Yazarlar hiçbir katkıda bulunanlar var.

1. örnek plaka Önkoşullanma
<ol><li>
Örnek plakası temizleme
	<ol>
<li>Temizleme sırasında kontaminasyon örnek plaka için nitril eldiven giymek.</li>
		<li>El-yıkama deterjan solüsyonu (1,0 mg/mL) 100,0 mL örnek plakalı.</li>
		<li>El-distile-deiyonize su (DDW) ile örnek tabak yıkama.</li>
		<li>Örnek plaka yüzeyi MeOH 30,0 mL ile yıkayın.</li>
		<li>Örnek plaka tamamen suya batırılır kadar 600 mL kabı ve dolgu ile DDW örnek plaka koymak.</li>
		<li>Kabı bir ultrasonik koymak ( tablo malzemelerigörmek) banyo ve örnek plaka 15 dk (200 W, 40 kHz) için solüsyon içeren temizleyicide.</li>
		<li>Örnek plaka almak ve basınçlı azot kullanarak su damlaları darbe.</li>
		<li>MeOH 0.2 µL MeOH diğer noktalar yayılır olup olmadığını denetlemek için örnek plaka üzerinde mevduat. Not: MeOH diğer noktalar ile birleştirir adımları 1.1.3-1.1.5 işlemini yineleyin; Eğer değilse, sonraki adıma geçin.</li>
	</ol>
</li>
	<li>
Kurutma odası sıcaklık düzenleme
	<ol>
<li>Kurutma odası istikrarlı koşullar altında damlacıkları,11,12,13daha önce açıklandığı gibi kuru için kullanın. Özellikle, Ou, Y.-M 2.1-2,5 adımlarda açıklandığı ayrıntılı yordamı kullanın. vd. 201612. Kısaca:<ol>
<li>Kurutma odası oda sıcaklığında azot düşük bağıl nem ortam sağlamak için sürekli akış hızında tarafından tasfiye.</li>
			<li>Isı kontrollü bakır bloğunda kurutma odası tarafından düzenlenen örnek plaka sıcaklık sabiti normal - (25 ° C) veya hızlı kurutma koşulları (50 ° C), devam et.</li>
		</ol>
</li>
	</ol>
</li>
	<li>
Matris ve analit çözümleri hazırlanması
	<ol>
<li>
Matris çözümleri hazırlanması
		<ol>
<li>DHB % 50 Asetonitril (ACN) dağıtılması: %50 0.1 M çözüm hazırlamak için DDW.</li>
		</ol>
</li>
		<li>
Karbonhidrat analitler hazırlanması
		<ol>
<li>SLeA 10-4 M çözüm hazırlamak için DDW geçiyoruz.</li>
			<li>Bir 10-4 M çözüm hazırlamak için DDW MH geçiyoruz.</li>
		</ol>
</li>
	</ol>
</li>
</ol>2. örnek ifade ve rekristalizasyon
Not: Küçük ve düzenli miktarlarda örnekleri analiz etmek için en iyi duruma getirilmiş yordamlar burada açıklanmıştır. Çözümler yatırma önce numune plaka sıcaklığı istenilen sıcaklıkta stabil emin olun. Örnek diğer örnek noktalar rekristalizasyon sırasında karşılamak için büyük bir alana yayılırsa, yeni bir örnek hazırlamak veya 1.1 arasındaki adımları yineleyin.
<ol><li>
Küçük bir miktar (0.1 µL) örnek analizi için Not: örnek ve zaman tüketimi en aza indirmek için aşağıdaki adımları geliştirilmiştir. Kantitatif analiz sınırlı veya miktar analiz için hızlı IMS ile gerçek örnekleri için uygundur.<ol>
<li>DHB çözüm ve 0,25 SLeA µL veya MH çözüm microcentrifuge tüp içinde 0.25 µL premix.</li>
		<li>Girdap 3 için bir girdap Mikser kullanarak karma çözüm s.</li>
		<li>Spin aşağı 2 için mini santrifüj karışık çözümde s (2000 x g).</li>
		<li>Premix çözüm 0.1 µL çizmek ve hemen örnek plaka üzerinde mevduat bir pipet kullanın. Not: Örnek küçük bir miktar yatırırken, Yapmak değil tutmak premix çözüm için 10 pipet ucu s.</li>
		<li>Örnek kurumasını bekleyin. Tipik kurutma kez Tablo 1' de listelenmiştir.</li>
		<li>MeOH 0.2 µL şu kurutulmuş örnek nokta yatırmak için bir pipet kullanın. Örnek alacak ıslak ve kuru hemen. Not: İfade yordamı 3'te tamamlandığını emin olmak MeOH önemli buharlaşma kaybı olmaması için s.</li>
		<li>Bir mikroskop kullanarak örnek inceleyin. Kristal türleri morfoloji olarak beklenen (bakınız Şekil 1 örneğin istenen sonuçları) yoksa, yeni bir örnek hazırlamak için 2.1.1-2.1.6 kadar olan adımları tekrarlayın.</li>
		<li>Nitril eldiven giymek ve dikkatle kurutma odası örnek tabaktan dışarı almak.</li>
	</ol>
</li>
	<li>
Düzenli bir miktar (1 µL) örnek analizi için Not: Aşağıdaki adım karbonhidrat örnekleri ile genellikle kullanılan maldı örnek miktar polimerlerin en üst düzeye çıkarma geliştirilir. Rutin ve kantitatif analizleri için uygun bir süreçtir. Rekristalizasyon işlemi örnekleri ve matrisleri büyük alanlar için eşit olarak yeniden dağıtır.<ol>
<li>DHB çözüm ve 2.5 µL SLeA veya bir microcentrifuge tüp çözümde MH 2.5 µL premix.</li>
		<li>Girdap 5 için bir girdap karıştırıcılı premix çözüm s.</li>
		<li>Spin aşağı 2 için mini santrifüj karışık çözümde s (2000 x g).</li>
		<li>Premix çözüm 1.0 µL çizmek ve hemen örnek plaka üzerinde mevduat bir pipet kullanın. Not: yok kalan kullanım kılavuzuyla çözüm tekrar örnekleri yatırma sonra.</li>
		<li>Örnek kurumasını bekleyin. Tipik kurutma kez Tablo 1' de listelenmiştir.</li>
		<li>Şu kurutulmuş örnek nokta MeOH 1,5 µL yatırmak bir pipet kullanın. Örnek alacak ıslak ve kuru hemen. Not: Yüksek örnek plaka sıcaklık (50 ° C) durumlarda, rekristalizasyon adım 5 içinde yapılmalıdır s MeOH buharlaşma pipet ipucunda en aza indirmek için.</li>
		<li>Bir mikroskop kullanarak örnek inceleyin. Kristal türleri morfoloji olarak beklenen (bakınız Şekil 1 örneğin istenen sonuçları) yoksa, yeni bir örnek hazırlamak için 2.2.1-2.2.6 kadar olan adımları tekrarlayın.</li>
		<li>Nitril eldiven giymek ve dikkatle kurutma odası örnek tabaktan dışarı almak.</li>
	</ol>
</li>
</ol>3. kütle spektrometresi veri toplama ve Analizi
Not: Analiz bir maldı İyon kaynağı ile donatılmış bir ticari uçuş zaman kütle spektrometre (Tablo reçetesi) kullanılarak gerçekleştirilir. Araç ön en iyi duruma getirilmiş ayıklama gecikme ve lazer enerjisi ile belirli kontrol yazılımı (Tablo reçetesi) tarafından işletilmektedir. Spectra m/z kitlesel bir dizi ile doğrusal modu kaydedilir = 0-1500. Örnek potansiyel ±25 plakadır keV ve her spektrum ortalama 10 lazer çekim. Kullanıcıların araç optimizasyonu ve uyumlu yazılım kullanarak örnek analiz yapmak ve araç üreticisinin yönergelerini izleyin.
<ol><li>Araç kontrol yazılımı açın (bkz. Tablo malzeme).</li>
	<li>Örnek plaka kütle spektrometre yerleştirin.</li>
	<li>Önceden en iyi duruma getirilmiş veri toplama yöntemi yazılımı seçin.</li>
	<li>Tüm örnek bölge görüntüleme yazılımı kullanarak IMS için kayıt ( Tablo malzemelerigörmek). Not: IMS yapıyor yoksa bu adımı atlayın.</li>
	<li>Veri toplama kontrol yazılımı toplu modunda başlatın.</li>
	<li>İyon görüntüleri veri toplama işlemi tamamlandıktan sonra görüntüleme yazılımı kullanarak çizmek.</li>
	<li>Kitle spectra analiz yazılımı kullanarak analiz ( Tablo malzemelerigörmek) verileri bir iyon görüntü kaydedilirse.</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. 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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. 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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. 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Yazarlar ifşa gerek yok.

Örnek heterojenite maldı-Bayan DD çok önemli bir sorun en sık kullanılan örnek hazırlama yöntemi, ama sonuç kristalleri son derece heterojen olmasıdır. Böyle örnekleri zavallı atış-to-shot ve örnek örnek sinyal tekrarlanabilirlik göster. Bu nedenle, veri toplama sırasında "tatlı noktalar" örnek alanlarda arama ortak bir maldı deneylerde yöntemidir. Miktar rutin analizleri için uygun olmayan türdeş olmayan böyle örneklerindendir.
Mevcut çalışmada, maldı örnek...

Karbonhidrat analiz önemli ve zorlu bir konudur. Karbonhidrat ve onların türevleri yaşayan organizmalar1,2,3önemli roller oyun olarak. Bu moleküllerin yapıları karmaşık ve çürümeye yatkındır. Birçoğu açıkça ayrılık ve algılama zorluklar nedeniyle karakterize olamaz. Matris yardımlı Lazer Desorpsiyon/iyonlaşma (MALDI) kütle spektrometresi (MS) biomolecules, duyarlılık ve anlaşılır sonuçları4, geniş bir analizi uygulanmış olduğundan ne kadar karbonhidratlar MALDI-MS kullanarak analiz devam ediyor Böyle molekülleri5düşük iyonlaşma verimliliği nedeniyle büyük bir meydan okuma olacak. Kimyasal derivatization karbonhidrat6,7iyonlaşma verimliliğini artırmak için sık kullanılan bir yöntemdir, ancak zaman ve örnek tüketen tür yordamlar vardır. Ayrıca, derivatized karbonhidrat iyonlaşma verimliliğini hala proteinlerin daha düşüktür. Bu nedenle, karmaşık prosedürler olmaksızın MALDI-MS karbonhidrat sinyallerinin geliştirilmesi için yöntemler geliştirilmesi gereklidir.
Kantitatif analiz MALDI-MS uygulamaya zor bir konudur. Bir büyük MALDI-MS onun duyarlılığı ve veri tekrarlanabilirlik eleştirel örnek hazırlama protokolleri ve deneysel parametreleri kullanır bir sorundur. Birçok durumda, kantitatif analiz MALDI-MS tarafından türdeş olmayan örnek türleri Morfoloji ve analit dağıtım nedeniyle güvenilmez olduğunu. 2,5-dihidroksibenzoik asit (DHB) maldı matris ile hazırlanan örnekleri iyi bilinen bir örnektir. DHB yavaş yavaş ortam bir ortamda kristalize zaman, çünkü sonuç örnekleri düzensiz türleri morfoloji ölçüde matris kristalleri içine analit şirketleşme, tahmin edilemez. Böyle örnekleri normalde büyük iğne şekilli ve güzel kristalleri oluşur. DHB uçucu solvent ve/veya ısıtmalı örnek plaka kullanılarak hazırlanan zaman hızlı bir kurutma işlemi daha homojen iyi kristaller ve daha iyi nicel sonuçları8,9,10ile sonuçlanır. Bu teknik maldı örnekleri "rekristalizasyon" bilinir. İyileşme analitler hızlı kristalizasyon işlemi sırasında daha iyi birleşme iyi matris kristalleri içine atfedilir. Biz de örnek hazırlama ortamını ayarlama karbonhidrat sinyal ve geliştirilmiş nicel sonuçları11,12heterojen azalma göstermiştir. Bu çalışmaların bulguları örnek morfoloji karbonhidrat sinyal kalitesini belirlemede önemli bir faktör olduğunu göstermektedir. Günlük analiz için genel bir strateji geliştirmek için geliştirilmiş karbonhidrat duyarlılık sağlayan bir verimli örnek reformasyon yöntemi gereklidir.
Sistematik olarak örnek Morfoloji ve karbonhidrat duyarlılık MALDI-MS bir son raporu13arasında korelasyon inceledik. Birkaç önemli karbonhidrat kullanılarak sonuçlar elde ve en iyi sinyal geliştirme recrystallizing tarafından yerine getirilir matrisleri Haritayı maldı örnekleri kurutulmuş. Geleneksel kurutulmuş damlacık (DD) yöntemi kullanılarak hazırlanan örnekleri morfolojisi metanol (MeOH) ile hızlı rekristalizasyon tarafından reform. Detaylı örnek hazırlama protokolleri burada gösterildiği. Protokol örnek plaka Önkoşullanma, örnek ifade ve rekristalizasyon ve kütle spektrometresi analizleri de dahil olmak üzere üç ana adımlardan oluşur. Kullanılan karbonhidrat sialyl-lewis bir (SLeA) ve maltoheptaose (MH) içerir. DHB modeli matris kullanılır. Karbonhidrat sinyal yoğunluğu ve kayma dağıtım belirgin rekristalizasyon sonra geliştirilmiş sonuçlar gösterir. Böyle bir yöntem örnekleri 2,4,6-trihydroxyacetophenone (THAP) ve α- cyano-4-hydroxycinnamic asit gibi diğer popüler matrisleri ile uygulanabilir. Bu yöntem kolayca laboratuvar rutin karbonhidrat analiz için entegre edilebilir genel bir yaklaşım olarak hizmet vermektedir.

<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

Numune hazırlama karbonhidrat kütle spektrometresi (MS) analizi kritik bir süreçtir. Matris yardımlı Lazer Desorpsiyon/iyonlaşma (MALDI) MS seçtiğiniz karbonhidrat analiz yöntemi olsa da, zavallı iyon sinyal ve veri tekrarlanabilirlik karbonhidrat örnekleri ciddi sorunlar olmaya devam etmektedir. Karbonhidrat kantitatif analiz için üstün veri kalite sağlayan etkili bir analitik Protokolü gereklidir. Bu video sinyal yoğunluğu artırmak ve veri değişim karbonhidrat MALDI-MS en aza indirmek için örnek hazırlanması protokolleri gösteriyor. Kurutma ve örnek damlacıkları kristalleşme sonra kristal morfoloji metanol kitle spektrometrik analiz önce reformlar yaptı. Karbonhidrat sinyalinde geliştirme maldı görüntüleme kütle spektrometresi ile (IMS) incelenir. Deneysel sonuçlar kristal reformasyon kristal yapıları ayarlar ve karbonhidrat analitler yeniden dağıtan gösterir. Geleneksel MALDI-MS, reform karbonhidrat kristal türleri morfoloji metanol gösterir önemli ölçüde daha iyi sinyal yoğunluğu, iyon görüntü dağıtım ve veri istikrar ile kurutulmuş damlacık hazırlama yöntemi ile karşılaştırıldığında. Burada gösterildiği protokollerin örnek kompozisyon değişiklikleri içermeyen beri onlar çeşitli karbonhidrat ve matrisleri genellikle tabidir.

SLeA temsilcisi SEM görüntüleri DD kullanılarak hazırlanan DHB ile karışık ve rekristalizasyon Yöntemler Şekil 1' de gösterilmektedir. Tipik bir DHB morfoloji DD yöntemi tarafından hazırlanan büyük iğne biçimli kristaller RIM ve örnek noktalar ortasına ince kristal yapıları var. İğne şeklindeki böyle kristallerinin tipik uzunluğu ~ 100 µm ' dir. MeOH tarafından rekristalizasyon sonra örnek eşit iyi pul gibi kristaller...

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

Karbonhidrat iyon sinyalleri maldı kütle spektrometresi reform kristal yapılar tarafından örnek hazırlama sürecinde güçlendirmeye yönelik bir protokol gösterilmiştir.

Matris yardımlı Lazer Desorpsiyon/iyonlaşma kütle spektrometresi karbonhidrat iyon sinyalleri geliştirmek için bir verimli örnek hazırlama yöntemi

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 Görüntüleme.  Academia Sinica. Karbonhidrat iyon sinyalleri maldı kütle spektrometresi reform kristal yapılar tarafından örnek hazırlama sürecinde güçlendirmeye yönelik bir protokol gösterilmiştir.

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.

Kantitatif Uygulamalar için Homojen MALDI Örneklerin Hazırlanması

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

Biyokimya

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.

Uçuş lazer desorpsiyon / iyonizasyon zaman sağlam proteinler (MALDI-TOF) kütle spektrometrik analizi Matris destekli daha büyük 100 kDa

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

Kimya

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.

Biyolojik Numune metabolik profil için Sheathless Kapiler Elektroforez-Kütle Spektrometre

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).

MALDI Numune Hazırlama: Ultra İnce Tabaka Yöntemi

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

Biyoloji

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.

Biyomarker Discovery için Mezogözenekli Silika İnce Filmler üzerine düşük molekül ağırlıklı Protein Zenginleştirme

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

Biyomühendislik

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.

Örnekleri için matris peptit esaslı tamir Formalin en iyi hazırlık lazer desorpsiyon/iyonlaşma kütle spektrometresi iş akışlarını görüntüleme destekli

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.

Matris ile sentetik polimerler karakterizasyonu Lazer Desorpsiyon iyonlaşma zaman uçuş (MALDI-TOF) kütle spektrometresi destekli

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.

Tandem İyon Hareketlilik Deneyleri için Döngüsel İyon Hareketlilik Spektrometresi Kullanma

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.

Ekstrasellüler polisakkaritler Oligo Kitle Profil (Olimp)

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.

Bir Fourier Matrix Yardımlı Lazer Dezorpsivon / İyonizasyon Görüntüleme için bir Matrix olarak ditranol İyon Cyclotron Rezonans Kütle Spektrometresi Dönüşümü

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