Name: semi-quantitative analysis of peptidoglycan by liquid chromatography mass spectrometry and bioinformatics
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Description: Watch this Scientific Journal Video about Semi-Quantitative Analysis of Peptidoglycan by Liquid Chromatography Mass Spectrometry and Bioinformatics at JoVE.com

저자는 이 프로토콜을 개선하는 데 기여한 Jennifer Geddes-McAlister 박사와 Anthony Clarke 박사에게 감사를 표합니다. 이 작업은 C.M.K(PJT 156111)에 수여된 CIHR의 운영 보조금과 E.M.A.에 수여된 NSERC Alexander Graham Bell CGS D의 지원을 받았습니다. 피규어는 BioRender.com 에 만들어졌습니다.

<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>

저자는 이해 상충을 선언하지 않습니다.

이 프로토콜은 박테리아 배양에서 펩티도글리칸을 정제하고, LC-MS 검출을 처리하고, 생물정보학 기술을 사용하여 조성을 분석하는 방법을 설명합니다. 여기서는 그람 음성 박테리아에 초점을 맞추고 그람 양성 박테리아를 분석하려면 약간의 수정이 필요합니다.
muropeptides의 제조는 1960 년대 9,11,15 년에 처음 생산 된 이<sup cl...

펩티도글리칸(Peptidoglycan, PG)은 단백질 및 기타 세포 성분에 대한 구조적 지지를 제공하면서 세포 형태를 유지하는 역할을 하는 박테리아의 특징이다 1,2. PG의 백본은 교대로 β-1,4-연결된 N-아세틸 무라믹산(MurNAc)과 N-아세틸 글루코사민(GlcNAc)1,2로 구성됩니다. 각 MurNAc는 인접한 이당류 결합 펩타이드에 가교될 수 있는 ᴅ-락틸 잔기에 결합된 짧은 펩타이드를 가지고 있습니다(그림 1A, B). 이 가교결합은 전체 세포를 둘러싸고 있는 그물망과 같은 구조를 생성하며 종종 천골이라고 합니다(그림 1C). PG 합성 동안, 전구체는 세포질에서 생성되고, 플리파스에 의해 세포질 막을 가로질러 운반된다. 전구체는 후속적으로 트랜스글리코실라제와 트랜스펩티다아제 효소에 의해 성숙한 PG에 혼입되며, 이는 각각 글리코시드 및 펩티드 결합을 생성한다3. 그러나 일단 조립되면 성장 및 분열을 포함한 여러 세포 과정을 수행하기 위해 PG를 수정 및/또는 분해하는 박테리아에 의해 생성되는 수많은 효소가 있습니다. 또한, PG의 다양한 변형은 균주, 성장 조건 및 환경 스트레스에 특이적인 적응을 부여하는 것으로 나타났으며, 이는 세포 신호 전달, 항생제 내성 및 숙주 면역 회피와 관련이 있다4. 예를 들어, 일반적인 변형은 PG 4,5,6을 분해하는 숙주 생산 리소자임 효소에 대한 글리칸 β-1,4 결합에 대한 접근을 제한함으로써 저항성을 부여하는 MurNAc에 C6 아세틸기를 추가하는 것입니다. 장구균에서 펩타이드 측쇄의 말단 ᴅ-Ala를 ᴅ-Lac로 치환하면 항균제인 반코마이신 7,8에 대한 내성이 더 커집니다.
PG 분리 및 정제를 위한 일반적인 절차는 1960년대에 기술된 이래로 비교적 변하지 않았다9. 박테리아 막은 SDS로 열처리를 통해 용해된 후 결합된 단백질, 당지질 및 나머지 DNA를 효소로 제거합니다. 정제된 온전한 천골은 GlcNAc와 MurNAc 사이의 β-1,4 결합의 가수분해에 의해 개별 성분으로 후속적으로 분해될 수 있습니다. 이 소화는 구조적 변형 및/또는 가교결합이 손상되지 않은 GlcNAc-MurNAc 이당류를 생성하며 이를 무로펩티드라고 합니다(그림 1B).
PG의 조성 분석은 초기에 고압 액체 크로마토그래피 분리(HPLC)를 통해 수행되어 각 무로펩티드를 정제한 후 무로펩티드10,11을 수동으로 식별했습니다. 이는 이후 액체 크로마토그래피 탠덤 질량분석법(LC-MS)으로 대체되어 검출 감도를 높이고 각 개별 무로펩타이드를 정제하는 수동 작업량을 줄입니다. 그러나 muropeptides의 수동 식별의 시간 소모적이고 복잡한 특성은 여전히 제한 요소로 남아 수행된 연구의 수를 줄입니다. 최근 몇 년 동안 "omic" 기술의 출현으로 자동화된 LC-MS 특징 추출이 강력한 도구가 되어 매우 큰 데이터 세트의 복잡한 샘플에서 개별 화합물을 신속하게 검출하고 식별할 수 있습니다. 특징이 식별되면 생물정보학 소프트웨어는 복잡한 데이터 세트 간의 최소한의 차이도 분리하여 사용자에게 그래픽으로 표시하는 차등 분석을 사용하여 샘플 간의 변동을 통계적으로 비교합니다. PG 조성의 분석을 위한 특징 추출 소프트웨어의 적용은 이제 막 탐구되기 시작했고(12,13,14) 생물정보 학적 분석(bioinformatic analysis)12과 결합되었다. 펩타이드 단편화를 예측하여 완전히 자동화된 식별을 가능하게 하는 쉽게 사용할 수 있는 단백질 데이터베이스의 이점을 제공하는 단백질체 분석과 달리 현재 펩티도글리코믹스에 대한 단편화 라이브러리는 존재하지 않습니다. 그러나, 특징 추출은 무로펩티드 식별을 예측하기 위해 알려지고 예측된 구조 데이터베이스와 결합될 수 있다12. 여기에서는 PG 조성의 자동 식별 및 생물정보학적 차등 분석을 위해 무로펩타이드 라이브러리와 결합된 LC-MS 기반 특징 추출의 사용에 대한 자세한 프로토콜을 제시합니다(그림 2).

<table><tbody><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</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 &amp;micro;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&amp;deg;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 &amp;micro;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>&lt;strong&gt;Software&lt;/strong&gt;</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>&lt;strong&gt;Material&lt;/strong&gt;</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 (&gt;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

펩티도글리칸은 박테리아 세포벽의 중요한 구성 요소이며 항균제의 일반적인 세포 표적입니다. 펩티도글리칸 구조의 측면은 모든 박테리아에 걸쳐 상당히 보존되어 있지만 그람 양성/음성 및 종 간에도 상당한 차이가 있습니다. 또한, 성장 단계 및/또는 환경 자극에 대한 반응으로 박테리아 종 내에서 발생할 수 있는 펩티도글리칸에 대한 수많은 알려진 변이, 변형 또는 적응이 있습니다. 이러한 변이는 성장/분열, 항생제 내성 및 숙주 방어 회피를 포함한 많은 세포 기능에 참여하는 것으로 알려진 매우 역동적인 구조를 생성합니다. 펩티도글리칸 내의 변이를 이해하려면 전체 구조를 구성 부분(무로펩티드로 알려짐)으로 분해하고 전체 세포 구성에 대해 평가해야 합니다. Peptidoglycomics는 고성능 생물정보학 데이터 분석과 결합된 고급 질량분석법을 사용하여 펩티도글리칸 조성을 세밀하게 조사합니다. 다음 프로토콜은 박테리아 배양에서 펩티도글리칸의 정제, 액체 크로마토그래프(질량 분석기)를 통한 무로펩티드 강도 데이터 획득, 생물정보학을 사용한 펩티도글리칸 조성의 차등 분석에 대해 설명합니다.

고성능 피크 인식 소프트웨어와 결합된 MS 기계의 향상된 검출 감도는 복잡한 샘플의 물질 조성을 매우 세밀하게 분리, 모니터링 및 분석하는 기능을 향상시켰습니다. 이러한 기술적 진보를 이용하여, 펩티도글리칸 조성물에 대한 최근 연구들은 이전의 HPLC-기반 방법론 11,25,26,27,28,29,30,31에 비해 자동화된 LC-MS 특징 추출 기술12,13,14,24<s...

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

이 프로토콜은 고급 특징 추출 및 생물정보학 분석 소프트웨어와 결합된 액체 크로마토그래피 질량분석법을 사용하여 펩티도글리칸 조성에 대한 자세한 분석을 다룹니다.

액체 크로마토그래피, 질량 분석법 및 생물정보학에 의한 펩티도글리칸의 반정량 분석

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

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

4.2K 조회수.  University of Guelph. 이 프로토콜은 고급 특징 추출 및 생물정보학 분석 소프트웨어와 결합된 액체 크로마토그래피 질량분석법을 사용하여 펩티도글리칸 조성에 대한 자세한 분석을 다룹니다.

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

Subtyping of Campylobacter jejuni ssp. doylei Isolates Using Mass Spectrometry-based PhyloProteomics (MSPP)

MALDI-TOF MS offers the possibility to differentiate some bacteria not only at the species and subspecies level but even below, at the strain level. Allelic isoforms of the detectable biomarker ions result in isolate-specific mass shifts. Mass spectrometry-based phyloproteomics (MSPP) is a novel technique that combines the mass spectrometric detectable biomarker masses in a scheme that allows deduction of phyloproteomic relations from isolate specific mass shifts compared to a genome sequenced reference strain. The deduced amino acid sequences are then used to calculate MSPP-based dendrograms.
Here we describe the workflow of MSPP by typing a Campylobacter jejuni ssp. doylei isolate collection of seven strains. All seven strains were of human origin and multilocus sequence typing (MLST) demonstrated their genetic diversity. MSPP-typing resulted in seven different MSPP sequence types, sufficiently reflecting their phylogenetic relations.
The C. jejuni ssp. doylei MSPP scheme includes 14 different biomarker ions, mostly ribosomal proteins in the mass range of 2 to 11 kDa. MSPP can in principle, be adapted to other mass spectrometric platforms with an extended mass range. Therefore, this technique has the potential to become a useful tool for strain level microbial typing.

Subtyping of Campylobacter jejuni ssp. doylei Isolates Using Mass Spectrometry-based PhyloProteomics MSPP

Mass spectrometry-based phyloproteomics (MSPP) was used to type a collection of Campylobacter jejuni ssp. doylei isolates at the strain level in comparison to multilocus sequence typing (MLST).

의 하위 유형<em&gt; 캄 필로 박터 jejuni와</em&gt; SSP.<em&gt; doylei</em&gt;은 질량 분석 기반 PhyloProteomics를 사용하여 분리 (MSPP)

MALDI-TOF MS offers the possibility to differentiate some bacteria not only at the species and subspecies level but even below, at the strain level. ...

연구

유전학

Isolation and Preparation of Bacterial Cell Walls for Compositional Analysis by Ultra Performance Liquid Chromatography

The bacterial cell wall is critical for the determination of cell shape during growth and division, and maintains the mechanical integrity of cells in the face of turgor pressures several atmospheres in magnitude. Across the diverse shapes and sizes of the bacterial kingdom, the cell wall is composed of peptidoglycan, a macromolecular network of sugar strands crosslinked by short peptides. Peptidoglycan&#8217;s central importance to bacterial physiology underlies its use as an antibiotic target and has motivated genetic, structural, and cell biological studies of how it is robustly assembled during growth and division. Nonetheless, extensive investigations are still required to fully characterize the key enzymatic activities in peptidoglycan synthesis and the chemical composition of bacterial cell walls. High Performance Liquid Chromatography (HPLC) is a powerful analytical method for quantifying differences in the chemical composition of the walls of bacteria grown under a variety of environmental and genetic conditions, but its throughput is often limited. Here, we present a straightforward procedure for the isolation and preparation of bacterial cell walls for biological analyses of peptidoglycan via HPLC and Ultra Performance Liquid Chromatography (UPLC), an extension of HPLC that utilizes pumps to deliver ultra-high pressures of up to 15,000 psi, compared with 6,000 psi for HPLC. In combination with the preparation of bacterial cell walls presented here, the low-volume sample injectors, detectors with high sampling rates, smaller sample volumes, and shorter run times of UPLC will enable high resolution and throughput for novel discoveries of peptidoglycan composition and fundamental bacterial cell biology in most biological laboratories with access to an ultracentrifuge and UPLC.

The bacterial cell wall is composed of peptidoglycan, a macromolecular network of sugar strands crosslinked by peptides. Ultra Performance Liquid Chromatography provides high resolution and throughput for novel discoveries of peptidoglycan composition. We present a procedure for the isolation of cell walls (sacculi) and their subsequent preparation for analysis via UPLC.

초성능 액체 크로마토그래피에 의한 조성 분석을 위한 세균세포벽의 격리 및 준비

The bacterial cell wall is critical for the determination of cell shape during growth and division, and maintains the mechanical integrity of cells in the ...

화학

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.

블루 기본 페이지와 단백질 상관 관계 프로파일 링에 의한 선호도 정제 단백질 복합체 해결

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

생화학

A Quantitative Glycomics and Proteomics Combined Purification Strategy

There is a growing desire in the biological and clinical sciences to integrate and correlate multiple classes of biomolecules to unravel biology, define pathways, improve treatment, understand disease, and aid biomarker discovery. N-linked glycosylation is one of the most important and robust post-translational modifications on proteins and regulates critical cell functions such as signaling, adhesion, and enzymatic function. Analytical techniques to purify and analyze N-glycans have remained relatively static over the last decade. While accurate and effective, they commonly require significant expertise and resources. Though some high-throughput purification schemes have been developed, they have yet to find widespread adoption and often rely on the enrichment of glycopeptides. One promising method, developed by Thomas-Oates et al., filter aided N-glycan separation (FANGS), was qualitatively demonstrated on tissues. Herein, we adapted FANGS to plasma and coupled it to the individuality normalization when labeling with glycan hydrazide tags strategy in order to achieve accurate relative quantification by liquid chromatography mass spectrometry and enhanced electrospray ionization. Furthermore, we designed new functionality to the protocol by achieving tandem, shotgun proteomics and glycosylation site analysis on hen plasma. We showed that N-glycans purified on filter and derivatized by hydrophobic hydrazide tags were comparable in terms of abundance and class to those by solid phase extraction (SPE); the latter is considered a gold standard in the field. Importantly, the variability in the two protocols was not statistically different. Proteomic data that was collected in-line with glycomic data had the same depth compared to a standard trypsin digest. Peptide deamidation is minimized in the protocol, limiting non-specific deamidation detected at glycosylation motifs. This allowed for direct glycosylation site analysis, though the protocol can accommodate 18O site labeling as well. Overall, we demonstrated a new in-line high-throughput, unbiased, filter based protocol for quantitative glycomics and proteomics analysis.

A high-throughput protocol was developed for combined proteomics and glycomics purification and LC-MS/MS quantification in plasma. Deamidation analysis of N-linked glycosylation motifs was specific to deglycosylated sites. Accurate quantitation of N-glycans was achieved by coupling filter aided N-glycan separation to the individuality normalization when labeling with glycan hydrazide tags strategy.

양적 글리코 믹스와 단백질 체학 복합 정제 전략

There is a growing desire in the biological and clinical sciences to integrate and correlate multiple classes of biomolecules to unravel biology, define ...

Synthesis and Mass Spectrometry Analysis of Oligo-peptoids

Peptoids are sequence-controlled peptide-mimicking oligomers consisting of N-alkylated glycine units. Among many potential applications, peptoids have been thought of as a type of molecular information storage. Mass spectrometry analysis has been considered the method of choice for sequencing peptoids. Peptoids can be synthesized via solid phase chemistry using a repeating two-step reaction cycle. Here we present a method to manually synthesize oligo-peptoids and to analyze the sequence of the peptoids using tandem mass spectrometry (MS/MS) techniques. The sample peptoid is a nonamer consisting of alternating N-(2-methyloxyethyl)glycine (Nme) and N-(2-phenylethyl)glycine (Npe), as well as an N-(2-aminoethyl)glycine (Nae) at the N-terminus. The sequence formula of the peptoid is Ac-Nae-(Npe-Nme)4-NH2, where Ac is the acetyl group. The synthesis takes place in a commercially available solid-phase reaction vessel. The rink amide resin is used as the solid support to yield the peptoid with an amide group at the C-terminus. The resulting peptoid product is subjected to sequence analysis using a triple-quadrupole mass spectrometer coupled to an electrospray ionization source. The MS/MS measurement produces a spectrum of fragment ions resulting from the dissociation of charged peptoid. The fragment ions are sorted out based on the values of their mass-to-charge ratio (m/z). The m/z values of the fragment ions are compared against the nominal masses of theoretically predicted fragment ions, according to the scheme of peptoid fragmentation. The analysis generates a fragmentation pattern of the charged peptoid. The fragmentation pattern is correlated to the monomer sequence of the neutral peptoid. In this regard, MS analysis reads out the sequence information of the peptoids.

A protocol is described for the manual synthesis of oligo-peptoids followed by sequence analysis by mass spectrometry.

합성 및 올리고 peptoids의 질량 분석 분석

Peptoids are sequence-controlled peptide-mimicking oligomers consisting of N-alkylated glycine units. Among many potential applications, peptoids have ...

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.

탠덤 질량 분석과 결합 된 액체 크로마토그래피를 사용하여 사카로 미세화세세비시아의 세포 지질의 정량분석

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

Synthesis of Masarimycin, a Small Molecule Inhibitor of Gram-Positive Bacterial Growth

Peptidoglycan (PG) in the cell wall of bacteria is a unique macromolecular structure that confers shape, and protection from the surrounding environment. Central to understanding cell growth and division is the knowledge of how PG degradation influences biosynthesis and cell wall assembly. Recently, the metabolic labeling of PG through the introduction of modified sugars or amino acids has been reported. While chemical interrogation of biosynthetic steps with small molecule inhibitors is possible, chemical biology tools to study PG degradation by autolysins are underdeveloped. Bacterial autolysins are a broad class of enzymes that are involved in the tightly coordinated degradation of PG. Here, a detailed protocol is presented for preparing a small molecule probe, masarimycin, which is an inhibitor of N-acetylglucosaminidase LytG in Bacillus subtilis, and cell wall metabolism in Streptococcus pneumoniae. Preparation of the inhibitor via microwave-assisted and classical organic synthesis is provided. Its applicability as a tool to study Gram-positive physiology in biological assays is presented.

A detailed protocol is presented for preparing the bacteriostatic diamide masarimycin, a small molecule probe that inhibits the growth of Bacillus subtilis and Streptococcus pneumoniae by targeting cell wall degradation. Its application as a chemical probe is demonstrated in synergy/antagonism assays and morphological studies with B. subtilis and S. pneumoniae.

그람 양성 박테리아 성장의 소분자 억제제 인 Masarimycin의 합성

Peptidoglycan (PG) in the cell wall of bacteria is a unique macromolecular structure that confers shape, and protection from the surrounding environment. ...

The Application of Open Searching-based Approaches for the Identification of Acinetobacter baumannii O-linked Glycopeptides

Protein glycosylation is increasingly recognized as a common modification within bacterial organisms, contributing to prokaryotic physiology and optimal infectivity of pathogenic species. Due to this, there is increasing interest in characterizing bacterial glycosylation and a need for high-throughput analytical tools to identify these events. Although bottom-up proteomics readily enables the generation of rich glycopeptide data, the breadth and diversity of glycans observed in prokaryotic species make the identification of bacterial glycosylation events extremely challenging.
Traditionally, the manual determination of glycan compositions within bacterial proteomic datasets made this a largely bespoke analysis restricted to field-specific experts. Recently, open searching-based approaches have emerged as a powerful alternative for the identification of unknown modifications. By analyzing the frequency of unique modifications observed on peptide sequences, open searching techniques allow the identification of common glycans attached to peptides within complex samples. This article presents a streamlined workflow for the interpretation and analysis of glycoproteomic data, demonstrating how open searching techniques can be used to identify bacterial glycopeptides without prior knowledge of the glycan compositions.
Using this approach, glycopeptides within samples can rapidly be identified to understand glycosylation differences. Using Acinetobacter baumannii as a model, these approaches enable the comparison of glycan compositions between strains and the identification of novel glycoproteins. Taken together, this work demonstrates the versatility of open database-searching techniques for the identification of bacterial glycosylation, making the characterization of these highly diverse glycoproteomes easier than ever before.

The Application of Open Searching-based Approaches for the Identification of Acinetobacter baumannii O-linked Glycopeptides

Open searching enables the identification of glycopeptides decorated with previously unknown glycan compositions. Within this article, a streamlined approach for undertaking open searching and subsequent glycan-focused glycopeptide searches are presented for bacterial samples using Acinetobacter baumannii as a model.

Acinetobacter baumannii O 결합 글리코 펩타이드의 확인을위한 개방형 검색 기반 접근법의 적용

Protein glycosylation is increasingly recognized as a common modification within bacterial organisms, contributing to prokaryotic physiology and optimal ...

Enrichment of Bacterial Lipoproteins and Preparation of N-terminal Lipopeptides for Structural Determination by Mass Spectrometry

Lipoproteins are important constituents of the bacterial cell envelope and potent activators of the mammalian innate immune response. Despite their significance to both cell physiology and immunology, much remains to be discovered about novel lipoprotein forms, how they are synthesized, and the effect of the various forms on host immunity. To enable thorough studies on lipoproteins, this protocol describes a method for bacterial lipoprotein enrichment and preparation of N-terminal tryptic lipopeptides for structural determination by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Expanding on an established Triton X-114 phase partitioning method for lipoprotein extraction and enrichment from the bacterial cell membrane, the protocol includes additional steps to remove non-lipoprotein contaminants, increasing lipoprotein yield and purity. Since lipoproteins are commonly used in Toll-like receptor (TLR) assays, it is critical to first characterize the N-terminal structure by MALDI-TOF MS. Herein, a method is presented to isolate concentrated hydrophobic peptides enriched in N-terminal lipopeptides suitable for direct analysis by MALDI-TOF MS/MS. Lipoproteins that have been separated by Sodium Dodecyl Sulfate Poly-Acrylamide Gel Electrophoresis (SDS-PAGE) are transferred to a nitrocellulose membrane, digested in situ with trypsin, sequentially washed to remove polar tryptic peptides, and finally eluted with chloroform-methanol. When coupled with MS of the more polar trypsinized peptides from wash solutions, this method provides the ability to both identify the lipoprotein and characterize its N-terminus in a single experiment. Intentional sodium adduct formation can also be employed as a tool to promote more structurally informative fragmentation spectra. Ultimately, enrichment of lipoproteins and determination of their N-terminal structures will permit more extensive studies on this ubiquitous class of bacterial proteins.

The enrichment of bacterial lipoproteins using a non-ionic surfactant phase partitioning method is described for direct use in TLR assays or other applications. Further steps are detailed to prepare N-terminal tryptic lipopeptides for structural characterization by mass spectrometry.

세균성 단백질 질량 분석에 의하여 구조 결심에 대 한 N 맨끝 Lipopeptides의 준비의 농축

Lipoproteins are important constituents of the bacterial cell envelope and potent activators of the mammalian innate immune response. Despite their ...

면역학 및 감염병학

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.

액체 크로마토그래피, 질량 분석법 및 생물정보학에 의한 펩티도글리칸의 반정량 분석

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