Bu eser kısmen Nihon ilaç Üniversitesi Araştırma Bursu Nihon ilaç Üniversitesi (2016) ve MEXT KAKENHI Grant numarası JP17854179 tarafından desteklenmiştir.

Hayvan deneysel protokoller Nihon ilaç Üniversitesi Etik Komitesi tarafından onaylanmış ve bakım ve kullanım Laboratuvar hayvanları Nihon ilaç Üniversitesi kılavuzlarınıza uygun olarak yürüttü. Protokol doku özleri ICR fareler türetilmiş için ayarlandı. ICR fareler Nippon SLC Ltd. (Shizuoka, Japonya) satın alınan
1. Iminobiotin etiketli substrat hazırlanması
9-fluorenylmethyloxycarbonyl grubu (Fmoc) kullanarak katı fazlı sentez substrat uğrar-11açıklandığı gibi amino asitler korumalı. Tablo 2 , istenen amino asit ve sentez aşamasına bağlı olarak kullanılan Fmoc bileşik belirlemek için kullanın.
<ol><li>Fmoc-NH-SAL reçine 0.2 g N, N-dimethylformamide (DMF) bir sentez sütunda 10 mL ile açgözlü.</li>
	<li>5 mL % 30 Piperodin DMF içinde ekleyin. Fmoc-grup ayırmak için 10 dakika için çözüm rock.</li>
	<li>
Kontrol Fmoc bölünme trinitrobenzenesulfonic asit (TNBS) ile test12: TNBS DMF ve diisopropylethylamine (DIPEA) 10 x 75 mm2 cam tüp sonra cam reçine az miktarda aktarmak için 10 µL eklemek 10 µL % 1'lik tüp. Fmoc i ciddi reçine turuncu renk geliştirmeniz gerekir.
	<ol>
<li>Bu sonuç olumsuz ise 1.2-1.3 adımları yineleyin.</li>
	</ol>
</li>
	<li>Reçine üç kez DMF 5 mL ile yıkayın.</li>
	<li>
5 mL Fmoc bahçedeki 0,3 mmol içeren DMF de tablo 2'de (Etap 1 kullanılan bileşik ile başlayan), O-(6-Chloro-1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HCTU), 1-0,3 mmol 0,3 mmol listelenen Ekle Hidroksi-1H-benzotriazol, monohidrat (HOBt) ve diisopropylethylamine (DIPEA) 0.6 mmol. Reçine ile çift 30 dk için çözüm rock.
	<ol>
<li>Bu adım TNBS testi ile kontrol edin. Sonuç pozitif çıkarsa, bu tepki değil yeterince ilerlemiştir ve bu adımı tekrarlanması gerekir anlamına gelir.</li>
	</ol>
</li>
	<li>Reçine üç kez DMF 5 mL ile yıkayın.</li>
	<li>Uzama tepki adımları yineleyerek 1.2-1.6, değiştirme Tablo 2göre 1.5 adımda kullandığınız Fmoc bileşik yineleyin.</li>
	<li>% 2.5 su, 1 mL ekleyin %1 triisopropylsilane (TIS) ve % 2.5 ethanedithiol (EDT) trifluoroacetic asit peptidlerin reçine ve koruma grupları ayırmak için. Rock 60 dk için çözüm.</li>
	<li>Filtre ve filtrate 50 mL tüp içinde toplamak.</li>
	<li>40 mL soğuk dietil etanol ve mağaza gecede-20 ° C'de ekleyin</li>
	<li>-20 ° C'de 30 dakika süreyle santrifüj 4.000 x g Pelet toplamak.</li>
	<li>3 h için bir desiccator dietil eter kaldırmak için kullanın.</li>
	<li>% 50 Asetonitril 1 mL su ekleyin ve ham peptidler geçiyoruz. Trifluoroacetic asit kaldırmak için çözüm lyophilize. Bu adımı birkaç kez yineleyin.</li>
	<li>
C18 kartuş sütun kullanarak peptidler arındırmak.
	<ol>
<li>3 mL Asetonitril (ACN) kullanarak C18 kartuş sütunundaki reçine etkinleştirin.</li>
		<li>5 mL %5 ACN, % 0,1 TFA H2O. ile equilibrate</li>
		<li>Ham sentetik peptidler C18 kartuş sütuna yerleştirin.</li>
		<li>10 mL %5 ACN, % 0,1 TFA H2o yıkayın</li>
		<li>%60 3 mL örnekleriyle elute ACN, % 0,1 TFA H2O.</li>
	</ol>
</li>
	<li>
Peptidler MALDI-TOF Kütle spektrometresi kullanarak kendi moleküler ağırlığı ölçerek kontrol edin.
	<ol>
<li>Eluent bir hedef plaka üzerinde 1 µL pipet. Hemen % 0,1 trifluoroacetic asit % 50 Asetonitril doymuş α-cyano-4-hydroxycinnamic asit (CHCA) çözüm ekleyin</li>
		<li>Karışımı air-drying tarafından kristalize.</li>
		<li>Araç üreticisinin protokolüne göre örneklerin kitle spectra elde etmek.<ol>
<li>El ile ayarlamak için olumlu bir MALDI-TOF Kütle Spektrometre modu yansıtmak.</li>
			<li>CHCA kullanarak kütle spektrometre kalibre, bradikinin parçası 1-7 (monoisotopic molekül ağırlığı 756.3997 Da =) ve Anjiyotensin II (monoisotopic molekül ağırlığı 1,045.5423 Da =).</li>
			<li>Sentetik peptidler kitle spectra alacak ve kuramsal moleküler ağırlığı ile karşılaştırarak değerlendirmek.</li>
		</ol>
</li>
	</ol>
</li>
</ol>2. doku özleri hazırlanması
<ol><li>Erkek ICR fareler bir isoflurane inhalasyon odasında anestezi ve anestezi derinliği ayak tutam ile doğrulayın. Fareler tarafından servikal çıkığı ötenazi.</li>
	<li>Makas kullanarak, cilt xiphoid süreci kadar uzanan aracılığıyla bir karın ensizyon olun.</li>
	<li>Diyafram kesin ve tüm akciğer doku toplamak. Doku küçük parçalar halinde kesin.</li>
	<li>Doku buz gibi 50 mM Tris arabelleğe alınmış serum fizyolojik (pH 7,4) ile üç kez yıkayın.</li>
	<li>Doku ve bir 12 x 75 mm2 cam tüp 100 mg dokuların aktarmak tartın.</li>
	<li>Ayıklama arabellek (50 mM Tris-arabellek, % 0,1 poli (oxyethylene) octylphenyl eter içeren pH 7.4,) 0.3 mL ekleyin.</li>
	<li>
Bir karıştırma homogenizer kullanarak akciğer doku örnekleri lunaparkçı.
	<ol>
<li>Buz üzerinde örnekleri ne yapıyorsun?</li>
		<li>Örnekleri için 30 20.000 rpm'de homojenize bir karıştırıcıda s. Tamamen homojen olduğu kadar bu adımı yineleyin. Not: sıcaklık artışı önlemek amacıyla sürekli olarak daha fazla 1 dakikadır homojenize değil.</li>
	</ol>
</li>
	<li>Homogenate 1000 µL 1,5 mL tüp ve santrifüj 10.000 x g 4 ° C'de, 5 min için transfer</li>
	<li>500 µL süpernatant ile yeni bir 1,5 mL plastik tüp aktarın.</li>
	<li>Örnek 10 µL protein konsantrasyonu, bicinchoninic asit (BCA) gibi yöntemler veya Coomassie parlak mavi (CBB) yöntemini kullanarak belirlemek için kullanın.</li>
	<li>Donma önlemek için örnekleri % 50 gliserol son bir konsantrasyon için % 80 gliserol ekleyin.</li>
	<li>-80 ° C'de örnekleri daha fazla kullanılmasını kadar saklayın.</li>
</ol>3. sindirim yüzeylerde modeli proteaz ve doku ayıklar
<ol><li>
0.1 µg/µL N-tosyl-L-Fenilalanin chloromethyl keton (TPCK) 10 µL eklemek-tripsin, 0.1 µg/µL Staphylococcus aureus V8 proteaz veya 10 µg/µL doku özler bir sindirim arabellek 890 µL içine.
	<ol>
<li>Proteaz özgüllük bağlı olarak pH farkı açıklamak için pH 3, 5, 7 ve 9 ayarlanabilir arabelleklerini kullan. Sindirim arabellek bileşimi Tablo 3' te gösterilmiştir.</li>
		<li>Etkin olmayan proteaz doku özleri, 10 dakikadır kuluçka kaynar su (veya ısıtma 100 ° c) tarafından hazırlanan kullanarak bir negatif kontrol yerine getirir.</li>
	</ol>
</li>
	<li>İminobiotin etiketli yüzeylerde 10 µL ekleyin (dimetil sülfoksit (DMSO), nihai toplama çözünmüş 100 pmol/10 µL =).</li>
	<li>37 ° C'de iminobiotin etiketli yüzeylerde sindirmek için 3 saat kuluçkaya.</li>
	<li>Kuluçka sonra yüzeylerde kaynar su ile (veya ısıtma 100 ° c) sindirim 10 dakikadır durdurmak.</li>
	<li>Sindirilir yüzeylerde, 10.000 x g 4 ° C'de 5 dakika santrifüj kapasitesi</li>
	<li>Süpernatant yeni 1,5 mL tüp içine aktarın.</li>
</ol>4. Iminobiotin etiketli substrat saflaştırılması
<ol><li>
50 µL % 50 streptavidin sepharose boncuk 1 M Tris-tampon (pH 9) kapsayan %0,1 poly(oxyethylene) octylphenyl eter içinde ekleyin.
	<ol>
<li>Çözüm pH 9 olması için pH test kağıt kullanın. Çözüm pH düşükse, pH yaklaşık 9 1 M Tris-tampon (pH 9) kapsayan %0,1 poly(oxyethylene) octylphenyl eter ekleyerek ayarlayın. Bu adım önemlidir, iminobiotin etiketli substrat streptavidin için bağlama karıştırmak gibi.</li>
	</ol>
</li>
	<li>Gecede 4 ° C'de sürekli nazik iminobiotin etiketli yüzeyler için streptavidin bağlamak için bir rotator tekerlek üzerinde karıştırma ile kuluçkaya. Boncuk kuluçka asılı kalması gerekir.</li>
	<li>10.000 x g 4 ° C'de, 1 dk santrifüj Süpernatant kaldırın.</li>
	<li>
Boncuk aşağıdaki gibi beş kez yıkama:
	<ol>
<li>Bir yıkama arabellek (50 mM Tris-tampon, pH 9) 1.000 µL boncuk için ekleyin.</li>
		<li>4 ° C'de sürekli nazik bir rotator tekerlek üzerinde karıştırma ile 10 dakika yıkayın. Boncuk kuluçka asılı kalması gerekir.</li>
		<li>10.000 x g 4 ° c de 1 dk santrifüj kapasitesi ve süpernatant kaldırın.</li>
	</ol>
</li>
	<li>
100 µL % 0,2 trifluoroacetic asit için boncuk ekleyin.
	<ol>
<li>Çözüm pH 2 olması için pH test kağıt kullanın. Çözüm pH yüksek ise, için pH 2 aşağıda %1 trifluoroacetic asit ekleyerek ayarlayın.</li>
	</ol>
</li>
	<li>Sürekli nazik bir rotator tekerlek üzerinde karıştırma ile Oda sıcaklığında 30 dk için kuluçkaya.</li>
	<li>10.000 x g 4 ° c de 1 dk santrifüj kapasitesi, sonra yeni bir 1,5 mL tüp süpernatant transfer.</li>
</ol>5. örnek hazırlama ve matris yardımlı Lazer Desorpsiyon/iyonlaşma uçuş zaman kütle spektrometresi (MALDI-TOF Kütle spektrometresi)
Not: Aşağıdaki adımlarda tüm çözümleri ile yüksek performanslı sıvı kromatografi (HPLC) hazırlanmalıdır-sınıf veya amino asit sıralama-grade su.
<ol><li>Asetonitril etkinleştirmek için C18 reçine üzerine 500 µL pipet. Kullanılan Asetonitril kaldırın. Bu üç kez tekrarlayın.</li>
	<li>C18 reçine 100 µL % 0,1 trifluoroacetic asit ile equilibrate. Eluent kaldırın. Bu üç kez tekrarlayın.</li>
	<li>Örnekleri yüklemek için bir pipet kullanarak reçine örnekleri bağlayın.</li>
	<li>Reçine 20 µL % 0,1 trifluoroacetic asit ile yıkayın. Bu beş kez tekrarlayın.</li>
	<li>Örnekleri 3 µL % 60 Asetonitril %0,1 trifluoroacetic asit ile elute. Eluent MALDI-TOF Kütle spektrometresi için hedef tabakta pipette. Hemen doymuş α-cyano-4-hydroxycinnamic asit (CHCA) çözüm % 50 Asetonitril %0,1 trifluoroacetic asit ekleyin.</li>
	<li>Karışımı air-drying tarafından kristalize.</li>
	<li>
Araç üreticisinin protokolüne göre örneklerin kitle spectra elde etmek.
	<ol>
<li>El ile ayarlamak için olumlu bir MALDI-TOF Kütle Spektrometre modu yansıtmak.</li>
		<li>CHCA kullanarak kütle spektrometre kalibre, bradikinin parçası 1-7 (monoisotopic molekül ağırlığı 756.3997 Da =) ve Anjiyotensin II (monoisotopic molekül ağırlığı 1,045.5423 Da =).</li>
		<li>Örnekleri yakın kitle spectra 700 üzerinden 900 savcıya biotin etiketli yüzeylerde cleaved form için dikkat, kitle spectra elde etmek.</li>
	</ol>
</li>
	<li>Tablo 1kullanarak algılanan doruklarına tanımlayın. Uygun moleküler ağırlık tespiti C-terminus ilgili amino asit, bölünme gösterir.</li>
</ol>

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+detection+of+casein+zymography+of+matrix+metalloproteinases.">Enhanced detection of casein zymography of matrix metalloproteinases.</a> Anal Biochem. 224 (1), 434-435 (1995).</li><li>Erlanger, B. F., Kokowsky, N., Cohen, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+preparation+and+properties+of+two+new+chromogenic+substrates+of+trypsin.">The preparation and properties of two new chromogenic substrates of trypsin.</a> Arch Biochem Biophys. 95, 271-278 (1961).</li><li>Smith, R. E., Bissell, E. R., Mitchell, A. R., Pearson, K. 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C., 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=Profiling+constitutive+proteolytic+events+in+vivo.">Profiling constitutive proteolytic events in vivo.</a> Biochem J. 407 (1), 41-48 (2007).</li><li>Biniossek, M. L., 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=Identification+of+protease+specificity+by+combining+proteome-derived+peptide+libraries+and+quantitative+proteomics.">Identification of protease specificity by combining proteome-derived peptide libraries and quantitative proteomics.</a> Mol Cell Proteomics. 15 (7), 2515-2524 (2016).</li><li>Yamamoto, H., Saito, S., Sawaguchi, Y., Kimura, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+protease+specificity+using+biotin-labeled+substrates.">Identification of protease specificity using biotin-labeled substrates.</a> Open Biochem J. 11, 27-35 (2017).</li><li>Hofmann, K., Wood, S. W., Brinton, C. C., Montibeller, J. A., Finn, F. 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Yazarlar hiçbir çıkar çatışması beyan ederim.

Bu iletişim kuralı MALDI-TOF Kütle spektrometresi proteaz özgüllük doku özler elde edilen ham örneklerinde tanımlamak için kullanır ve kolayca birden çok örnek işleme için ölçekli. Özellikle, pH substrat özgüllüğü bir değişiklik neden manipüle.
Yaygın Biyokimya onun bağlama özgüllüğü nedeniyle kullanılmıştır, avidin-biotin karmaşık (ABC) yöntemi, bizim iletişim kuralında kullanılmıştır. ABC güçlü yakınlığı nedeniyle, avidin biotin için bağ...

Proteaz proteinler fizyon tarafından fizyolojik süreçleri düzenleyen ve belirli zamanlarda ve yerlerde proteaz aktivitesinin başlangıcında düzenlenmiş1. Bu nedenle ifade ve yerel olarak düzenlenmiştir dokuların aktivite tanımlama ve proteaz algılamak için yeni bir yöntem geliştirmek için önemlidir. Önerilen yöntem bu çalışma proteaz özgüllük proteaz tespit için paralel olarak tanımlamak amaçlamaktadır.
Proteaz özgüllük algılamak için bazı yöntemler vardır. Azocasein yönteminin kullanımı ile proteaz2 algılanması için iyi bilinen ama onun yetenek daha fazla bilgi almak için sınırlıdır. Zymography, proteaz3molekül ağırlığı belirlemek için kullanılan başka bir kapsamlı proteaz algılama yöntemi olmakla birlikte, substrat özgüllüğü incelemek için kullanılamaz. Proteaz özgüllük spektrometrik deneyleri, p-nitroanilide4gibi yüzeylerde kullanarak ve Fluorometrik deneyleri, coumarin yüzeylerde5 veya floresan rezonans enerji transferi (FRET) deneyleri6kullanarak tarafından belirlenebilir. Bu yöntemler tek bir kuyuda bulunan yüzeylerde tek-özgüllük algılanmasını etkinleştirin. Bu özler birden çok proteaz içeren çalışmanın, doku özleri proteaz özgüllük çeşitli koşullar altında incelenir. Proteaz aktivitesi pH, koenzimler, prostetik gruplar ve iyon gücü de dahil olmak üzere çeşitli faktörler tarafından düzenlenmiştir. Son zamanlarda, proteomik tabanlı yöntemleri proteaz özgüllük tanımlamak için kullanılmıştır; Örneğin, Biniossek ve arktarafından bildirilen yöntemi. 7 Proteom substrat özgüllüğü bile içinde ham özleri belirlemek için kullanır ve birden çok amino asit dizileri8tanımak proteaz bölünme etkinliğinin doğru belirlenmesi sağlar. Ancak, bu yöntem çok sayıda örnekleri analiz için uygun değildir. Buna ek olarak, bizim Yöntem birden çok örneği aynı anda işleme ve kullanım Matrix-Assisted Lazer Desorpsiyon/iyonlaşma sağlar saat uçuş (MALDI-TOF) örnek analiz için kütle spektrometresi i ciddi, hızlı ve kolay algılama kolaylaştırır yüzeylerde.
Bu kağıt proteaz özgüllük benzersiz yüzeylerde molekül ağırlığı ölçmek için MALDI-TOF Kütle spektrometresi kullanılarak değerlendirmek için bir yöntem özetliyor. Onların kuramsal moleküler ağırlıkları birlikte i ciddi yüzeylerde moleküler formları Şekil 1 ve Tablo 1' de gösterilmiştir. Önceki çalışmalarda polyglycine çubukları ve biotin9içeren yüzeylerde kullanılan; Ancak, polyglycine sıra glisin sıra tanımak enzimler tarafından i ciddi çünkü bu yüzeylerde hatalı. Ayrıca, yüksek afinite avidin ve biotin arasında düşük kurtarma oranı için neden olabilir. Polietilen glikol (PEG), iminobiotin ve bölünme sitesi (Şekil 1) teşhis edebilecek bir amino asit oluşan benzersiz bir substrat sentezlenmiş Bu çalışmada bu sakıncaları üzerine geliştirmek için. Benzer moleküler ağırlık amino asitler arasında ayırımcılık için D-serin PEG spacer ve amino asit cleaved sitesinde arasında eklendi.
N-terminal substrat, benzeşim arıtma ham örneklerinden sağlayan iminobiotin ile etiketli oldu. İminobiotin yerine biotin kullanımı önemlidir; biotin avidin avidin için iminobiotin'ın ilgi pH tarafından değiştirilebilir iken hangi biotin etiketli yüzeylerde avidin reçine, düşük oranda düşük kurtarma sonuçlar ile güçlü bir ilgi vardır. İminobiotin etiketli yüzeylerde pH 4 aşağıdaki koşullarda avidin bültenleri iken Iminobiotin etiketli yüzeylerde avidin pH 9, yukarıdaki koşullarda bağlayacaksınız. Bu nedenle, iminobiotin benzeşme arıtma10için kullanıldı. Özetle, biz proteaz özgüllük benzersiz yüzeylerde kullanarak tespit için detaylı bir protokol tanımlamak.

<table><tbody><tr><td>Fmoc-NH-SAL Resin(-[2,4-Dimethoxyphenyl-N-(9-fluorenylmethoxycarbonyl)&lt;br/&gt;aminomethyl]phenoxy resin)</td><td>Watanabe Chemical Co., Ltd.</td><td>A00102</td><td></td></tr><tr><td>N,N-dimethylformamide (DMF)</td><td>Watanabe Chemical Co., Ltd.</td><td>A00185</td><td></td></tr><tr><td>piperidine</td><td>Watanabe Chemical Co., Ltd.</td><td>A00176</td><td></td></tr><tr><td>trinitrobenzenesulfonic acid (TNBS)</td><td>WAKO Chemical</td><td>209-1483</td><td></td></tr><tr><td>O-(6-Chloro-1H-benzotriazol-1-yl)-N,N,N&#039;,N&#039;-tetramethyluronium hexafluorophosphate (HCTU)</td><td>Watanabe Chemical Co., Ltd.</td><td>A00067</td><td></td></tr><tr><td>1-Hydroxy-1H-benzotriazole,monohydrate (HOBt)</td><td>Watanabe Chemical Co., Ltd.</td><td>A00014</td><td></td></tr><tr><td>diisopropylethylamine (DIPEA)</td><td>Watanabe Chemical Co., Ltd.</td><td>A00030</td><td></td></tr><tr><td>triisopropylsilane (TIS)</td><td>Watanabe Chemical Co., Ltd.</td><td>A00170</td><td></td></tr><tr><td>ethanedithiol (EDT)</td><td>Watanabe Chemical Co., Ltd.</td><td>A00057</td><td></td></tr><tr><td>trifluoroacetic acid (TFA)</td><td>Millipore</td><td>S6612278 403</td><td></td></tr><tr><td>acetonitrile</td><td>Kokusan Chemical</td><td>2153025</td><td></td></tr><tr><td>C18 cartridge column</td><td>Waters</td><td>WAT051910</td><td></td></tr><tr><td>poly(oxyethylene) octylphenyl ether</td><td>WAKO Chemical</td><td>168-11805</td><td>Triton X-100</td></tr><tr><td>mixing homogenizer</td><td>Kinematica</td><td>PT3100</td><td>polytron homogenizer</td></tr><tr><td>Coomassie Brilliant Blue (CBB) -G250</td><td>Nakarai Chemical</td><td>094-09</td><td></td></tr><tr><td>glycerol</td><td>Nakarai Chemical</td><td>170-18</td><td></td></tr><tr><td>N-tosyl-L-phenylalanine chloromethyl ketone (TPCK)-trypsin</td><td>Sigma-Aldrich</td><td>T1426</td><td></td></tr><tr><td>dimethyl sulfoxide (DMSO)</td><td>WAKO Chemical</td><td>043-07216</td><td></td></tr><tr><td>streptavidin sepharose</td><td>GE Healthcare</td><td>17-511-01</td><td></td></tr><tr><td>ZipTipC18</td><td>Millipore</td><td>ZTC18S096</td><td></td></tr><tr><td>&amp;alpha;-cyano-4-hydroxycinnamic acid (CHCA)</td><td>Sigma-Aldrich</td><td>C2020</td><td></td></tr><tr><td>MALDI-TOF mass spectrometry</td><td>Bruker Daltonics, Germany</td><td>autoflex</td><td></td></tr><tr><td>2-iminobiotin</td><td>Sigma-Aldrich</td><td>I4632</td><td></td></tr><tr><td>Fmoc-amino acids</td><td>Watanabe Chemical Co., Ltd.</td><td></td><td></td></tr></tbody></table>

the determination of protease specificity in mouse tissue extracts by maldi-tof mass spectrometry: manipulating ph to cause specificity changes

Proteaz protein harekete geçirmek/inactivation ve gıda sindirimi de dahil olmak üzere birçok biyolojik fonksiyonları var. Proteaz özgüllük tanımlayan proteaz işlevi ifşa için önemlidir. Bu çalışmada önerilen yöntem matris yardımlı Lazer Desorpsiyon/iyonlaşma kullanarak benzersiz yüzeylerde molekül ağırlığı ölçerek proteaz özgüllüğü belirler saat uçuş (MALDI-TOF) kütle spektrometresi. Cleaved site amino asitleri oluşur ve boşluk Ekleyici Polietilen glikol oluşur yüzeylerde iminobiotin, içerir. Cleaved substrat cleaved bir amino asit kullanarak benzersiz bir moleküler ağırlığı oluşturur. Bu yöntem yararları bu ham numuneleri kullanılarak bir tencereye yürütülen ve aynı zamanda birden fazla örnekleri değerlendirmek için uygundur biridir. Bu makalede, biz basit bir deneysel Yöntem doku çıkarma, sindirim yüzeylerde yerleşimini farklı pH koşullarında sindirim yüzeylerde arıtma örnekleri, içine dahil olmak üzere fare akciğer dokusundan elde örnekleri ile optimize tarif ve yüzeylerde moleküler ağırlık MALDI-TOF Kütle spektrometresi kullanılarak ölçümü. Özet olarak, bu teknik proteaz özgüllük ham örneklerinde kolayca birden çok örnek işleme için ölçekli MALDI-TOF Kütle spektrometresi kullanılarak doku özleri elde tanımlanması için izin verir.

Proteaz özgüllük modeli proteaz tarafından tanımlanması için yöntem değerlendirilmesi
Bu sistemin verimliliği TPCK-tripsin ve V8 proteaz olan substrat özelliklerine elde edilmiştir, kullanılarak değerlendirilmiştir. TPCK-tripsin ve V8 proteaz amino asit dizisi lizin ve arginin kalıntılarının C terminalinde ve carboxy yanında aspartik asit ve glutamik asit kalıntıları eklenmesi, sırasıyla ayırmak için tahm...

Watch this Scientific Journal Video about The Determination of Protease Specificity in Mouse Tissue Extracts by MALDI-TOF Mass Spectrometry: Manipulating PH to Cause Specificity Changes at JoVE.com

The Determination of Protease Specificity in Mouse Tissue Extracts by MALDI-TOF Mass Spectrometry: Manipulating PH to Cause Specificity Changes

Proteaz özgüllük fare doku belirlenmesi MALDI-TOF Kütle spektrometresi tarafından ayıklar: PH özgüllük değişiklikler neden manipüle

Burada, proteaz özgüllük ham fare doku belirlemek için bir protokol MALDI-TOF spektrometresi kullanılarak özleri mevcut.

Proteases have several biological functions, including protein activation/inactivation and food digestion. Identifying protease specificity is important ...

the-determination-protease-specificity-mouse-tissue-extracts-maldi

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

6.7K Görüntüleme.  Nihon Pharmaceutical University. Burada, proteaz özgüllük ham fare doku belirlemek için bir protokol MALDI-TOF spektrometresi kullanılarak özleri mevcut.

Video: The Determination of Protease Specificity in Mouse Tissue Extracts by MALDI-TOF Mass Spectrometry: Manipulating PH to Cause Specificity Changes

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

MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson's Disease

MALDI imaging mass spectrometry (IMS) is a powerful approach that facilitates the spatial analysis of molecular species in biological tissue samples2 (Fig.1). A 12 &mu;m thin tissue section is covered with a MALDI matrix, which facilitates desorption and ionization of intact peptides and proteins that can be detected with a mass analyzer, typically using a MALDI TOF/TOF mass spectrometer. Generally hundreds of peaks can be assessed in a single rat brain tissue section. In contrast to commonly used imaging techniques, this approach does not require prior knowledge of the molecules of interest and allows for unsupervised and comprehensive analysis of multiple molecular species while maintaining high molecular specificity and sensitivity2. Here we describe a MALDI IMS based approach for elucidating region-specific distribution profiles of neuropeptides in the rat brain of an animal model Parkinson's disease (PD). 
PD is a common neurodegenerative disease with a prevalence of 1% for people over 65 of age3,4. The most common symptomatic treatment is based on dopamine replacement using L-DOPA5. However this is accompanied by severe side effects including involuntary abnormal movements, termed L-DOPA-induced dyskinesias (LID)1,3,6. One of the most prominent molecular change in LID is an upregulation of the opioid precursor prodynorphin mRNA7. The dynorphin peptides modulate neurotransmission in brain areas that are essentially involved in movement control7,8. However, to date the exact opioid peptides that originate from processing of the neuropeptide precursor have not been characterized. Therefore, we utilized MALDI IMS in an animal model of experimental Parkinson's disease and L-DOPA induced dyskinesia. 
MALDI imaging mass spectrometry proved to be particularly advantageous with respect to neuropeptide characterization, since commonly used antibody based approaches targets known peptide sequences and previously observed post-translational modifications. By contrast MALDI IMS can unravel novel peptide processing products and thus reveal new molecular mechanisms of neuropeptide modulation of neuronal transmission. While the absolute amount of neuropeptides cannot be determined by MALDI IMS, the relative abundance of peptide ions can be delineated from the mass spectra, giving insights about changing levels in health and disease. In the examples presented here, the peak intensities of dynorphin B, alpha-neoendorphin and substance P were found to be significantly increased in the dorsolateral, but not the dorsomedial, striatum of animals with severe dyskinesia involving facial, trunk and orolingual muscles (Fig. 5). Furthermore, MALDI IMS revealed a correlation between dyskinesia severity and levels of des-tyrosine alpha-neoendorphin, representing a previously unknown mechanism of functional inactivation of dynorphins in the striatum as the removal of N-terminal tyrosine reduces the dynorphin's opioid-receptor binding capacity9. This is the first study on neuropeptide characterization in LID using MALDI IMS and the results highlight the potential of the technique for application in all fields of biomedical research.

Dopamine replacement pharmacotherapy using L-DOPA is the most commonly used symptomatic treatment of Parkinson&#x2019;s disease, but is accompanied by side effects including involuntary abnormal movements, termed dyskinesia 1. Here, a protocol for MALDI imaging mass spectrometry is presented that detects changes in rat brain neuropeptide levels related to dyskinesia.

Parkinson Hastalığında nöropeptitler MALDI Görüntüleme Kütle Spektrometresi

MALDI imaging mass spectrometry (IMS) is a powerful approach that facilitates the spatial analysis of molecular species in biological tissue samples2 ...

Tıp

Detection of Protease Activity by Fluorescent Peptide Zymography

The purpose of this method is to measure the proteolytic activity of complex biological samples. The samples are separated by molecular weight using electrophoresis through a resolving gel embedded with a degradable substrate. This method differs from traditional gel zymography in that a quenched fluorogenic peptide is covalently incorporated into the resolving gel instead of full length proteins, such as gelatin or casein. Use of the fluorogenic peptides enables direct detection of proteolytic activity without additional staining steps. Enzymes within the biological samples cleave the quenched fluorogenic peptide, resulting in an increase in fluorescence. The fluorescent signal in the gels is then imaged with a standard fluorescent gel scanner and quantified using densitometry. The use of peptides as the degradable substrate greatly expands the possible proteases detectable with zymographic techniques.

Here, we present a detailed protocol for a modified zymographic technique in which fluorescent peptides are used as the degradable substrate in place of native proteins. Electrophoresis of biological samples in fluorescent peptide zymograms enables detection of a wider range of proteases than previous zymographic techniques.

Floresan peptid Zymography tarafından proteaz aktivitesinin algılama

The purpose of this method is to measure the proteolytic activity of complex biological samples. The samples are separated by molecular weight using ...

Kanser Araştırmaları

Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ischemia and reperfusion using the isolated perfused mouse heart coupled with polysome profiling. To further understand the dynamic changes in protein translation, we characterized the mRNAs that were loaded with cytosolic ribosomes (polyribosomes or polysomes) and also recovered mitochondrial polysomes and compared mRNA and protein distribution in the high-efficiency fractions (numerous ribosomes attached to mRNA), low-efficiency (fewer ribosomes attached) which also included mitochondrial polysomes, and the non-translating fractions. miRNAs can also associate with mRNAs that are being translated, thereby reducing the efficiency of translation, we examined the distribution of miRNAs across the fractions. The distribution of mRNAs, miRNAs, and proteins was examined under basal perfused conditions, at the end of 30 min of global no-flow ischemia, and after 30 min of reperfusion. Here we present the methods used to accomplish this analysis&#8212;in particular, the approach to optimization of protein extraction from the sucrose gradient, as this has not been described before&#8212;and provide some representative results.

Here we present a protocol to perform polysome profiling on the isolated perfused mouse heart. We describe methods for heart perfusion, polysome profiling, and analysis of the polysome fractions with respect to mRNAs, miRNAs, and the polysome proteome.

Dinamik proteomik ve miRNA analiz polizomlar izole fare kalp sonra Langendorff perfüzyon üzerinden

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ...

Biyokimya

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ubiquitinated proteins, peptides with a diglycine remnant conjugated to the epsilon amino group of lysine (&#39;K-&#949;-diglycine&#39; or simply &#39;diGly&#39;) can be used to track back the original modification site. Efficient immunopurification of diGly peptides combined with sensitive detection by mass spectrometry has resulted in a huge increase in the number of ubiquitination sites identified up to date. We have made several improvements to this workflow, including offline high pH reverse-phase fractionation of peptides prior to the enrichment procedure, and the inclusion of more advanced peptide fragmentation settings in the ion routing multipole. Also, more efficient cleanup of the sample using a filter-based plug in order to retain the antibody beads results in a greater specificity for diGly peptides. These improvements result in the routine detection of more than 23,000 diGly peptides from human cervical cancer cells (HeLa) cell lysates upon proteasome inhibition in the cell. We show the efficacy of this strategy for in-depth analysis of the ubiquitinome profiles of several different cell types and of in vivo samples, such as brain tissue. This study presents an original addition to the toolbox for protein ubiquitination analysis to uncover the deep cellular ubiquitinome.

We present a method for the purification, detection, and identification of diGly peptides that originate from ubiquitinated proteins from complex biological samples. The presented method is reproducible, robust, and outperforms published methods with respect to the level of depth of the ubiquitinome analysis.

Peptit Zenginleştirme ve Kütle Spektrometresi ile Protein Ubiquitinasyon Sitelerinin Saptanması

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

A Mass Spectrometry-Based Proteomics Approach for Global and High-Confidence Protein R-Methylation Analysis

Protein Arginine (R)-methylation is a widespread protein post-translational modification (PTM) involved in the regulation of several cellular pathways, including RNA processing, signal transduction, DNA damage response, miRNA biogenesis, and translation.
In recent years, thanks to biochemical and analytical developments, mass spectrometry (MS)-based proteomics has emerged as the most effective strategy to characterize the cellular methyl-proteome with single-site resolution. However, identifying and profiling in vivo protein R-methylation by MS remains challenging and error-prone, mainly due to the substoichiometric nature of this modification and the presence of various amino acid substitutions and chemical methyl-esterification of acidic residues that are isobaric to methylation. Thus, enrichment methods to enhance the identification of R-methyl-peptides and orthogonal validation strategies to reduce False Discovery Rates (FDR) in methyl-proteomics studies are required.
Here, a protocol specifically designed for high-confidence R-methyl-peptides identification and quantitation from cellular samples is described, which couples metabolic labeling of cells with heavy isotope-encoded Methionine (hmSILAC) and dual protease in-solution digestion of whole cell extract, followed by off-line High-pH Reversed Phase (HpH-RP) chromatography fractionation and affinity enrichment of R-methyl-peptides using anti-pan-R-methyl antibodies. Upon high-resolution MS analysis, raw data are first processed with the MaxQuant software package and the results are then analyzed by hmSEEKER, a software designed for the in-depth search of MS peak pairs corresponding to light and heavy methyl-peptide within the MaxQuant output files.

Protein Arginine (R)-methylation is a wide-spread post-translational modification regulating multiple biological pathways. Mass spectrometry is the best technology to globally profile the R-methyl-proteome, when coupled to biochemical approaches for modified peptide enrichment. The workflow designed for the high confidence identification of global R-methylation in human cells is described here.

Global ve Yüksek Güvenilirlikli Protein R-Metilasyon Analizi için Kütle Spektrometrisine Dayalı Proteomik Yaklaşım

Protein Arginine (R)-methylation is a widespread protein post-translational modification (PTM) involved in the regulation of several cellular pathways, ...

Identification of Antibacterial Immunity Proteins in Escherichia coli using MALDI-TOF-TOF-MS/MS and Top-Down Proteomic Analysis

This protocol identifies the immunity proteins of the bactericidal enzymes: colicin E3 and bacteriocin, produced by a pathogenic Escherichia coli strain using antibiotic induction, and identified by MALDI-TOF-TOF tandem mass spectrometry and top-down proteomic analysis with software developed in-house. The immunity protein of colicin E3 (Im3) and the immunity protein of bacteriocin (Im-Bac) were identified from prominent b- and/or y-type fragment ions generated by the polypeptide backbone cleavage (PBC) on the C-terminal side of aspartic acid, glutamic acid, and asparagine residues by the aspartic acid effect fragmentation mechanism. The software rapidly scans in silico protein sequences derived from the whole genome sequencing of the bacterial strain. The software also iteratively removes amino acid residues of a protein sequence in the event that the mature protein sequence is truncated. A single protein sequence possessed mass and fragment ions consistent with those detected for each immunity protein. The candidate sequence was then manually inspected to confirm that all detected fragment ions could be assigned. The N-terminal methionine of Im3 was post-translationally removed, whereas Im-Bac had the complete sequence. In addition, we found that only two or three non-complementary fragment ions formed by PBC are necessary to identify the correct protein sequence. Finally, a promoter (SOS box) was identified upstream of the antibacterial and immunity genes in a plasmid genome of the bacterial strain.

Identification of Antibacterial Immunity Proteins in Escherichia coli using MALDI-TOF-TOF-MS/MS and Top-Down Proteomic Analysis

Here we present a protocol for the rapid identification of proteins produced by genomically sequenced pathogenic bacteria using MALDI-TOF-TOF tandem mass spectrometry and top-down proteomic analysis with software developed in-house. Metastable protein ions fragment because of the aspartic acid effect and this specificity is exploited for protein identification.

MALDI-TOF-TOF-MS/MS ve Yukarıdan Aşağıya Proteomik Analiz kullanılarak Escherichia coli'de Antibakteriyel Bağışıklık Proteinlerinin Tanımlanması

This protocol identifies the immunity proteins of the bactericidal enzymes: colicin E3 and bacteriocin, produced by a pathogenic Escherichia coli strain ...

A Mass Spectrometry-Based Approach to Identify Phosphoprotein Phosphatases and their Interactors

Most cellular processes are regulated by dynamic protein phosphorylation. More than three-quarters of proteins are phosphorylated, and phosphoprotein phosphatases (PPPs) coordinate over 90% of all cellular serine/threonine dephosphorylation. Deregulation of protein phosphorylation has been implicated in the pathophysiology of various diseases, including cancer and neurodegeneration. Despite their widespread activity, the molecular mechanisms controlling PPPs and those controlled by PPPs are poorly characterized. Here, a proteomic approach termed phosphatase inhibitor beads and mass spectrometry (PIB-MS) is described to identify and quantify PPPs, their posttranslational modifications, and their interactors in as little as 12 h using any cell line or tissue. PIB-MS utilizes a non-selective PPP inhibitor, microcystin-LR (MCLR), immobilized on sepharose beads to capture and enrich endogenous PPPs and their associated proteins (termed the PPPome). This method does not require the exogenous expression of tagged versions of PPPs or the use of specific antibodies. PIB-MS offers an innovative way to study the evolutionarily conserved PPPs and expand our current understanding of dephosphorylation signaling.

Here, we present a protocol for the enrichment of endogenous phosphoprotein phosphatases and their interacting proteins from cells and tissues and their identification and quantification by mass spectrometry-based proteomics.

Fosfoprotein fosfatazları ve bunların interaktörlerini tanımlamak için kütle spektrometrisine dayalı bir yaklaşım

Most cellular processes are regulated by dynamic protein phosphorylation. More than three-quarters of proteins are phosphorylated, and phosphoprotein ...

Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water

Stable isotopes are essential tools in biological mass spectrometry. Historically, 18O-stable isotopes have been extensively used to study the catalytic mechanisms of proteolytic enzymes1-3. With the advent of mass spectrometry-based proteomics, the enzymatically-catalyzed incorporation of 18O-atoms from stable isotopically enriched water has become a popular method to quantitatively compare protein expression levels (reviewed by Fenselau and Yao4, Miyagi and Rao5 and Ye et al.6). 18O-labeling constitutes a simple and low-cost alternative to chemical (e.g. iTRAQ, ICAT) and metabolic (e.g. SILAC) labeling techniques7. Depending on the protease utilized, 18O-labeling can result in the incorporation of up to two 18O-atoms in the C-terminal carboxyl group of the cleavage product3. The labeling reaction can be subdivided into two independent processes, the peptide bond cleavage and the carboxyl oxygen exchange reaction8. In our PALeO (protease-assisted labeling employing 18O-enriched water) adaptation of enzymatic 18O-labeling, we utilized 50% 18O-enriched water to yield distinctive isotope signatures. In combination with high-resolution matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS/MS), the characteristic isotope envelopes can be used to identify cleavage products with a high level of specificity. We previously have used the PALeO-methodology to detect and characterize endogenous proteases9 and monitor proteolytic reactions10-11. Since PALeO encodes the very essence of the proteolytic cleavage reaction, the experimental setup is simple and biochemical enrichment steps of cleavage products can be circumvented. The PALeO-method can easily be extended to (i) time course experiments that monitor the dynamics of proteolytic cleavage reactions and (ii) the analysis of proteolysis in complex biological samples that represent physiological conditions. PALeO-TimeCourse experiments help identifying rate-limiting processing steps and reaction intermediates in complex proteolytic pathway reactions. Furthermore, the PALeO-reaction allows us to identify proteolytic enzymes such as the serine protease trypsin that is capable to rebind its cleavage products and catalyze the incorporation of a second 18O-atom. Such "double-labeling" enzymes can be used for postdigestion 18O-labeling, in which peptides are exclusively labeled by the carboxyl oxygen exchange reaction. Our third strategy extends labeling employing 18O-enriched water beyond enzymes and uses acidic pH conditions to introduce 18O-stable isotope signatures into peptides.

Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water

Stable isotope labeling workflows employing 18O-enriched water (LeO-workflows) are versatile tools for quantitative and qualitative proteomics studies. In protease-assisted (PALeO) workflows, 18O-atoms are introduced by proteolytic cleavage and carboxyl oxygen exchange reactions mediated by proteases. In the acid-catalyzed (ALeO) workflow, 18O-atoms are introduced by carboxyl oxygen exchange at low pH.

Istihdam Proteaz-ve Asit-katalizli Etiketleme İş Akışları<sup&gt; 18</sup&gt; O-zenginleştirilmiş Su

Stable isotopes are essential tools in biological mass spectrometry. Historically, 18O-stable isotopes have been extensively used to study the catalytic ...

Biyoloji

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis (diguanylate cyclases) and degradation (phosphodiesterases) of the second messenger c-di-GMP. In contrast, little information is available regarding the molecular mechanisms and cellular components through which this signaling molecule regulates a diverse range of cellular processes. Most of the known effector proteins belong to the PilZ family or are degenerated diguanylate cyclases or phosphodiesterases that have given up on catalysis and have adopted effector function. Thus, to better define the cellular c-di-GMP network in a wide range of bacteria experimental methods are required to identify and validate novel effectors for which reliable in silico predictions fail.
We have recently developed a novel Capture Compound Mass Spectrometry (CCMS) based technology as a powerful tool to biochemically identify and characterize c-di-GMP binding proteins. This technique has previously been reported to be applicable to a wide range of organisms1. Here we give a detailed description of the protocol that we utilize to probe such signaling components. As an example, we use Pseudomonas aeruginosa, an opportunistic pathogen in which c-di-GMP plays a critical role in virulence and biofilm control. CCMS identified 74% (38/51) of the known or predicted components of the c-di-GMP network. This study explains the CCMS procedure in detail, and establishes it as a powerful and versatile tool to identify novel components involved in small molecule signaling.

The ubiquitous second messenger c-di-GMP controls growth and behavior of many bacteria. We have developed a novel Capture Compound Mass Spectrometry based technology to biochemically identify and characterize c-di-GMP binding proteins in virtually any bacterial species.

Proteaz özgüllük fare doku belirlenmesi MALDI-TOF Kütle spektrometresi tarafından ayıklar: PH özgüllük değişiklikler neden manipüle

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