Yazarlar, aşağıdaki kurumlar tarafından mali destek kabul Ulusal Bilim Vakfı (Grant 0.719.232), Enerji Departmanı (Grant DEFG02-01ER15129), WM Keck Vakfı (Grant 041.904), ve George Washington Üniversitesi Araştırma Geliştirme Fonu. Yazarlar cömertçe Kızılötesi Fiber Sistemleri (Silver Spring, MD) yanı sıra Mark E. Reeves ve George Washington Joan A. Hoffmann tarafından fiber dağlama ile ilgili ilk tartışma için protokol tarafından sağlanan GeO2 tabanlı optik fiberler için minnettar Üniversitesi. Yazarlar ayrıca, protokol videoya sırasında ona yardım Jessica A. Stolee teşekkür etmek istiyorum.

1. Optik bileşenler <ol><li> Lazer ablasyon elektrosprey iyonizasyon (LAESI) kütle spektrometresi (MS) için lazer ablasyon 5 ns 2.94 mikron dalga boyunda lazer darbe tarafından üretilmektedir. Nd sürülen ayarlanabilir optik parametrik osilatör: YAG lazer (Opolette 100, Opotek A.Ş., Carlsbad, CA), bu çalışmada ablasyon için kullanılır. Tekrarlama oranı 5 ve 100 Hz arasında seçilir. Bu, doğrudan maruz kalması üzerine, göz veya deri ciddi kalıcı hasara neden olabilir bir sınıfı IV lazer. Cisimden yansımalı lazer ışını da cilt veya göz için tehlikeli olabilir. Korunması için uygun bir lazer koruyucu gözlük takın. </li><li> Germanyum dioksit tabanlı cam fiber optik kablo, Kızılötesi Fiber Systems, Inc (Silver Spring, MD) elde edilmiştir. Orada Hytrel ve fiber kaplamalar ucundan bunları kaldırmak için aşağıdaki yönergeleri izleyin poliimid. Isı 1-metil-2-pyrrolidinone 130-150 ° C davlumbaz. Lif, yaklaşık 1 dakika süreyle ya da kaplama kadar yumuşamaya başlar ve soyulabilir bu sıvı içine şerit istediğiniz biter batırın. Yumuşatılmış kaplama metanol ya da bir dakika izopropanol içine batırın ve kalan herhangi bir kaplama kapalı tüy bırakmayan bir peçete ile silin. Kaplama çıkardıktan sonra, cleave bir safir puanlama ve nazikçe onları yakalamaya bıçak (KITCO Fiber Optik, Virginia Beach, VA) fiber iki ucunda. </li><li> % 1 (v / v) nitrik asit (saflıkta) çözümü, bir ucunu fiber ucu kimyasal aşındırma istenilen netliği elde etmek için. Dikey seçilen ilk temastan sonra 300 ila 500 mm derinliğe kadar asit çözeltisi içine fiber sonuna bırakın. Gravür yaklaşık 15 dakika sonra, ucu kendiliğinden asit yüzeyinden ayırır. Bu sonuçlar ~ 15 mikron eğrilik yarıçapı ile fiber ucu sivriltilmiş. Herhangi bir asit artıklarını kaldırmak için deiyonize su ile durulayın. </li><li> Kazınmış sonuna bir micromanipulator lif (MN-151, Narishige, Tokyo, Japonya) Mount ve verimli enerji birikimi için hücre yüzey yakınlığı (~ 20 mm) yakın getirmek. </li><li> Mandren (BFC300, Siskiyou Corporation, Grants Pass, OR) tarafından çıplak fiber optik fiber olmayan kazınmış sonunda tutun. Optik uyum sağlanabilmesi için, fiber mandreni beş-eksenli bir tercüman (BFT-5, Siskiyou Corporation, Grants Pass, OR) monte edilir. Lazer enerjisi (0,5 - 1 mJ), non-kazınmış ucuna bir yüzü düz diğeri dışbükey kalsiyum florür veya yansıma önleyici kaplama ZnSe objektif (Kızılötesi Optik Ürünler, Farmingdale, NY) ışın odaklama elyaf haline birleştirilmiştir. Korumalı altın aynalar (PF10-03-M01, Thorlabs, Newton, NJ) odaklama lens üzerine orta-kızılötesi lazer ışını yönlendirmek için kullanılır. </li></ol> 2. Elektrosprey Kurulum <ol><li> Elektrosprey sistemi aşağıdaki bileşenleri kullanılarak inşa edilebilir iletken perfluoroelastomer halkalı, bağlantı parçaları, boru kol, iğne bağlantı noktası (örneğin, U-435, M215, F-331Nx, F-242x, 9013, IDEX Sağlık ve Bilim, metal sendikası: Oak Harbor, WA) yanı sıra erimiş silika tüp (örneğin, CT360-100-50-5, Yeni Amaç A.Ş., Woburn, MA). Tavsiye edilen yayıcı paslanmaz çelikten imal edilmiş ve 50 mikron id (örneğin, MT320-50-5-5, Yeni Amaç A.Ş., Woburn, MA), konik bir uç vardır. </li><li> Elektrosprey için% 0.1 asetik asit (v / v)% 50 metanol çözeltisi hazırlayın. Çözümünü kullanarak bir şırınga pompası (Harvard Apparatus, Holliston, MA) tarafından 200 ile 300 nl / dak hızında pompalanır, örneğin, 500 ul şırınga (81.222 Hamilton Şirket, Reno, NV). </li><li> 2.800 ve 3.000 V arasındaki yüksek gerilim, sürekli bir elektrosprey oluşturmak için düzenlenmiş bir güç kaynağı (PS350, Stanford Araştırma Sistemleri A.Ş., Sunnyvale, CA) tarafından metal sendika uygulayın. Tüm elektrik bağlantılarını güvenli ve düzgün topraklanmış koruma korumalı olduğundan emin olun. Maruz kalan yüksek gerilim ile doğrudan temas, elektrik çarpmasına neden olabilir. </li></ol> 3. Numune Alma <ol><li> Uygun bir kaynaktan canlı doku ya da hücre örneği elde etmek ve bunları analiz yapılması tavsiye tutmak. Bu gösteri için Allium cepa ampuller yerel bir mağazadan satın alındı. Uzunlamasına bir ampul bir cerrahi neşter tarafından kesin. Bir ölçekte birkaç santimetre segment bir şerit içine kesitli oldu. Iç epidermal dokusunun sağlam bir tek tabaka sıyrılır. </li><li> Epidermisin ıslak yüzey doku önceden temizlenmiş cam mikroskop lamı üzerine monte etmek için kullanılır. Konumlama için üç eksenli bir çeviri sahnede monte genel amaçlı bir plaka tutucu (FP01, Thorlabs A.Ş., Newton, NJ) slayt tutun. Hücre bozulmasını önlemek için örnek montaj hemen sonra kütle spektrumları edinin. </li></ol> 4. Görüntüleme Sistemi <ol><li> İki uzun mesafe mikroskoplar analiz için hücreleri seçin ve bilenmiş fiber ucu ve hücre yüzeyi arasındaki mesafeyi korumak için kullanılır. Sonuncusu yüzeyinden ölçülen 15 ° -30 ° açı altında monte edilir. Bu sistem al5x sonsuz ong mesafe video mikroskobu (7x hassas optik zum, Edmund Optik, Barrington, NJ) düzeltilmiş bir CCD kamera ile donatılmıştır (Marlin F131, Müttefik Vizyon Teknolojileri objektif lens (M 5x Planı Apo, Mitutoyo Co, Kanagawa, Japonya) Stadtroda, Almanya), izleme ve görüntü yakalamak için. </li><li> Ikinci video mikroskop üst hücreler görselleştirmek için örnek yüzeyine dik açıda monte edilir. Bu sistem, ablasyon ve analiz için seçilen hücrenin üzerine fiber ucu hizalamak için görsel geribildirim sağlar. Uzun çalışma mesafesi objektif lens (10x M Planı Apo, Mitutoyo Co, Kanagawa, Japonya) ve bir CCD kamera, 10x sonsuza ile donatılmış 7x hassas zum optik (Edmund Optik, Barrington, NJ), oluşur düzeltildi. </li></ol> 5. Kitle Spectra alımı <ol><li> Lazer, elektrosprey ve kütle spektrometresi, örneğin, bir quadrupole zaman uçuş sistemi (S-TOF Premier, Waters, Milford, MA) açın. Ablasyon spot ve elektrosprey birbirlerine göre verici ve maksimum iyon yoğunlukları ulaşmak için kütle spektrometresi ağzı pozisyonunu ayarlayarak LAESI iyon kaynağı geometri optimize edin. </li><li> Üst görünümü görüntüleme sistemi kullanılarak analiz için faiz hücreyi seçin. Seçimden sonra, doğrudan fiber optik ucu sivriltilmiş altında seçili hücre getirmek için örnek sahne taşıyın. ~ 20 mm ucu-hücre yüzeyinde mesafe yan mikroskop kullanarak tekrar ayarlayın. </li><li> Yangın kazınmış fiber ucu ile seçili hücre lazer bakliyat. Bu sonuç, hücre duvarı ve partiküler madde şeklinde sitoplazma ejeksiyon delinme. Elektrosprey postionization sonra, üretilen iyonlar kütle spektrometresi ve kütle spektrumları tarafından toplanan kaydedilir. </li><li> Veri toplama sonra, lazer, elektrosprey ve bekleme modunda kütle spektrometresi koymak veya bunları kapatın. Ablasyon A. tutun cepa, ışık mikroskobu ile isteğe bağlı gözlem için doku örneği. </li></ol> 6. Temsilcisi Sonuçlar Başarılı ablasyon, hücre duvarı ve bir LAESI kütle spektrumu toplanması patlama tek bir hücre. Başarısız bir deney, genellikle hiçbir ablasyon ve / veya kütle spektrumu sonuçları. Gösteri amaçlı, biz tek bir gömülü A. epidermal hücre LAESI-MS analiz sonuçlarını sunmak cepa ampul. Şekil 2a, bir hücrenin örnekleme sonra, temsili bir optik mikroskop görüntü gösterir. Hücre duvarı delikli ve perforasyonu ölçüde komşu hücrelerle sınırları ile sınırlıdır. Bitişik hücreleri sağlam gibi görünüyor. Şekil 2b hücreden üretilen bir temsilcisi LAESI kütle spektrumu gösterir. Iyonları çok çeşitli A. tespit cepa hücre ve onların doğru kitlelere göre, izotop dağılım şekilleri ve bazı durumlarda, tandem kütle spektrumları, onlar geçici endojen metabolitler atanır. Tek bir hücrenin tespit iyonları birden fazla hücre geleneksel LAESI-MS ile aynı doku analizi gözlenen olanlara benzerdi. 13 <img src="/files/ftp_upload/2144/2144fig1.jpg" alt="Şekil 1" /> Şekil 1. LAESI-MS ile tek bir hücre analizi şematik. Orta-IR lazer darbesi bir fiber optik birleştiğinde cam slayt üzerine yerleştirilen numune teslim edilir. Bilenmiş lif ucu hedef hücre içine ışık odaklanır ve yatırılan hücrenin enerji patlamaları. Üretilen ablasyon tüy (kırmızı noktalar) örnek spesifik iyon üretim damlacıkları kaynasabilirler bir elektrosprey tüy (siyah noktalar) (yeşil nokta) ile birleşti. Bu iyonlar analiz ve kütle spektrometresi (MS) tarafından tespit edilir. İki uzun mesafe Video mikroskoplar, Mikroskop 1 ve Mikroskop 2, fiber ucu ve hücre yüzeyi arasındaki mesafe izlemek ve analiz için bir hücre sırasıyla seçmek için kullanılmaktadır. <img src="/files/ftp_upload/2144/2144fig2a.jpg" alt="Şekil 2a" /> A. tek bir epidermal hücre Şekil 2a. Ablasyon işareti cepa ampul. Bitişik hücreleri görünür bir hasar göstermezler ve daha sonra ayrı ayrı analiz edilebilir. <img src="/files/ftp_upload/2144/2144fig2b.jpg" alt="Şekil 2b" /> Bir tek A. ablasyon karşılık gelen Şekil 2b. Mass Spektrum cepa epidermal hücre birincil ve ikincil metabolitleri varlığını gösterir.

<ol>
	<li>Cooks, R. G., Ouyang, Z., Takats, Z., Wiseman, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ambient+Mass+Spectrometry.">Ambient Mass Spectrometry.</a> Science. 311, 1566-1570 (2006).</li><li>Chen, Z. Y., 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=Early+plume+expansion+in+atmospheric+pressure+midinfrared+laser+ablation+of+water-rich+targets.">Early plume expansion in atmospheric pressure midinfrared laser ablation of water-rich targets.</a> Physical Review E. E77, 036316-036316 (2008).</li><li>Li, Y., Shrestha, B., 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=Atmospheric+Pressure+Molecular+Imaging+by+Infrared+MALDI.">Atmospheric Pressure Molecular Imaging by Infrared MALDI.</a> Mass Spectrometry. Analytical Chemistry. 79, 523-532 (2006).</li><li>Li, Y., Shrestha, B., 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=Atmospheric+Pressure+Infrared+MALDI+Imaging+Mass+Spectrometry+for+Plant+Metabolomics.">Atmospheric Pressure Infrared MALDI Imaging Mass Spectrometry for Plant Metabolomics.</a> Analytical Chemistry. 80, 407-420 (2007).</li><li>Nemes, P., 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=Laser+Ablation+Electrospray+Ionization+for+Atmospheric+Pressure+in+Vivo,+and+Imaging+Mass+Spectrometry.">Laser Ablation Electrospray Ionization for Atmospheric Pressure in Vivo, and Imaging Mass Spectrometry.</a> Analytical Chemistry. 79, 8098-8106 (2007).</li><li>Shrestha, B., Li, Y., 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=Rapid+analysis+of+pharmaceuticals+and+excreted+xenobiotic+and+endogenous+metabolites+with+atmospheric+pressure+infrared+MALDI+mass+spectrometry.">Rapid analysis of pharmaceuticals and excreted xenobiotic and endogenous metabolites with atmospheric pressure infrared MALDI mass spectrometry.</a> Metabolomics. 4, 297-311 (2008).</li><li>Nemes, P., Barton, A. A., Li, Y., 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=Ambient+Molecular+Imaging+and+Depth+Profiling+of+Live+Tissue+by+Infrared+Laser+Ablation+Electrospray+Ionization+Mass+Spectrometry.">Ambient Molecular Imaging and Depth Profiling of Live Tissue by Infrared Laser Ablation Electrospray Ionization Mass Spectrometry.</a> Analytical Chemistry. 80, 4575-4582 (2008).</li><li>Nemes, P., Barton, A. A., 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=Three-Dimensional+Imaging+of+Metabolites+in+Tissues+under+Ambient+Conditions+by+Laser+Ablation+Electrospray+Ionization+Mass+Spectrometry.">Three-Dimensional Imaging of Metabolites in Tissues under Ambient Conditions by Laser Ablation Electrospray Ionization Mass Spectrometry.</a> Analytical Chemistry. 81, 6668-6675 (2009).</li><li>Nemes, P., Woods, A. S., 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=Simultaneous+Imaging+of+Small+Metabolites+and+Lipids+in+Rat+Brain+Tissues+at+Atmospheric+Pressure+by+Laser+Ablation+Electrospray+Ionization+Mass+Spectrometry.">Simultaneous Imaging of Small Metabolites and Lipids in Rat Brain Tissues at Atmospheric Pressure by Laser Ablation Electrospray Ionization Mass Spectrometry.</a> Analytical Chemistry. 82, 982-988 (2010).</li><li>Shrestha, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+analysis+of+lipids+and+small+metabolites+in+mouse+brain+tissue+by+AP+IR-MALDI+and+reactive+LAESI+mass+spectrometry.">Direct analysis of lipids and small metabolites in mouse brain tissue by AP IR-MALDI and reactive LAESI mass spectrometry.</a> Analyst. , (2010).</li><li>Sripadi, P., Nazarian, J., Hathout, Y., Hoffman, E., 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=In+vitro+analysis+of+metabolites+from+the+untreated+tissue+of+Torpedo+californica+electric+organ+by+mid-infrared+laser+ablation+electrospray+ionization+mass+spectrometry.">In vitro analysis of metabolites from the untreated tissue of Torpedo californica electric organ by mid-infrared laser ablation electrospray ionization mass spectrometry.</a> Metabolomics. 5, 263-276 (2009).</li><li>Shrestha, B., 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=In+Situ+Metabolic+Profiling+of+Single+Cells+by+Laser+Ablation+Electrospray+Ionization+Mass+Spectrometry.">In Situ Metabolic Profiling of Single Cells by Laser Ablation Electrospray Ionization Mass Spectrometry.</a> Analytical Chemistry. 81, 8265-8271 (2009).</li><li>Shrestha, B., Nemes, P., 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=Ablation+and+Analysis+of+a+Single+Cell+by+Consecutive+Laser+Pulses.">Ablation and Analysis of a Single Cell by Consecutive Laser Pulses.</a> Applied Physics A. , (2010).</li></ol>

Hücre bir sitoplazma ve hücre duvarı ya da membran çekme dayanımı, su içeriği LAESI-MS analizi sırasında tek hücre ablasyon düzenleyen kritik faktörlerdir. Lazer fluens farklı hücre tiplerinin ablasyon eşikleri ulaşmak için ayarlanmalıdır. Teslim lazer fluens, lazer darbe enerji ve fiber ucu ve hücre yüzeyi arasındaki mesafeye bağlıdır. Komşu hücreleri tek bir hücre analizi sırasında pertürbasyon en aza indirilmesi önemlidir. Ablasyon eşiğinde lazer enerjisi tutulması komşu hücreleri üzerindeki etkisini analiz sınırlar. Potansiyel uygulamalardan biri tek hücre MS kimyasal görüntüleme için doğal hacimsel piksel (vokseller) olarak kullanıyor. Yapay genellikle dikdörtgen bir ızgara üzerinde veri toplama ile karşılaştırıldığında, bir defada bir doku bir hücre kimyasal görüntüleme hücreleri arasındaki biyokimyasal etkileşimler mekansal organizasyon korumak için daha olasıdır.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Acetic acid</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>45727</td>
<td>&nbsp;</td>
<tr>
<td>Methanol</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>65548</td>
<td>&nbsp;</td>
<tr>
<td>1-methyl-2-pyrrolidinon</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>M79603</td>
<td>&nbsp;</td>
<tr>
<td>Water</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>39259</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

direct analysis of single cells by mass spectrometry at atmospheric pressure

Tek hücre Biyokimyasalların analizi, aynı hücre türleri bile kendi fizyolojik koşullar ve çevre ile etkileşim bağlı olarak heterojen bir biyokimyasal makyaj sergiler hücre metabolizması, hücre döngüsü, adaptasyon, hastalık durumları, vb anlamak için önemlidir. Kütle spektrometresi (MS), tek hücre Biyomoleküllerin analizi için kullanılan geleneksel yöntemlerin, kapsamlı bir numune hazırlama güveniyor. Kendi doğal ortamı ve geniş numune işleme hücrelerin çıkarılması hücresel bileşimindeki değişikliklere yol açabilir. Ortam iyonizasyon yöntemleri kendi doğal ortamında ve geniş numune hazırlama olmadan numunelerin analizi sağlar. 1 2.94 mikron dalga boyunda biyolojik maddeler (orta-IR) orta kızılötesi lazer ablasyon dayalı teknikler su ani uyarma kullanan faz sonuçları patlama 2 lazer ablasyon elektrosprey iyonizasyon (LAESI) ve atmosferik basınç kızılötesi matris yardımlı lazer desorpsiyon iyonizasyon (AP IR-MALDI) olarak orta-IR lazer radyasyonu dayalı Ortam iyonizasyon teknikleri, başarılı bir şekilde doğrudan analiz etmek için su yeteneğini göstermişlerdir atmosferik basınç zengin doku ve biofluids LAESI, çoğunlukla iyonları üretmek için yüksek ücret elektrosprey damlacıkları ile örnek pekişen nötr partikül madde oluşur orta-IR lazer ablasyon tüy 3-11 . Son zamanlarda, tek hücre orta-IR ablasyon kazınmış bir fiber üzerinden orta-IR radyasyon sunarak yapıldı. Bu ablasyon üretilen tüy LAESI-MS tek hücreleri çeşitli metabolitlerin analizi sağlayan bir elektrosprey postionized 12 Bu makale LAESI-MS kullanarak tek bir hücre analizi için detaylı bir protokol açıklar . Sunulan video Allium cepa ampul cilde tek bir epidermal hücre analizi göstermektedir. Sisteminin şematik Şekil 1&#39;de gösterilmiştir. Hücreden tek bir hücre ablasyon ve bir LAESI kütle spektrumu bir temsilci örnek Şekil 2&#39;de verilmektedir.

Watch this Scientific Journal Video about Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure at JoVE.com

Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure

Lazer ablasyon elektrosprey iyonizasyon (LAESI) kütle spektrometresi hücreleri örnek bilenmiş bir fiber optik kablo kullanılarak, atmosferik basınç, bitki ve hayvan hücreleri üzerinde kütle spektrometresi ile tek bir hücre analizi yapılır.

Doğrudan Atmosferik Basınç Kütle Spektrometresi Tek Hücre Analizi

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell ...

direct-analysis-single-cells-mass-spectrometry-at-atmospheric

Research

JoVE Journal

Biology

15.9K Görüntüleme.  George Washington University. Lazer ablasyon elektrosprey iyonizasyon (LAESI) kütle spektrometresi hücreleri örnek bilenmiş bir fiber optik kablo kullanılarak, atmosferik basınç, bitki ve hayvan hücreleri üzerinde kütle spektrometresi ile tek bir hücre analizi yapılır.

Video: Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure

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

Analyzing Large Protein Complexes by Structural Mass Spectrometry

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an array of dynamic, multi-protein complexes1. To gain a mechanistic understanding of the various cellular processes, it is crucial to determine the structure of such protein complexes, and reveal how their structural organization dictates their function. Many aspects of multi-protein complexes are, however, difficult to characterize, due to their heterogeneous nature, asymmetric structure, and dynamics. Therefore, new approaches are required for the study of the tertiary levels of protein organization.
One of the emerging structural biology tools for analyzing macromolecular complexes is mass spectrometry (MS)2-5. This method yields information on the complex protein composition, subunit stoichiometry, and structural topology. The power of MS derives from its high sensitivity and, as a consequence, low sample requirement, which enables examination of protein complexes expressed at endogenous levels. Another advantage is the speed of analysis, which allows monitoring of reactions in real time. Moreover, the technique can simultaneously measure the characteristics of separate populations co-existing in a mixture. 
Here, we describe a detailed protocol for the application of structural MS to the analysis of large protein assemblies. The procedure begins with the preparation of gold-coated capillaries for nanoflow electrospray ionization (nESI). It then continues with sample preparation, emphasizing the buffer conditions which should be compatible with nESI on the one hand, and enable to maintain complexes intact on the other. We then explain, step-by-step, how to optimize the experimental conditions for high mass measurements and acquire MS and tandem MS spectra. Finally, we chart the data processing and analyses that follow. Rather than attempting to characterize every aspect of protein assemblies, this protocol introduces basic MS procedures, enabling the performance of MS and MS/MS experiments on non-covalent complexes. Overall, our goal is to provide researchers unacquainted with the field of structural MS, with knowledge of the principal experimental tools.

Mass spectrometry has proven to be a valuable tool for analyzing large protein complexes. This method enables insights into the composition, stoichiometry and overall architecture of multi-subunit assemblies. Here, we describe, step-by-step, how to perform a structural mass spectrometry analysis, and characterize macromolecular structures.

Yapısal Kütle Spektrometresi büyük protein kompleksleri Analiz

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an ...

T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. The speed by which the ions traverse the drift region depends on their size: large ions will experience a greater number of collisions with the background inert gas (usually N2) and thus travel more slowly through the IM device than those ions that comprise a smaller cross-section. In general, the time it takes for the ions to migrate though the dense gas phase separates them, according to their collision cross-section (&Omega;). 
Recently, IM spectrometry was coupled with mass spectrometry and a traveling-wave (T-wave) Synapt ion mobility mass spectrometer (IM-MS) was released. Integrating mass spectrometry with ion mobility enables an extra dimension of sample separation and definition, yielding a three-dimensional spectrum (mass to charge, intensity, and drift time). This separation technique allows the spectral overlap to decrease, and enables resolution of heterogeneous complexes with very similar mass, or mass-to-charge ratios, but different drift times. Moreover, the drift time measurements provide an important layer of structural information, as &Omega; is related to the overall shape and topology of the ion. The correlation between the measured drift time values and &Omega; is calculated using a calibration curve generated from calibrant proteins with defined cross-sections1.
The power of the IM-MS approach lies in its ability to define the subunit packing and overall shape of protein assemblies at micromolar concentrations, and near-physiological conditions1. Several recent IM studies of both individual proteins2,3 and non-covalent protein complexes4-9, successfully demonstrated that protein quaternary structure is maintained in the gas phase, and highlighted the potential of this approach in the study of protein assemblies of unknown geometry. Here, we provide a detailed description of IMS-MS analysis of protein complexes using the Synapt (Quadrupole-Ion Mobility-Time-of-Flight) HDMS instrument (Waters Ltd; the only commercial IM-MS instrument currently available)10. We describe the basic optimization steps, the calibration of collision cross-sections, and methods for data processing and interpretation. The final step of the protocol discusses methods for calculating theoretical &Omega; values. Overall, the protocol does not attempt to cover every aspect of IM-MS characterization of protein assemblies; rather, its goal is to introduce the practical aspects of the method to new researchers in the field.

Ion mobility-mass spectrometry is an emerging gas-phase technology that separates ions, based on their collision cross-section and mass. The method provides three-dimensional information on the overall topology and shape of protein complexes. Here, we outline a basic procedure for instrument setting and optimization, calibration of drift times, and data interpretation.

T-dalgası İyon Hareketlilik-Kütle Spektrometresi: Protein Karmaşık Analiz Temel Deneysel Prosedürler

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. ...

Atmospheric-pressure Molecular Imaging of Biological Tissues and Biofilms by LAESI Mass Spectrometry

Ambient ionization methods in mass spectrometry allow analytical investigations to be performed directly on a tissue or biofilm under native-like experimental conditions. Laser ablation electrospray ionization (LAESI) is one such development and is particularly well-suited for the investigation of water-containing specimens. LAESI utilizes a mid-infrared laser beam (2.94 &mu;m wavelength) to excite the water molecules of the sample. When the ablation fluence threshold is exceeded, the sample material is expelled in the form of particulate matter and these projectiles travel to tens of millimeters above the sample surface. In LAESI, this ablation plume is intercepted by highly charged droplets to capture a fraction of the ejected sample material and convert its chemical constituents into gas-phase ions. A mass spectrometer equipped with an atmospheric-pressure ion source interface is employed to analyze and record the composition of the released ions originating from the probed area (pixel) of the sample. A systematic interrogation over an array of pixels opens a way for molecular imaging in the microprobe analysis mode. A unique aspect of LAESI mass spectrometric imaging is depth profiling that, in combination with lateral imaging, enables three-dimensional (3D) molecular imaging. With current lateral and depth resolutions of ~100 &mu;m and ~40 &mu;m, respectively, LAESI mass spectrometric imaging helps to explore the molecular structure of biological tissues. Herein, we review the major elements of a LAESI system and provide guidelines for a successful imaging experiment.

Laser ablation electrospray ionization (LAESI) is an atmospheric-pressure ion source for mass spectrometry. In the imaging mode, a mid-infrared laser probes the distributions of molecules across a tissue section or a biofilm. This technique presents a new approach for diverse bioanalytical studies carried out under native experimental conditions.

Atmosferik basınç Moleküler Görüntüleme LAESI Kütle Spektrometresi Biyolojik Dokular ve Biyofilmler

Ambient ionization methods in mass spectrometry allow analytical investigations to be performed directly on a tissue or biofilm under native-like ...

Amide Hydrogen/Deuterium Exchange &amp; MALDI-TOF Mass Spectrometry Analysis of Pak2 Activation

Amide hydrogen/deuterium exchange (H/D exchange) coupled with mass spectrometry has been widely used to analyze the interface of protein-protein interactions, protein conformational changes, protein dynamics and protein-ligand interactions. H/D exchange on the backbone amide positions has been utilized to measure the deuteration rates of the micro-regions in a protein by mass spectrometry1,2,3. The resolution of this method depends on pepsin digestion of the deuterated protein of interest into peptides that normally range from 3-20 residues. Although the resolution of H/D exchange measured by mass spectrometry is lower than the single residue resolution measured by the Heteronuclear Single Quantum Coherence (HSQC) method of NMR, the mass spectrometry measurement in H/D exchange is not restricted by the size of the protein4. H/D exchange is carried out in an aqueous solution which maintains protein conformation. We provide a method that utilizes the MALDI-TOF for detection2, instead of a HPLC/ESI (electrospray ionization)-MS system5,6. The MALDI-TOF provides accurate mass intensity data for the peptides of the digested protein, in this case protein kinase Pak2 (also called &gamma;-Pak). Proteolysis of Pak 2 is carried out in an offline pepsin digestion. This alternative method, when the user does not have access to a HPLC and pepsin column connected to mass spectrometry, or when the pepsin column on HPLC does not result in an optimal digestion map, for example, the heavily disulfide-bonded secreted Phospholipase A2 (sPLA2). Utilizing this method, we successfully monitored changes in the deuteration level during activation of Pak2 by caspase 3 cleavage and autophosphorylation7,8,9.

MALDI-TOF mass spectrometry was successfully utilized to monitor the amide hydrogen/deuterium exchange in protein kinase Pak2 activation.

Pak2 Etkinleştirme Amid Hidrojen / Döteryum Alım Satım &amp; MALDI-TOF Kütle Spektrometre Analiz

Amide hydrogen/deuterium exchange (H/D exchange) coupled with mass spectrometry has been widely used to analyze the interface of protein-protein ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Ününüz Aşağıdaki Tek Hücre Düzeyinde Küresel RNA Sentezi Analizi

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of cell proliferation, survival, migration, etc. The ECM plays important roles in development and in diverse pathologies including cardio-vascular and musculo-skeletal diseases, fibrosis, and cancer. Thus, characterizing the composition of ECMs of normal and diseased tissues could lead to the identification of novel prognostic and diagnostic biomarkers and potential novel therapeutic targets. However, the very nature of ECM proteins (large in size, cross-linked and covalently bound, heavily glycosylated) has rendered biochemical analyses of ECMs challenging. To overcome this challenge, we developed a method to enrich ECMs from fresh or frozen tissues and tumors that takes advantage of the insolubility of ECM proteins. We describe here in detail the decellularization procedure that consists of sequential incubations in buffers of different pH and salt and detergent concentrations and that results in 1) the extraction of intracellular (cytosolic, nuclear, membrane and cytoskeletal) proteins and 2) the enrichment of ECM proteins. We then describe how to deglycosylate and digest ECM-enriched protein preparations into peptides for subsequent analysis by mass spectrometry.

This protocol describes a procedure for enriching ECM proteins from tissues or tumors and deglycosylating and digesting the ECM-enriched preparations into peptides to analyze their protein composition by mass spectrometry.

Kütle Spektrometresi Analizi için Peptitler içine Dokular ve Sindirim gelen Ekstrasellüler Matriks Proteinleri zenginleştirilmesi

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of ...

Analysis of LINE-1 Retrotransposition at the Single Nucleus Level

Long interspersed nuclear element-1 (Line-1 or L1) accounts for approximately 17% of the DNA present in the human genome. While the majority of L1s are inactive due to 5' truncations, ~80-100 of these elements remain retrotransposition competent and propagate to different locations throughout the genome via RNA intermediates. While older L1s are believed to target AT rich regions of the genome, the chromosomal targets of newer, more active L1s remain poorly defined. Here we describe fluorescence in situ hybridization (FISH) methodology that can be used to track patterns of L1 insertion and rates of ectopic L1 incorporation at the single nucleus level. In these experiments, fluorescein isothiocyanate/cyanine-3 (FITC/CY3) labeled neomycin probes were employed to track L1 retrotransposition in vitro in HepG2 cells stably expressing ectopic L1. This methodology prevents errors in the estimation of rates of retrotransposition posed by toxicity and account for the occurrence of multiple insertions into a single nucleus.

Here we employ FISH methodology to track LINE-1 retrotransposition at the single nuclei level in chromosome spreads of HepG2 cell lines stably expressing synthetic LINE-1.

Tek Çekirdek Düzeyde HAT-1 Retrotransposition Analizi

Long interspersed nuclear element-1 (Line-1 or L1) accounts for approximately 17% of the DNA present in the human genome. While the majority of L1s are ...

Applications of the Single-probe: Mass Spectrometry Imaging and Single Cell Analysis under Ambient Conditions

Mass spectrometry imaging (MSI) and in-situ single cell mass spectrometry (SCMS) analysis under ambient conditions are two emerging fields with great potential for the detailed mass spectrometry (MS) analysis of biomolecules from biological samples. The single-probe, a miniaturized device with integrated sampling and ionization capabilities, is capable of performing both ambient MSI and in-situ SCMS analysis. For ambient MSI, the single-probe uses surface micro-extraction to continually conduct MS analysis of the sample, and this technique allows the creation of MS images with high spatial resolution (8.5 &#181;m) from biological samples such as mouse brain and kidney sections. Ambient MSI has the advantage that little to no sample preparation is needed before the analysis, which reduces the amount of potential artifacts present in data acquisition and allows a more representative analysis of the sample to be acquired. For in-situ SCMS, the single-probe tip can be directly inserted into live eukaryotic cells such as HeLa cells, due to the small sampling tip size (&lt; 10 &#181;m), and this technique is capable of detecting a wide range of metabolites inside individual cells at near real-time. SCMS enables a greater sensitivity and accuracy of chemical information to be acquired at the single cell level, which could improve our understanding of biological processes at a more fundamental level than previously possible. The single-probe device can be potentially coupled with a variety of mass spectrometers for broad ranges of MSI and SCMS studies.

Here, we present protocols to perform both ambient mass spectrometry imaging (MSI) of tissues and in-situ live single cell MS (SCMS) analysis using the single-probe, which is a miniaturized multifunctional device for MS analysis.

Tek prob Uygulamalar: Ortam Koşullarında Kütle Spektrometre Görüntüleme ve Tek Hücre Analizi

Mass spectrometry imaging (MSI) and in-situ single cell mass spectrometry (SCMS) analysis under ambient conditions are two emerging fields with great ...

Sample Preparation for Mass-spectrometry-based Proteomics Analysis of Ocular Microvessels

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly expanded our understanding of certain diseases. Mass spectrometry (MS)-based proteomics has emerged as a powerful tool to unravel alterations in the molecular mechanisms and protein signaling pathways in the vascular beds in health and disease. However, sample preparation steps prior to MS analyses are crucial to obtain reproducible results and in-depth elucidation of the complex proteome. This is particularly important for preparation of ocular microvessels, where the amount of sample available for analyses is often limited and thus, poses a challenge for optimum protein extraction. This article endeavors to provide an efficient, rapid and robust protocol for sample preparation from an exemplary retrobulbar ocular vascular bed employing the porcine short posterior ciliary arteries. The present method focuses on protein extraction procedures from both the supernatant and pellet of the sample following homogenization, sample cleaning with centrifugal filter devices prior to one-dimensional gel electrophoresis and peptide purification steps for label-free quantification in a liquid chromatography-electrospray ionization-linear ion trap-Orbitrap MS system. Although this method has been developed specifically for proteomics analyses of ocular microvessels, we have also provided convincing evidence that it can also be readily employed for other tissue-based samples.

Proteome characterization of ocular microvascular beds is pivotal for in-depth understanding of many ocular pathologies in humans. This study demonstrates an effective, rapid, and robust method for protein extraction and sample preparation from small blood vessels employing the porcine short posterior ciliary arteries as model vessels for mass-spectrometry-based proteomics analyses.

Oküler yüzdeki kütle spektrometresi esaslı proteomik analiz için numune hazırlama

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly ...