desorption Mass-Spectrometry.</a> Org. Mass Spectrom. 27, 156-158 (1992).</li><li>Ehring, H., 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=Role+of+Photoionization+and+Photochemistry+in+Ionization+Processes+of+Organic-Molecules+and+Relevance+for+Matrix-Assisted+Laser+Desorption+Ionization+Mass-Spectrometry.">Role of Photoionization and Photochemistry in Ionization Processes of Organic-Molecules and Relevance for Matrix-Assisted Laser Desorption Ionization Mass-Spectrometry.</a> Org. 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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Baseret på tidligere teoretiske forudsigelser, kan temperatur-induceret hydrodynamiske strømme inden dråberne overvindes udad kapillære strømme induceret af opløsningsmiddelafdampning. Effektiviteten af ​​en sådan intern recirkulation af molekyler forøges, når temperaturen gradienter inden en stigning dråbe. Ifølge de forudsagte resultater, når holde prøven plade temperatur under 5 ° C under opretholdelse dens omgivelser ved omgivelsestemperatur, den gennemsnitlige hastighed af recirkuleringsanlæg str...

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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		<tr height="21">
			<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.

De lyse-field billeder samt MS billeder af maltotriose og bradykinin-fragment (1-7) fremstillet med prøveplade temperatur på 5 og 25 ° C er vist i figur 1. For sodiated maltotriose, ion signal hovedsagelig udfylder ved periferien af ​​prøven, da den er fremstillet med en prøveplade temperatur på 25 ° C. Ved at nedsætte prøvepladen temperaturen til 5 ° C, signalet udfylder homogent over hele prøven området. Den eneste mærkbare ulempe ved udarbejdelsen pr?...

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

En protokol til at reducere rumlige heterogeniteter af ion signaler i MALDI massespektrometri ved at regulere substrat temperatur under prøven tørringsprocesser demonstreres.

Udarbejdelse af Homogene MALDI Prøver til Kvantitative applikationer

This protocol demonstrates a simple sample preparation to reduce spatial heterogeneity in ion signals during matrix-assisted laser desorption/ionization ...

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