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

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.

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	<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. 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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. 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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>&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

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

4.2K 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

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).

alt türlerinin<em&gt; Campylobacter jejuni</em&gt; Ssp.<em&gt; doylei</em&gt; Kütle Spektrometre tabanlı PhyloProteomics kullanma izolatlar (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. ...

Genetik

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.

Ultra Performanslı Sıvı Kromatografisi ile Bileşimsel Analiz için Bakteriyel Hücre Duvarlarının İzolasyonu ve Hazırlanması

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 ...

Kimya

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 ...

Biyokimya

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.

Nicel Glycomics ve Proteomik Kombine Arıtma Stratejisi

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.

Sentez ve kütle spektrometresi analizleri Oligo-peptoids

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.

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 ...

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.

Gram-pozitif bakteriyel büyümenin küçük moleküllü bir inhibitörü olan masarimisinin sentezi

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-bağlantılı Glikopeptitlerlerin Tanımlanması için Açık Arama Tabanlı Yaklaşımların Uygulanması

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.

Zenginleştirme bakteriyel lipoproteinler ve kütle spektrometresi tarafından yapısal kararlılık için N-terminal Lipopeptides hazırlanması

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

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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.

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

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