Yazarlar, bu protokolün rafine edilmesindeki katkılarından dolayı Dr. Jennifer Geddes-McAlister ve Dr. Anthony Clarke'a teşekkür eder. Bu çalışma, CIHR'den C.M.K'ya (PJT 156111) verilen işletme hibeleri ile desteklendi ve E.M.A. Rakamları'na verilen NSERC Alexander Graham Bell CGS D, BioRender.com'da oluşturuldu.

<ol>
	<li>Vollmer, W., Blanot, D., de Pedro, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptidoglycan+structure+and+architecture.">Peptidoglycan structure and architecture.</a> FEMS Microbiology Reviews. 32, 149-167 (2007).</li><li>Pazos, M., Peters, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptidoglycan.">Peptidoglycan.</a> Sub-cellular Biochemistry. 92, 127-168 (2019).</li><li>Typas, A., Banzhaf, M., Gross, C. A., Vollmer, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+the+regulation+of+peptidoglycan+synthesis+to+bacterial+growth+and+morphology.">From the regulation of peptidoglycan synthesis to bacterial growth and morphology.</a> Nature Reviews. Microbiology. 10 (2), 123-136 (2011).</li><li>Yadav, A. K., Espaillat, A., Cava, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+strategies+to+preserve+cell+wall+integrity+against+environmental+threats.">Bacterial strategies to preserve cell wall integrity against environmental threats.</a> Frontiers in Microbiology. 9, 2064 (2018).</li><li>Bera, A., Herbert, S., Jakob, A., Vollmer, W., G&#246;tz, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+are+pathogenic+staphylococci+so+lysozyme+resistant?+The+peptidoglycan+O-acetyltransferase+OatA+is+the+major+determinant+for+lysozyme+resistance+of+Staphylococcus+aureus.">Why are pathogenic staphylococci so lysozyme resistant? The peptidoglycan O-acetyltransferase OatA is the major determinant for lysozyme resistance of Staphylococcus aureus.</a> Molecular Microbiology. 55 (3), 778-787 (2005).</li><li>Brott, A. S., Clarke, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptidoglycan+O-acetylation+as+a+virulence+factor:+its+effect+on+lysozyme+in+the+innate+immune+system.">Peptidoglycan O-acetylation as a virulence factor: its effect on lysozyme in the innate immune system.</a> Antibiotics. 8 (3), 94 (2019).</li><li>Putty, S., Vemula, H., Bobba, S., Gutheil, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+liquid+chromatography-tandem+mass+spectrometry+assay+for+D-Ala-D-Lac:+A+key+intermediate+for+vancomycin+resistance+in+vancomycin-resistant+Enterococci.">A liquid chromatography-tandem mass spectrometry assay for D-Ala-D-Lac: A key intermediate for vancomycin resistance in vancomycin-resistant Enterococci.</a> Analytical Biochemistry. 442 (2), 166-171 (2013).</li><li>Arthur, 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=Evidence+for+in+vivo+incorporation+of+D-lactate+into+peptidoglycan+precursors+of+vancomycin-resistant+Enterococci.">Evidence for in vivo incorporation of D-lactate into peptidoglycan precursors of vancomycin-resistant Enterococci.</a> Antimicrobial Agents and Chemotherapy. 36 (4), 867-869 (1992).</li><li>Mardarowicz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolierung+und+Charakterisierung+des+Murein-Sacculus+von+Brucella.">Isolierung und Charakterisierung des Murein-Sacculus von Brucella.</a> Z. Naturforsdig. 21, 1006-1007 (1966).</li><li>Glauner, B., H&#246;ltje, J. V., Schwarz, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+composition+of+the+murein+of+Escherichia+coli.">The composition of the murein of Escherichia coli.</a> The Journal of Biological Chemistry. 263 (21), 10088-10095 (1988).</li><li>Glauner, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+and+quantification+of+muropeptides+with+high-performance+liquid+chromatography.">Separation and quantification of muropeptides with high-performance liquid chromatography.</a> Analytical Biochemistry. 172, 451-464 (1988).</li><li>Anderson, E. 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=Peptidoglycomics+reveals+compositional+changes+in+peptidoglycan+between+biofilm-+and+planktonic-derived+Pseudomonas+aeruginosa.">Peptidoglycomics reveals compositional changes in peptidoglycan between biofilm- and planktonic-derived Pseudomonas aeruginosa.</a> The Journal of Biological Chemistry. 295 (2), 504-516 (2020).</li><li>van der Aart, L. T., 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=High-resolution+analysis+of+the+peptidoglycan+composition+in+Streptomyces+coelicolor.">High-resolution analysis of the peptidoglycan composition in Streptomyces coelicolor.</a> Journal of Bacteriology. 200 (20), 00290 (2018).</li><li>Bern, M., Beniston, R., Mesnage, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+an+automated+analysis+of+bacterial+peptidoglycan+structure.">Towards an automated analysis of bacterial peptidoglycan structure.</a> Analytical and Bioanalytical Chemistry. 409, 551-560 (2017).</li><li>Brott, A. S., Sychantha, D., Clarke, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assays+for+the+enzymes+catalyzing+the+O-acetylation+of+bacterial+cell+wall+polysaccharides.">Assays for the enzymes catalyzing the O-acetylation of bacterial cell wall polysaccharides.</a> Methods in Molecular Biology. 1954, 115-136 (2019).</li><li>Clarke, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compositional+analysis+of+peptidoglycan+by+high-performance+anion-exchange+chromatography.">Compositional analysis of peptidoglycan by high-performance anion-exchange chromatography.</a> Analytical Biochemistry. 212 (2), 344-350 (1993).</li><li>Rusconi, F., Douard Valton, &. #. 2. 0. 1. ;., Nguyen, R., Dufourc, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+sodium+dodecyl+sulfate+in+microliter-volume+biochemical+samples+by+visible+light+spectroscopy.">Quantification of sodium dodecyl sulfate in microliter-volume biochemical samples by visible light spectroscopy.</a> Analytical Biochemistry. 295, 31-37 (2001).</li><li>Verwer, R. W. H., Beachey, E. H., Keck, W., Stoub, A. M., Poldermans, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oriented+fragmentation+of+Escherichia+coli+sacculi+by+sonication.">Oriented fragmentation of Escherichia coli sacculi by sonication.</a> Journal of Bacteriology. 141 (1), 327-332 (1980).</li><li>Benjamini, Y., Hochberg, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+the+false+discovery+rate:+A+practical+and+powerful+approach+to+multiple+testing.">Controlling the false discovery rate: A practical and powerful approach to multiple testing.</a> Journal of the Royal Statistical Society: Series B (Methodological). 57 (1), 289-300 (1995).</li><li>Article, R., Alonso, A., Marsal, S., Juli&#224;, A., James Carroll, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analytical+methods+in+untargeted+metabolomics:+state+of+the+art+in+2015.">Analytical methods in untargeted metabolomics: state of the art in 2015.</a> Frontiers in Bioengineering and Biotechnology. 3, 23 (2015).</li><li>Xi, B., Gu, H., Baniasadi, H., Raftery, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Statistical+analysis+and+modeling+of+mass+spectrometry-based+metabolomics+data.">Statistical analysis and modeling of mass spectrometry-based metabolomics data.</a> Methods in Molecular Biology. 1198, 333-353 (2014).</li><li>Tyanova, S., 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=The+Perseus+computational+platform+for+comprehensive+analysis+of+(prote)omics+data.">The Perseus computational platform for comprehensive analysis of (prote)omics data.</a> Nature Methods. 13 (9), 731-740 (2016).</li><li>Cox, J., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=1D+and+2D+annotation+enrichment:+a+statistical+method+integrating+quantitative+proteomics+with+complementary+high-throughput+data.">1D and 2D annotation enrichment: a statistical method integrating quantitative proteomics with complementary high-throughput data.</a> BMC Bioinformatics. 13, 12 (2012).</li><li>Chang, J. D., Foster, E. E., Thadani, A. N., Ramirez, A. J., Kim, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+Staphylococcus+aureus+cell+wall+biosynthesis+by+desleucyl-oritavancin:+A+quantitative+peptidoglycan+composition+analysis+by+mass+spectrometry.">Inhibition of Staphylococcus aureus cell wall biosynthesis by desleucyl-oritavancin: A quantitative peptidoglycan composition analysis by mass spectrometry.</a> Journal of Bacteriology. 199 (15), 00278 (2017).</li><li>Glauner, B., H&#246;ltje, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+pattern+of+the+murein+sacculus+of+Escherichia+coli.">Growth pattern of the murein sacculus of Escherichia coli.</a> The Journal of Biological Chemistry. 265 (31), 18988-18996 (1990).</li><li>Espaillat, A., 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=Chemometric+analysis+of+bacterial+peptidoglycan+reveals+atypical+modifications+that+empower+the+cell+wall+against+predatory+enzymes+and+fly+innate+immunity.">Chemometric analysis of bacterial peptidoglycan reveals atypical modifications that empower the cell wall against predatory enzymes and fly innate immunity.</a> Journal of the American Chemical Society. 138 (29), 9193-9204 (2016).</li><li>Quintela, J. C., Caparros, M., de Pedro, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variability+of+peptidoglycan+structural+parameters+in+Gram-negative+bacteria.">Variability of peptidoglycan structural parameters in Gram-negative bacteria.</a> FEMS Microbiology Letters. 125, 95-100 (1995).</li><li>Quintela, J. C., Zollner, P., Portillo, F. G., Allmaier, G., de Pedro, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+wall+structural+divergence+among+Thermus+spp.">Cell wall structural divergence among Thermus spp.</a> FEMS Microbiology Letters. 172, 223-229 (1999).</li><li>Torrens, G., 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=Comparative+analysis+of+peptidoglycans+from+Pseudomonas+aeruginosa+isolates+recovered+from+chronic+and+acute+infections.">Comparative analysis of peptidoglycans from Pseudomonas aeruginosa isolates recovered from chronic and acute infections.</a> Frontiers in Microbiology. 10, 0868 (2019).</li><li>Antignac, A., 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=Detailed+structural+analysis+of+the+peptidoglycan+of+the+human+pathogen+Neisseria+meningitidis.">Detailed structural analysis of the peptidoglycan of the human pathogen Neisseria meningitidis.</a> The Journal of Biological Chemistry. 278 (34), 31521-31528 (2003).</li><li>De Jonges, B. L. M., Changi, Y. S., Gage, D., Tomaszs, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptidoglycan+composition+of+a+highly+methicillin-resistant+Staphylococcus+aureus+strain.+The+role+of+penicillin+binding+protein+2A.">Peptidoglycan composition of a highly methicillin-resistant Staphylococcus aureus strain. The role of penicillin binding protein 2A.</a> The Journal of Biological Chemistry. 267 (16), 11248-11264 (1992).</li><li>Lee, 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=Muropeptides+in+Pseudomonas+aeruginosa+and+their+role+as+elicitors+of+&#946;-lactam-antibiotic+resistance.">Muropeptides in Pseudomonas aeruginosa and their role as elicitors of &#946;-lactam-antibiotic resistance.</a> Angewandte Chemie - International Edition. 55, 6882-6886 (2016).</li><li>Schumann, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptidoglycan+Structure.">Peptidoglycan Structure.</a> Methods in Microbiology. 38, 101-129 (2011).</li><li>Wientjes, F. B., Woldringh, C. L., Nanninga, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amount+of+peptidoglycan+in+cell+walls+of+Gram-negative+bacteria.">Amount of peptidoglycan in cell walls of Gram-negative bacteria.</a> Journal of Bacteriology. 173 (23), 7684-7691 (1991).</li></ol>

Yazarlar çıkar çatışması olmadığını beyan ederler.

Bu protokol, peptidoglikanı bakteri kültürlerinden arındırmak, LC-MS tespiti için işlem yapmak ve biyoinformatik teknikleri kullanarak kompozisyonu analiz etmek için bir yöntem açıklamaktadır. Burada, Gram-negatif bakterilere odaklanıyoruz ve Gram-pozitif bakterilerin analizini sağlamak için bazı hafif değişiklikler gerekecektir.
Muropeptitlerin hazırlanması, 1960'larda ilk kez üretildiğinden beri neredeyse aynı kalmıştır 9,11,15.<su...

Peptidoglikan (PG), proteinler ve diğer hücresel bileşenler için yapısal destek sağlarken, hücre morfolojisini korumaya yarayan bakterilerin tanımlayıcı bir özelliğidir 1,2. PG'nin omurgası alternatif β-1,4'e bağlı N-asetil muramik asit (MurNAc) ve N-asetil glukozamin (GlcNAc)1,2'den oluşur. Her MurNAc, bitişik disakkarit bağlantılı peptitlere çapraz bağlanabilen ᴅ-laktil kalıntısına bağlı kısa bir peptide sahiptir (Şekil 1A, B). Bu çapraz bağlama, tüm hücreyi kapsayan ve genellikle sakkül olarak adlandırılan ağ benzeri bir yapı üretir (Şekil 1C). PG sentezi sırasında, öncüller sitoplazmada üretilir ve sitoplazmik membran boyunca flippaslar tarafından taşınır. Öncüller daha sonra sırasıyla glikosidik ve peptid bağlarını üreten transglikozilaz ve transpeptidaz enzimleri tarafından olgun PG'ye dahil edilir3. Bununla birlikte, bir kez monte edildikten sonra, büyüme ve bölünme de dahil olmak üzere bir dizi hücresel işlemi gerçekleştirmek için PG'yi değiştiren ve / veya bozan bakteriler tarafından üretilen çok sayıda enzim vardır. Ek olarak, PG'nin çeşitli modifikasyonlarının, hücre sinyalizasyonu, antimikrobiyal direnç ve konakçı immün kaçınma4 ile ilişkili olan suş, büyüme koşulları ve çevresel strese özgü adaptasyonlar sağladığı gösterilmiştir. Örnek olarak, yaygın bir modifikasyon, MurNAc üzerine, PG 4,5,6'yı bozan konakçı tarafından üretilen lizozim enzimlerine glikan β-1,4 bağlantılarına erişimi sınırlayarak direnç kazandıran bir C6 asetil grubunun eklenmesidir. Enterokoklarda, peptit yan zincirinin ᴅ-Ala terminalinin ᴅ-Lac ile değiştirilmesi, antimikrobiyal, vankomisin 7,8'e karşı daha büyük bir direnç sağlar.
PG izolasyonu ve saflaştırma için genel prosedür, 1960'larda tanımlandığından beri nispeten değişmeden kalmıştır9. Bakteriyel membranlar SDS ile ısıl işlemle çözülür, ardından bağlı proteinlerin, glikolipidlerin ve kalan DNA'nın enzimatik olarak uzaklaştırılması sağlanır. Saflaştırılmış bozulmamış sakkül daha sonra GlcNAc ve MurNAc arasındaki β-1,4 bağlantısının hidrolizi ile bireysel bileşenlere sindirilebilir. Bu sindirim, herhangi bir yapısal modifikasyon ve / veya çapraz bağ bozulmadan GlcNAc-MurNAc disakkaritleri üretir ve muropeptitler olarak adlandırılır (Şekil 1B).
PG'nin bileşimsel analizi başlangıçta her bir muropeptidi saflaştırmak için yüksek basınçlı sıvı kromatografik separasyonu (HPLC) yoluyla gerçekleştirildi ve ardından muropeptitlerin manuel olarak tanımlanması10,11 yapıldı. O zamandan beri bunun yerini, algılama hassasiyetini artıran ve her bir muropeptidi saflaştırmanın manuel iş yükünü azaltan sıvı kromatografisi tandem kütle spektrometresi (LC-MS) almıştır. Bununla birlikte, muropeptitlerin manuel olarak tanımlanmasının zaman alıcı ve karmaşık doğası, yapılan çalışmaların sayısını azaltarak sınırlayıcı bir faktör olarak kalmıştır. Son yıllarda "omik" teknolojilerin ortaya çıkmasıyla birlikte, otomatik LC-MS özellik ekstraksiyonu, çok büyük veri kümelerinden karmaşık örneklerdeki bireysel bileşiklerin hızlı bir şekilde tespit edilmesini ve tanımlanmasını sağlayan güçlü bir araç haline gelmiştir. Özellikler tanımlandıktan sonra, biyoinformatik yazılım, karmaşık veri kümesi arasındaki minimum farklılıkları bile izole eden ve bunları kullanıcıya grafiksel olarak görüntüleyen diferansiyel analiz kullanarak örnekler arasındaki varyasyonu istatistiksel olarak karşılaştırır. PG kompozisyonunun analizi için özellik ekstraksiyon yazılımının uygulanması daha yeni keşfedilmeye başlanmıştır 12,13,14 ve biyoinformatik analiz 12 ile birleştirilmiştir. Tam otomatik tanımlamaya izin veren peptid parçalanmasını öngören hazır protein veritabanlarından yararlanan proteomik analizin aksine, peptidoglycomics için şu anda parçalanma kütüphanesi yoktur. Bununla birlikte, özellik ekstraksiyonu, muropeptid tanımlamasını tahmin etmek için bilinen ve tahmin edilen yapısal veritabanlarıyla birleştirilebilir12. Burada, PG kompozisyonunun otomatik tanımlanması ve biyoinformatik diferansiyel analizi için bir muropeptid kütüphanesi ile birlikte LC-MS tabanlı özellik ekstraksiyonunun kullanımı için ayrıntılı bir protokol sunuyoruz (Şekil 2).

<table>
	<tbody>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>C18 reverse phase column - AdvanceBio Peptide column (100 mm x 2.1 mm 2.7 &#181;m)</td>
			<td>Agilent</td>
			<td></td>
			<td>LC-MS data acquisition</td>
		</tr>
		<tr>
			<td>Heating mantle controller, Optichem</td>
			<td>Fisher</td>
			<td>50-401-788</td>
			<td>for 4% SDS boil</td>
		</tr>
		<tr>
			<td>Heating Mantle, 1000mL Hemispherical</td>
			<td>Fisher</td>
			<td>CG1000008</td>
			<td>for 4% SDS boil</td>
		</tr>
		<tr>
			<td>Incubator, 37&#176;C</td>
			<td></td>
			<td></td>
			<td>for sacculi purification and MS sample prep</td>
		</tr>
		<tr>
			<td>Leibig condenser, 300MM 24/40,</td>
			<td>Fisher</td>
			<td>CG121805</td>
			<td>for 4% SDS boil</td>
		</tr>
		<tr>
			<td>Lyophilizer</td>
			<td>Labconco</td>
			<td></td>
			<td>for lyophilization of sacculi</td>
		</tr>
		<tr>
			<td>Magentic stirrer</td>
			<td>Fisher</td>
			<td>90-691-18</td>
			<td>for 4% SDS boil</td>
		</tr>
		<tr>
			<td>mass spectrometer Q-Tof model UHD 6530</td>
			<td>Aglient</td>
			<td></td>
			<td>LC-MS data acquisition</td>
		</tr>
		<tr>
			<td>microcentrifuge filters, Nanosep MF 0.2 &#181;m</td>
			<td>Fisher</td>
			<td>50-197-9573</td>
			<td>cleanup of sample before MS injection</td>
		</tr>
		<tr>
			<td>Retort stand</td>
			<td>Fisher</td>
			<td>12-000-102</td>
			<td>for 4% SDS boil</td>
		</tr>
		<tr>
			<td>Retort clamp</td>
			<td>Fisher</td>
			<td>S02629</td>
			<td>for 4% SDS boil</td>
		</tr>
		<tr>
			<td>round bottom flask - 1 liter pyrex</td>
			<td>Fisher</td>
			<td>07-250-084</td>
			<td>for 4% SDS boil</td>
		</tr>
		<tr>
			<td>Sonicator model 120</td>
			<td>Fisher</td>
			<td>FB120</td>
			<td>for sacculi purification</td>
		</tr>
		<tr>
			<td>Sonicator - micro tip</td>
			<td>Fisher</td>
			<td>FB4422</td>
			<td>for sacculi purification</td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Beckman</td>
			<td></td>
			<td>sacculi wash steps</td>
		</tr>
		<tr>
			<td>Ultracentrifuge bottles, Ti45</td>
			<td>Fisher</td>
			<td>NC9691797</td>
			<td>sacculi wash steps</td>
		</tr>
		<tr>
			<td>Water supply</td>
			<td>City</td>
			<td></td>
			<td>for water cooled condenser</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chemdraw</td>
			<td>Cambridgesoft</td>
			<td></td>
			<td>molecular editor for muropeptide fragmentation prediction</td>
		</tr>
		<tr>
			<td>Excel</td>
			<td>Microsoft</td>
			<td></td>
			<td>viewing lists of annotated muropeptides, abundance, isotopic patterns, etc.</td>
		</tr>
		<tr>
			<td>MassHunter Acquisition</td>
			<td>Aglient</td>
			<td></td>
			<td>running QTOF instrument</td>
		</tr>
		<tr>
			<td>MassHunter Mass Profiler Professional</td>
			<td>Aglient</td>
			<td></td>
			<td>bioinformatic differential analysis</td>
		</tr>
		<tr>
			<td>MassHunter Personal Compound Database and Library Manager</td>
			<td>Aglient</td>
			<td></td>
			<td>muropeptide m/z MS database</td>
		</tr>
		<tr>
			<td>MassHunter Profinder</td>
			<td>Aglient</td>
			<td></td>
			<td>recursive feature extraction</td>
		</tr>
		<tr>
			<td>MassHunter Qualitative analysis</td>
			<td>Aglient</td>
			<td></td>
			<td>viewing MS and MS/MS chromatograms</td>
		</tr>
		<tr>
			<td>Prism</td>
			<td>Graphpad</td>
			<td></td>
			<td>Graphing software</td>
		</tr>
		<tr>
			<td>Perseus</td>
			<td>Max Plank Institute of Biochemistry</td>
			<td></td>
			<td>1D annotation</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Fisher</td>
			<td>A998-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium acetate</td>
			<td>Fisher</td>
			<td>A637</td>
			<td></td>
		</tr>
		<tr>
			<td>Amylase</td>
			<td>Sigma-Aldrich</td>
			<td>A6380</td>
			<td></td>
		</tr>
		<tr>
			<td>Boric acid</td>
			<td>Fisher</td>
			<td>BP168-1</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase</td>
			<td>Fisher</td>
			<td>EN0521</td>
			<td></td>
		</tr>
		<tr>
			<td>Formic acid</td>
			<td>Sigma-Aldrich</td>
			<td>27001-500ML-R</td>
			<td></td>
		</tr>
		<tr>
			<td>LC-MS tuning mix - HP0321</td>
			<td>Agilent</td>
			<td>G1969-85000</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>M7506</td>
			<td></td>
		</tr>
		<tr>
			<td>Mutanolysin from Streptomyces globisporus ATCC 21553</td>
			<td>Sigma-Aldrich</td>
			<td>M9901</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrogen gas (&#62;99% purity)</td>
			<td>Praxair</td>
			<td>NI 5.0UH-T</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphoric acid</td>
			<td>Fisher</td>
			<td>A242</td>
			<td></td>
		</tr>
		<tr>
			<td>Pronase E from Streptomyces griseus</td>
			<td>Sigma-Aldrich</td>
			<td>P5147</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase</td>
			<td>Fisher</td>
			<td>EN0531</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Fisher</td>
			<td>S0489</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium borohydride</td>
			<td>Sigma-Aldrich</td>
			<td>452890</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>Fisher</td>
			<td>BP166</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Fisher</td>
			<td>S318</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate (dibasic)</td>
			<td>Fisher</td>
			<td>S373</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate (monobasic)</td>
			<td>Fisher</td>
			<td>S369</td>
			<td></td>
		</tr>
		<tr>
			<td>Stains-all</td>
			<td>Sigma-Aldrich</td>
			<td>E9379</td>
			<td></td>
		</tr>
	</tbody>
</table>

semi-quantitative analysis of peptidoglycan by liquid chromatography mass spectrometry and bioinformatics

Peptidoglikan, bakteriyel hücre duvarlarının önemli bir bileşenidir ve antimikrobiyaller için ortak bir hücresel hedeftir. Peptidoglikan yapısının yönleri tüm bakterilerde oldukça korunmuş olsa da, Gram-pozitifler / negatifler arasında ve türler arasında da önemli farklılıklar vardır. Ek olarak, büyüme fazına ve / veya çevresel uyaranlara yanıt olarak bir bakteri türünde meydana gelebilecek peptidoglikan için bilinen çok sayıda varyasyon, modifikasyon veya adaptasyon vardır. Bu varyasyonlar, büyüme / bölünme, antibiyotik direnci ve konakçı savunmasından kaçınma dahil olmak üzere birçok hücresel fonksiyona katıldığı bilinen oldukça dinamik bir yapı üretir. Peptidoglikan içindeki varyasyonu anlamak için, genel yapı kurucu parçalarına (muropeptitler olarak bilinir) ayrılmalı ve genel hücresel kompozisyon için değerlendirilmelidir. Peptidoglycomics, peptidoglikan kompozisyonunu ince ayrıntılarla incelemek için yüksek güçlü biyoinformatik veri analizi ile birlikte gelişmiş kütle spektrometresi kullanır. Aşağıdaki protokol, peptidoglikanın bakteri kültürlerinden saflaştırılmasını, bir sıvı kromatograf - kütle spektrometresi aracılığıyla muropeptid yoğunluk verilerinin elde edilmesini ve biyoinformatik kullanılarak peptidoglikan bileşiminin diferansiyel analizini açıklamaktadır.

MS makinelerinin artan algılama hassasiyeti, yüksek güçlü tepe tanıma yazılımı ile birleştiğinde, karmaşık numunelerin madde bileşimlerini çok küçük ayrıntılarla izole etme, izleme ve analiz etme yeteneğini geliştirmiştir. Bu teknolojik gelişmeleri kullanarak, peptidoglikan bileşimi üzerine yapılan son çalışmalar, eski HPLC tabanlı metodoloji 11,25,26,27,28,29,30,31 üzerinde otomatik LC-MS özellik ekstraksiyon teknikleri <sup cla...

Watch this Scientific Journal Video about Semi-Quantitative Analysis of Peptidoglycan by Liquid Chromatography Mass Spectrometry and Bioinformatics at JoVE.com

Semi-Quantitative Analysis of Peptidoglycan by Liquid Chromatography Mass Spectrometry and Bioinformatics

Bu protokol, gelişmiş özellik ekstraksiyonu ve biyoinformatik analiz yazılımı ile birlikte sıvı kromatografi kütle spektrometresi kullanılarak peptidoglikan bileşiminin ayrıntılı bir analizini kapsar.

Peptidoglikanın Sıvı Kromatografisi, Kütle Spektrometrisi ve Biyoinformatik ile Yarı Kantitatif Analizi

Peptidoglycan is an important component of bacterial cell walls and a common cellular target for antimicrobials. Although aspects of peptidoglycan ...

semi-quantitative-analysis-peptidoglycan-liquid-chromatography-mass

Research

JoVE Journal

Biochemistry

4.1K Görüntüleme.  University of Guelph. Bu protokol, gelişmiş özellik ekstraksiyonu ve biyoinformatik analiz yazılımı ile birlikte sıvı kromatografi kütle spektrometresi kullanılarak peptidoglikan bileşiminin ayrıntılı bir analizini kapsar.

Video: Semi-Quantitative Analysis of Peptidoglycan by Liquid Chromatography Mass Spectrometry and Bioinformatics

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

Zaman çözülmesi elektro sprey iyonizasyon Hidrojen-döteryum Değişim Kütle Spektrometre Protein Yapısı ve Dinamikleri incelenmesi için

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Biyokimya

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

Mavi Yerli PAGE ve Protein İlişkisi Profil Yöntemi ile Affinity Saf protein Kompleksler çözümleniyor

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

A Tandem Liquid Chromatography&#8211;Mass Spectrometry-based Approach for Metabolite Analysis of Staphylococcus aureus

In an effort to thwart bacterial pathogens, hosts often limit the availability of nutrients at the site of infection. This limitation can alter the abundances of key metabolites to which regulatory factors respond, adjusting cellular metabolism. In recent years, a number of proteins and RNA have emerged as important regulators of virulence gene expression. For example, the CodY protein responds to levels of branched-chain amino acids and GTP and is widely conserved in low G+C Gram-positive bacteria. As a global regulator in Staphylococcus aureus, CodY controls the expression of dozens of virulence and metabolic genes. We hypothesize that S. aureus uses CodY, in part, to alter its metabolic state in an effort to adapt to nutrient-limiting conditions potentially encountered in the host environment. This manuscript describes a method for extracting and analyzing metabolites from S. aureus using liquid chromatography coupled with mass spectrometry, a protocol that was developed to test this hypothesis. The method also highlights best practices that will ensure rigor and reproducibility, such as maintaining biological steady state and constant aeration without the use of continuous chemostat cultures. Relative to the USA200 methicillin-susceptible S. aureus isolate UAMS-1 parental strain, the isogenic codY mutant exhibited significant increases in amino acids derived from aspartate (e.g., threonine and isoleucine) and decreases in their precursors (e.g., aspartate and O-acetylhomoserine). These findings correlate well with transcriptional data obtained with RNA-seq analysis: genes in these pathways were up-regulated between 10- and 800-fold in the codY null mutant. Coupling global analyses of the transcriptome and the metabolome can reveal how bacteria alter their metabolism when faced with environmental or nutritional stress, providing potential insight into the physiological changes associated with nutrient depletion experienced during infection. Such discoveries may pave the way for the development of novel anti-infectives and therapeutics.

A Tandem Liquid Chromatography&amp;#8211;Mass Spectrometry-based Approach for Metabolite Analysis of &lt;em&gt;Staphylococcus aureus&lt;/em&gt;

Here we describe a protocol for the extraction of metabolites from Staphylococcus aureus and their subsequent analysis via liquid chromatography and mass spectrometry.

metabolit analizi için bir Tandem Sıvı Kromatografi-Kütle Spektrometresi tabanlı yaklaşım<em&gt; Stafilokok aureus</em

In an effort to thwart bacterial pathogens, hosts often limit the availability of nutrients at the site of infection. This limitation can alter the ...

Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of virion morphogenesis. Quantitative lipid droplet proteome analysis can be used to identify proteins that localize to or are displaced from lipid droplets under conditions such as virus infections. Here, we describe a protocol that has been successfully used to characterize the changes in the lipid droplet proteome following infection with HCV. We use Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) and thus label the complete proteome of one population of cells with &#34;heavy&#34; amino acids to quantitate the proteins by mass spectrometry. For lipid droplet isolation, the two cell populations (i.e.&#160;HCV-infected/&#34;light&#34; amino acids and uninfected control/&#34;heavy&#34; amino acids) are mixed 1:1 and lysed mechanically in hypotonic buffer. After removing the nuclei and cell debris by low speed centrifugation, lipid droplet-associated proteins are enriched by two subsequent ultracentrifugation steps followed by three washing steps in isotonic buffer. The purity of the lipid droplet fractions is analyzed by western blotting with antibodies recognizing different subcellular compartments. Lipid droplet-associated proteins are then separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie staining. After tryptic digest, the peptides are quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Using this method, we identified proteins recruited to lipid droplets upon HCV infection that might represent pro- or antiviral host factors. Our method can be applied to a variety of different cells and culture conditions, such as infection with pathogens, environmental stress, or drug treatment.

Lipid droplets are important organelles for the replication of several pathogens, including the Hepatitis C Virus (HCV). We describe a method to isolate lipid droplets for quantitative mass spectrometry of associated proteins; it can be used under a variety of conditions, such as virus infection, environmental stress, or drug treatment.

Kantitatif Kütle Spektrometresi Analiz lipid Damlacık izolasyonu

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of ...

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) coupled to tandem mass spectrometry (LC-MS/MS) and isobaric labeling multiplexing capacity. Here, we introduce a deep-proteomics profiling protocol that combines 10-plex tandem mass tag (TMT) labeling with an extensive LC/LC-MS/MS platform, and post-MS computational interference correction to accurately quantitate whole proteomes. This protocol includes the following main steps: protein extraction and digestion, TMT labeling, 2-dimensional (2D) LC, high-resolution mass spectrometry, and computational data processing. Quality control steps are included for troubleshooting and evaluating experimental variation. More than 10,000 proteins in mammalian samples can be confidently quantitated with this protocol. This protocol can also be applied to the quantitation of post translational modifications with minor changes. This multiplexed, robust method provides a powerful tool for proteomic analysis in a variety of complex samples, including cell culture, animal tissues, and human clinical specimens.

We present a protocol to accurately quantitate proteins with isobaric labelling, extensive fractionation, bioinformatics tools, and quality control steps in combination with liquid chromatography interfaced to a high-resolution mass spectrometer.

Derin Proteom izobarik etiketleme, kapsamlı sıvı Kromatografi, kütle spektrometresi ve yazılım destekli miktar tarafından profil oluşturma

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) ...

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 ...

Nitropeptide Profiling and Identification Illustrated by Angiotensin II

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in eukaryotic cells. Precise identification of nitration sites on proteins is crucial for understanding the physiological and pathological processes related to protein nitration, such as inflammation, aging, and cancer. Since the nitrated proteins are of low abundance in cells even under induced conditions, no universal and efficient methods have been developed for the profiling and identification of protein nitration sites. Here we describe a protocol for nitropeptide enrichment by using a chemical reduction reaction and biotin labeling, followed by high resolution mass spectrometry. In our method, nitropeptide derivatives can be identified with high accuracy. Our method exhibits two advantages compared to the previously reported methods. First, dimethyl labeling is used to block the primary amine on nitropeptides, which can be used to generate quantitative results. Second, a disulfide bond containing NHS-biotin reagent is used for the enrichment, which can be further reduced and alkylated to enhance the detection signal on a mass spectrometer. This protocol has been successfully applied to the model peptide Angiotensin II in the current paper.

Proteomic profiling of tyrosine-nitrated proteins has been a challenging technique due to the low abundance of the 3-nitrotyrosine modification. Here we describe a novel approach for nitropeptide enrichment and profiling by using Angiotensin II as the model. This method can be extended for other in vitro or in vivo systems.

Anjiyotensin II tarafından gösterilen nitropeptid profil oluşturma ve tanımlama

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of ...

Identification of Inositol Phosphate or Phosphoinositide Interacting Proteins by Affinity Chromatography Coupled to Western Blot or Mass Spectrometry

Inositol phosphates and phosphoinositides regulate several cellular processes in eukaryotes, including gene expression, vesicle trafficking, signal transduction, metabolism, and development. These metabolites perform this regulatory activity by binding to proteins, thereby changing protein conformation, catalytic activity, and/or interactions. The method described here uses affinity chromatography coupled to mass spectrometry or Western blotting to identify proteins that interact with inositol phosphates or phosphoinositides. Inositol phosphates or phosphoinositides are chemically tagged with biotin, which is then captured via streptavidin conjugated to agarose or magnetic beads. Proteins are isolated by their affinity of binding to the metabolite, then eluted and identified by mass spectrometry or Western blotting. The method has a simple workflow that is sensitive, non-radioactive, liposome-free, and customizable, supporting the analysis of protein and metabolite interaction with precision. This approach can be used in label-free or in amino acid-labelled quantitative mass spectrometry methods to identify protein-metabolite interactions in complex biological samples or using purified proteins. This protocol is optimized for the analysis of proteins from Trypanosoma brucei, but it can be adapted to related protozoan parasites, yeast or mammalian cells.

This protocol focuses on the identification of proteins that bind to inositol phosphates or phosphoinositides. It uses affinity chromatography with biotinylated inositol phosphates or phosphoinositides that are immobilized via streptavidin to agarose or magnetic beads. Inositol phosphate or phosphoinositide binding proteins are identified by Western blotting or mass spectrometry.

Inositol fosfat veya Phosphoinositide etkileşim proteinleri yakınlık Kromatografi Batı Blot veya kütle spektrometresi ile bağlantılı olarak tanımlanması

Inositol phosphates and phosphoinositides regulate several cellular processes in eukaryotes, including gene expression, vesicle trafficking, signal ...

Absolute Quantification of Cell-Free Protein Synthesis Metabolism by Reversed-Phase Liquid Chromatography-Mass Spectrometry

Cell-free protein synthesis (CFPS) is an emerging technology in systems and synthetic biology for the in vitro production of proteins. However, if CFPS is going to move beyond the laboratory and become a widespread and standard just in time manufacturing technology, we must understand the performance limits of these systems. Toward this question, we developed a robust protocol to quantify 40 compounds involved in glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle, energy metabolism and cofactor regeneration in CFPS reactions. The method uses internal standards tagged with 13C-aniline, while compounds in the sample are derivatized with 12C-aniline. The internal standards and sample were mixed and analyzed by reversed-phase liquid chromatography-mass spectrometry (LC/MS). The co-elution of compounds eliminated ion suppression, allowing the accurate quantification of metabolite concentrations over 2-3 orders of magnitude where the average correlation coefficient was 0.988. Five of the forty compounds were untagged with aniline, however, they were still detected in the CFPS sample and quantified with a standard curve method. The chromatographic run takes approximately 10 min to complete. Taken together, we developed a fast, robust method to separate and accurately quantify 40 compounds involved in CFPS in a single LC/MS run. The method is a comprehensive and accurate approach to characterize cell-free metabolism, so that ultimately, we can understand and improve the yield, productivity and energy efficiency of cell-free systems.

Here, we present a robust protocol to quantify 40 compounds involved in central carbon and energy metabolism in cell-free protein synthesis reactions. The cell-free synthesis mixture is derivatized with aniline for effective separation using reversed-phase liquid chromatography and then quantified by mass spectrometry using isotopically labelled internal standards.

Ters Faz Sıvı Kromatografi-Kütle Spektrometresi Ile Hücresiz Protein Sentezmetabolizmasının Mutlak Niceliği

Cell-free protein synthesis (CFPS) is an emerging technology in systems and synthetic biology for the in vitro production of proteins. However, if CFPS is ...

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of biological membranes, energy storage and production, cellular signaling, vesicular transport of proteins, organelle biogenesis, and regulated cell death. Because the budding yeast Saccharomyces cerevisiae is a unicellular eukaryote amenable to thorough molecular analyses, its use as a model organism helped uncover mechanisms linking lipid metabolism and intracellular transport to complex biological processes within eukaryotic cells. The availability of a versatile analytical method for the robust, sensitive, and accurate quantitative assessment of major classes of lipids within a yeast cell is crucial for getting deep insights into these mechanisms. Here we present a protocol to use liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of major cellular lipids of S. cerevisiae. The LC-MS/MS method described is versatile and robust. It enables the identification and quantification of numerous species (including different isobaric or isomeric forms) within each of the 10 lipid classes. This method is sensitive and allows identification and quantitation of some lipid species at concentrations as low as 0.2 pmol/&#181;L. The method has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. The use of alternative mobile phase additives for electrospray ionization mass spectrometry in this method can increase the efficiency of ionization for some lipid species and can be therefore used to improve their identification and quantitation.

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol using liquid chromatography coupled with tandem mass spectrometry to identify and quantify major cellular lipids in Saccharomyces cerevisiae. The described method for a quantitative assessment of major lipid classes within a yeast cell is versatile, robust, and sensitive.

Tandem Kütle Spektrometresi Ile Birleşen Sıvı Kromatografi kullanarak Saccharomyces Cerevisiae'nin Hücresel Lipidomukan Analizi

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of ...