This research was supported by the Intramural Research Program of the NIH, National Institute of Allergy and Infectious Diseases.

The authors have nothing to disclose.

Mutlak Protein ölçümü bu biyobelirteç doğrulama ve sinyal iletim yolu modelleme gibi biyomedikal uygulamalarda bir çok farklı aralık için gereklidir. Son zamanlarda, LC-SRM kullanılarak hedeflenen proteomik peptid standart hazırlama, HPLC, qqq-MS ve LC-SRM veri analizi de dahil olmak üzere çok sayıda teknolojilere iyileştirmeler yararlanmıştır. Sonuç olarak, bağışıklık karşı güçlü bir alternatif haline gelmiştir. Immünoanalizler ve / veya uygun değildir multiplekslemesi için, son derece...

Kütle spektrometrisi (MS) kullanmak proteomik deneyler hedefli olmayan (av tüfeği) veya hedeflenen yöntemleri ya kullanmak için dizayn edilebilir. Keşif proteomik genellikle geleneksel veri bağımlı toplama modunu kullanarak, ya da (örneğin, MS E, SWATH) son zamanlarda geliştirilen veri bağımsız tekniklerden birini 1,2 kullanarak, ya aşağıdan yukarıya av tüfeği MS dayanır. Shotgun proteomik yüksek verimli peptit tanımlama ve nispi ölçümü için güçlü bir araçtır, ama mutlak ölçümü için veya proteinlerin küçük tanımlanan setler (~ onlarca) hedefleme için genellikle uygun değildir. En sık hedeflenen proteomik için kullanılan MS yöntemi nedeniyle yüksek hassasiyet, hız ve dinamik aralık 3-5 tepkisini izleme (SRM) seçilir. SRM alternatifleri yüksek çözünürlüklü, tam MS tarama 6 yararlanır paralel reaksiyon izleme içerir. SRM genellikle nano-akış Rever kullanılarak gerçekleştirilirSED-fazlı yüksek-performanslı sıvı kromatografisi (nano RP-LC) nano-elektrosprey iyonizasyon bağlanmış cihaz üçlü dört kutuplu kütle spektrometresi (qqq-MS) takılmış bir (daha nano-ESI) iyon kaynağı. Tipik bir deneyde, örnek proteinler proteolitik olarak sindirilir ve elde edilen peptitler, kromatografik ayrıldı, desorbe ve iyonize edilebilir. Elde edilen öncül iyonları m / ilk kuadruple (Q1) tarafından filtre z ve çarpışma gazı ile çarpışarak tarafından ikinci kuadropol (q2) parçalanmış bulunmaktadır. Elde edilen fragman, iyonlarıdır m / z-süzüldü üçüncü dört kutuplu (Q3) ve bir dynode ile nicelendirildi. Her bir ön-madde ve fragman iyon çifti bir geçiş olarak ifade edilir ve her bir geçiş belirli bir süre (kalma süresi tipik olarak 2-50 msn) izlenir. LC-SRM sırasında geçişler önceden tanımlanmış bir liste üzerinden qqq-MS döngüleri (görev döngüsü genellikle ≤3 sn olan) ve her bir geçiş kromatogram üretilir. Alternatif strategProtein ölçümü için ler, tipik olarak bu nokta, blotlar, Western blotlar, ELISA antikor mikrodizileri, ters fazlı protein mikrodizilerinin, mikroakışkan bağışıklık dijital ELISA ve mikroküre bazlı bağışıklık 7 olarak immün kullanımı. En iyi immünodeneyler LC-ARM önemli ölçüde daha fazla duyarlı olabilir ve bağışıklık örnek hacmi LC-SRM 5 önemli ölçüde daha yüksek olabilir. Ancak, gelişmekte olan immünoanalizler pahalı olabilir ve / veya zaman alıcı ve elde edilen analizler hücrenin / doku lizis / homojenizasyon yöntemleri ile uyumsuz, çapraz reaksiyon ve / veya müdahale savunmasız olabilir ve / veya uygun olmayan 5.8 multiplekslemesi için. Bu sorunlardan bazıları antikor ve MS tabanlı teknikler birleştirilmesi ele alınabilir. Örneğin, hedef proteinleri önce proteoliz ve LC-SRM 9-12 immunoprecipitation kullanılarak zenginleştirilebilir. Seçenek olarak ise, SISCAPA tekniği peptid leve de proteolize sonra imüno kullanmaktadırl 13,14. Buna ek olarak stratejiler immunoenrichment için, yüksek bolluk proteinlerin bağışıklık sistemine analitleri 15,16 elüsyona alarak paraziti azaltarak LC-SRM duyarlılığını artırmak için istihdam edilebilir. MS-bazlı protein ölçümü etiket özgür ve izotop etiketleme (örneğin, metabolik etiketleme, etiketleme kimyasal ve ağır etiketli protein ve peptid iç standartlar) içine de göreli ve mutlak ölçümü ayrılır ve yapılabilir. Etiket serbest teknikler göreli protein ölçümü için yararlı olabilir, ancak kesin mutlak ölçümü için uygun değildir olabilir. Karşılaştırma olarak, etiketleme teknikleri numune hazırlama ve MS varyans ile ilişkili hata azaltmıştır, ve çoğu zaman görece protein miktarının 17 kullanılmaktadır. Örneğin, izotop etiketli proteome (SILAP) standartları, insan serumu 18 LC-ARM üzerinden olası biyolojik belirteçler nispi miktarını etkin kültürlenmiş insan hücre hattı kullanılarak hazırlandı. MS ile Doğru mutlak protein ölçümü saflaştırılmış, sayısal, izotopik olarak etiketlenmiş protein ya da peptid iç standartlar çivili-içine olması MS öncesinde biyolojik örnekler gerektirir. LC-SRM iş akışına ağır izotop etiketli dahili standartların dahil son derece tekrarlanabilir ve laboratuarlar 16,19 arasında transfer olduğu gösterilmiştir mutlak kantifikasyonunu sağlar. MS ile saf protein ölçümü için izotop etiketli dahili standartlar peptid standartları, katı faz sentezi 20, birleştirilmiş proteaz klivaj peptid standartları 21 oluşan proteinler, ve tam uzunluktaki protein standartlarını 22 kullanılarak hazırlandı bulunmaktadır. Hedef protein kovalent modifikasyon ve eksik numune hazırlama (yani eksik örnek parçalama ve homojenizasyon ve eksik protein solübilizasyon, denatürasyonu, alkilasyon ve proteoliz) doğru kantifikasyon sarsar. İç protein standartlar bu potansiyel sorunların en etkilenecek en az olasıdır, ancak genellikle hazırlamak için en zor bulunmaktadır. Alternatif bir amino- ve karboksi-terminal ana kuşatan kalıntılarını içerecek şekilde tasarlanan çok sayıda dahili peptid standartları kullanılarak her bir hedef protein analiz etmektir. Ne olursa olsun, iç standart türü kullanıldığında bunlar, bu katkılı halinde gereken biyolojik örnekler gibi erken bir noktada mümkün olduğu numune hazırlama sırasında. Ayrıca, birden çok örnek hazırlama teknikleri (örneğin, farklı bir denatürasyon koşulları) test edilmelidir. Birden dik deneysel teknikler (deneysel çapraz doğrulama) kullanımı 23-25 ​​zorluklar çoğu potansiyel kantifikasyon üstesinden gelmek için uygun bir stratejidir. Proteinlerin LC-SRM miktar geniş bir uygulama yelpazesinde kullanılabilir olan son derece esnek bir tekniktir. Özellikle, bu içindeki peptid ve protein biyolojik işaretleri incelemek için kullanılmıştırBöyle serum, çekirdek biyopsiler ve ince iğne aspiratları 5 klinik örnekler. LC-SRM ayrıca, botulinum nörotoksinlerinin 27 tespit etmek için sinyal yolları 5 içinde protein fosforilasyon dinamikleri ölçmek için ve protein konformasyonuna 28 değişiklikleri ölçmek için, protein kompleksleri 5,26 stokiyometri ölçmek için kullanılmıştır. Laboratuvarımız kemotaksis yolu simülasyonları gelişimini desteklemek için makrofaj kemotaksisini arabuluculuk sinyal proteinleri ölçmek için LC-SRM kullanıyor. protokol genel şeması (Şekil 1) kesin olmayan hedef peptidleri sıralaması ile başlar. Daha sonra, ham dış peptid standartları sentezlendi ve biyolojik numunelerin nitel analiz için LC-SRM tahlilleri geliştirmek için kullanılır. Biyolojik numune türetilen hedef peptid tespit edilirse, saflaştırılmış ağır etiketli dahili peptid standartları niceliksel LC-ARM hazırlanır. Bu protokol, ac için kullanılabilircurately biyolojik örneklerin çeşitli proteinleri ölçmek ve protein hedeflerinin çok çeşitli araştırmalar desteklemek.

<table><tbody><tr><td>Acetonitrile (ACN), LC-MS grade</td><td>Fisher</td><td>A955-1</td><td></td></tr><tr><td>BCA (bicinchoninic acid) protein assay kit</td><td>Fisher</td><td>23235</td><td></td></tr><tr><td>Beads for bead beating, zirconia-silica, 0.1 mm</td><td>BioSpec Products</td><td>11079101z</td><td></td></tr><tr><td>Bestatin hydrochloride</td><td>Sigma</td><td>B8385-10MG</td><td></td></tr><tr><td>Cell culture DMEM (with glucose, without L-glutamine)</td><td>Lonza</td><td>12-614F</td><td></td></tr><tr><td>Cell culture EDTA, 500 mM, pH8</td><td>Gibco</td><td>15575</td><td></td></tr><tr><td>Cell culture fetal bovine serum (FBS)</td><td>Atlanta Biologicals</td><td>S11550</td><td></td></tr><tr><td>Cell culture L-glutamine</td><td>Sigma</td><td>G8540-25G</td><td></td></tr><tr><td>Cell culture phosphate buffered saline (PBS) pH 7.4</td><td>Gibco</td><td>10010-049</td><td></td></tr><tr><td>Cell culture Trypan Blue viability stain, 0.4% w/v</td><td>Lonza</td><td>17-942E</td><td></td></tr><tr><td>Cellometer Auto T4 cell counter</td><td>Nexcelom Bioscience</td><td>Cellometer Auto T4</td><td></td></tr><tr><td>Cellometer Auto T4 disposable counting chambers</td><td>Nexcelom Bioscience</td><td>CHT4-SD100-014</td><td></td></tr><tr><td>Dithiothreitol (DTT)</td><td>Sigma</td><td>D5545-5G</td><td></td></tr><tr><td>Formic acid, LC-MS grade, ampules</td><td>Fisher</td><td>A117-10X1AMP</td><td></td></tr><tr><td>Hemocytometer, Neubauer-improved, 0.1 mm deep</td><td>Marienfeld-Superior</td><td>0640030</td><td></td></tr><tr><td>HEPES, 1 M, pH 7.2</td><td>Mediatech</td><td>25-060-CI</td><td></td></tr><tr><td>Hydrochloric acid, 37% w/w</td><td>VWR</td><td>BDH3028-2.5LG</td><td></td></tr><tr><td>Iodoacetamide</td><td>Sigma</td><td>I1149-5G</td><td></td></tr><tr><td>Laser Based Micropipette Puller</td><td>Sutter Instrument Co.</td><td>P-2000</td><td></td></tr><tr><td>LC coated silica capillary, 50 &amp;micro;m id</td><td>Polymicro Technologies</td><td>1068150017</td><td></td></tr><tr><td>LC vial, autosampler, 12 mm x 32 mm polypropylene</td><td>SUN SRI</td><td>200-268</td><td></td></tr><tr><td>LC vial screw cap, autosampler, pre-slit PTFE/silicone</td><td>SUN SRI</td><td>500-061</td><td></td></tr><tr><td>Luciferase, from Photinus pyralis</td><td>Sigma</td><td>L9506-1MG</td><td></td></tr><tr><td>Pepstatin A</td><td>EMD Millipore</td><td>516481-25MG</td><td></td></tr><tr><td>pH strips colorpHast (pH 0.0-6.0)</td><td>EMD Chemicals</td><td>9586-1</td><td></td></tr><tr><td>PhosStop phosphatase inhibitor cocktail</td><td>Roche</td><td>04906837001</td><td></td></tr><tr><td>RapiGest SF</td><td>Waters</td><td>186001861</td><td></td></tr><tr><td>Sep-Pak SPE, C18 1 ml 100 mg cartridge</td><td>Waters</td><td>WAT023590</td><td></td></tr><tr><td>Sep-Pak SPE, extraction manifold, 20 position</td><td>Waters</td><td>WAT200609</td><td></td></tr><tr><td>Sep-Pak SPE, flat-surfaced rubber bulb</td><td>Fisher</td><td>03-448-25</td><td></td></tr><tr><td>Sodium hydroxide (NaOH)</td><td>Fisher</td><td>S318-500</td><td></td></tr><tr><td>SpeedVac vacuum concentrator</td><td>Fisher</td><td>SPD111V</td><td></td></tr><tr><td>Trifluoroacetic acid (TFA), LC-MS grade</td><td>Fisher</td><td>A116-50</td><td></td></tr><tr><td>Trypsin, sequencing grade, modified</td><td>Promega</td><td>V5113</td><td></td></tr><tr><td>Tube decapper for Micronic tubes</td><td>USA Scientific</td><td>1765-4000</td><td></td></tr><tr><td>Tubes, 2 ml microcentrifuge, o-ring screw-cap, sterile</td><td>Sarstedt</td><td>72.694.006</td><td></td></tr><tr><td>Urea</td><td>Sigma</td><td>U0631-500g</td><td></td></tr><tr><td>Water, LC-MS grade</td><td>Fisher</td><td>W6-1</td><td></td></tr></tbody></table>

selected reaction monitoring mass spectrometry for absolute protein quantification

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This protocol provides step-by-step instructions for the development and application of quantitative assays using selected reaction monitoring (SRM) mass spectrometry (MS). First, likely quantotypic target peptides are identified based on numerous criteria. This includes identifying proteotypic peptides, avoiding sites of posttranslational modification, and analyzing the uniqueness of the target peptide to the target protein. Next, crude external peptide standards are synthesized and used to develop SRM assays, and the resulting assays are used to perform qualitative analyses of the biological samples. Finally, purified, quantified, heavy isotope labeled internal peptide standards are prepared and used to perform isotope dilution series SRM assays. Analysis of all of the resulting MS data is presented. This protocol was used to accurately assay the absolute abundance of proteins of the chemotaxis signaling pathway within RAW 264.7 cells (a mouse monocyte/macrophage cell line). The quantification of Gi2 (a heterotrimeric G-protein &#945;-subunit) is described in detail.

sinyal iletim yollarının tahmini hesaplama modellerinin geliştirilmesi sistem biyolojisi 53 temel hedeflerinden biridir. Ne yazık ki, hatta yoğun çalışılan ve yüksek klinik öneme sahip olan sinyal yolları için, bu nicel tedirginlikler cevaben yolu davranışlarını tahmin etmek için genellikle hala mümkün değildir (örn bu MAPK / ERK yolu 54 için geçerlidir). Son zamanlarda, bir soruşturma yolunun 56 sinyal fare makrofaj kemotaksisini çalışmak için hedef...

Watch this Scientific Journal Video about Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification at JoVE.com

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

This protocol describes how to perform absolute quantification assays of target proteins within complex biological samples using selected reaction monitoring. It was used to accurately quantify proteins of the mouse macrophage chemotaxis signaling pathway. Target peptide selection, assay development, and qualitative and quantitative assays are described in detail.

Mutlak Protein Niceleme Kütle Spektrometresi İzleme Seçilen Tepki

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This ...

selected-reaction-monitoring-mass-spectrometry-for-absolute-protein

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

17.0K Görüntüleme.  National Institutes of Health. This protocol describes how to perform absolute quantification assays of target proteins within complex biological samples using selected reaction monitoring. It was used to accurately quantify proteins of the mouse macrophage chemotaxis signaling pathway. Target peptide selection, assay development, and qualitative and quantitative assays are described in detail.

Video: Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

A Simple Fractionated Extraction Method for the Comprehensive Analysis of Metabolites, Lipids, and Proteins from a Single Sample

Understanding of complex biological systems requires the measurement, analysis and integration of multiple compound classes of the living cell, usually determined by transcriptomic, proteomic, metabolomics and lipidomic measurements. In this protocol, we introduce a simple method for the reproducible extraction of metabolites, lipids and proteins from biological tissues using a single aliquot per sample. The extraction method is based on a methyl tert-butyl ether: methanol: water system for liquid: liquid partitioning of hydrophobic and polar metabolites into two immiscible phases along with the precipitation of proteins and other macromolecules as a solid pellet. This method, therefore, provides three different fractions of specific molecular composition, which are fully compatible with common high throughput 'omics' technologies such as liquid chromatography (LC) or gas chromatography (GC) coupled to mass spectrometers. Even though the method was initially developed for the analysis of different plant tissue samples, it has proved to be fully compatible for the extraction and analysis of biological samples from systems as diverse as algae, insects, and mammalian tissues and cell cultures.

A protocol for comprehensive extraction of lipids, metabolites and proteins from biological tissues using one sample is presented.

Tek Bir Numuneden Metabolitlerin, Lipidlerin ve Proteinlerin Kapsamlı Analizi için Basit Fraksiyonlu Ekstraksiyon Metodu

Understanding of complex biological systems requires the measurement, analysis and integration of multiple compound classes of the living cell, usually ...

Biyokimya

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) ...

An HS-MRM Assay for the Quantification of Host-cell Proteins in Protein Biopharmaceuticals by Liquid Chromatography Ion Mobility QTOF Mass Spectrometry

The analysis of low-level (1-100 ppm) protein impurities (e.g., host-cell proteins (HCPs)) in protein biotherapeutics is a challenging assay requiring high sensitivity and a wide dynamic range. Mass spectrometry-based quantification assays for proteins typically involve protein digestion followed by the selective reaction monitoring/multiple reaction monitoring (SRM/MRM) quantification of peptides using a low-resolution (Rs ~1,000) tandem quadrupole mass spectrometer. One of the limitations of this approach is the interference phenomenon observed when the peptide of interest has the "same" precursor and fragment mass (in terms of m/z values) as other co-eluting peptides present in the sample (within a 1-Da window). To avoid this phenomenon, we propose an alternative mass spectrometric approach, a high selectivity (HS) MRM assay that combines the ion mobility separation of peptide precursors with the high-resolution (Rs ~30,000) MS detection of peptide fragments. We explored the capabilities of this approach to quantify low-abundance peptide standards spiked in a monoclonal antibody (mAb) digest and demonstrated that it has the sensitivity and dynamic range (at least 3 orders of magnitude) typically achieved in HCP analysis. All six peptide standards were detected at concentrations as low as 0.1 nM (1 femtomole loaded on a 2.1-mm ID chromatographic column) in the presence of a high-abundance peptide background (2 &#181;g of a mAb digest loaded on-column). When considering the MW of rabbit phosphorylase (97.2 kDa), from which the spiked peptides were derived, the LOQ of this assay is lower than 50 ppm. Relative standard deviations (RSD) of peak areas (n = 4 replicates) were less than 15% across the entire concentration range investigated (0.1-100 nM or 1-1,000 ppm) in this study.

Here, we describe a chromatographic assay coupled with the ion mobility separation of peptide precursors followed by the high-resolution (~30,000) MS-detection of peptide fragments for the quantification of spiked peptide standards in a monoclonal antibody digest.

Bir HS-MRM tahlil için Protein Biyofarmasötikler sıvı Kromatografi iyon hareketlilik QTOF kütle spektrometresi tarafından Host-hücre proteinleri miktar

The analysis of low-level (1-100 ppm) protein impurities (e.g., host-cell proteins (HCPs)) in protein biotherapeutics is a challenging assay requiring ...

Kimya

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 ...

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 ...

Carrier-assisted One-pot Sample Preparation for Targeted Proteomics Analysis of Small Numbers of Human Cells

Protein analysis of small numbers of human cells is primarily achieved by targeted proteomics with antibody-based immunoassays, which have inherent limitations (e.g., low multiplex and unavailability of antibodies for new proteins). Mass spectrometry (MS)-based targeted proteomics has emerged as an alternative because it is antibody-free, high multiplex, and has high specificity and quantitation accuracy. Recent advances in MS instrumentation make MS-based targeted proteomics possible for multiplexed quantification of highly abundant proteins in single cells. However, there is a technical challenge for effective processing of single cells with minimal sample loss for MS analysis. To address this issue, we have recently developed a convenient protein carrier-assisted one-pot sample preparation coupled with liquid chromatography (LC) - selected reaction monitoring (SRM) termed cLC-SRM for targeted proteomics analysis of small numbers of human cells. This method capitalizes on using the combined excessive exogenous protein as a carrier and low-volume one-pot processing to greatly reduce surface adsorption losses as well as high-specificity LC-SRM to effectively address the increased dynamic concentration range due to the addition of exogeneous carrier protein. Its utility has been demonstrated by accurate quantification of most moderately abundant proteins in small numbers of cells (e.g., 10-100 cells) and highly abundant proteins in single cells. The easy-to-implement features and no need for specific devices make this method readily accessible to most proteomics laboratories. Herein we have provided a detailed protocol for cLC-SRM analysis of small numbers of human cells including cell sorting, cell lysis and digestion, LC-SRM analysis, and data analysis. Further improvements in detection sensitivity and sample throughput are needed towards targeted single-cell proteomics analysis. We anticipate that cLC-SRM will be broadly applied to biomedical research and systems biology with the potential of facilitating precision medicine.

A protein carrier-assisted one-pot sample preparation coupled with liquid chromatography (LC) - selected reaction monitoring (SRM) termed cLC-SRM is a convenient method for multiplexed targeted proteomics analysis of small numbers of cells, including single cells. It capitalizes on using excessive exogenous protein as a carrier and high-specificity LC-SRM for targeted quantification.

Protein analysis of small numbers of human cells is primarily achieved by targeted proteomics with antibody-based immunoassays, which have inherent ...

Microsampling in Targeted Mass Spectrometry-Based Protein Analysis of Low-Abundance Proteins

This paper presents a protocol with detailed descriptions for efficient sample cleanup of low-abundance proteins from dried samples. This is performed using bead-based proteolysis prior to proteotypic peptide affinity-capture and liquid chromatography tandem mass spectrometry (LC-MS/MS) determination. The procedure can be applied to both conventional dried samples using paper cards (e.g., dried blood spots [DBSs] and dried serum spots [DSSs]), as well as samples collected with newer sampling methods such as volumetric absorptive microsampling (VAMS). In addition to describing this procedure, the preparation of both trypsin beads and antibody-coated beads is presented in a step-by-step manner in this work. The advantages of the presented procedure are time-efficient proteolysis using beads and selective robust cleanup using peptide affinity-capture. The current procedure describes the determination of the low-abundance small-cell lung cancer (SCLC) biomarker, progastrin-releasing peptide (ProGRP), in dried serum (both DSSs and VAMS). Detailed procedures for bead preparation make it easier to implement the workflow in new applications or other laboratories. It is demonstrated that the results may be dependent on the sampling material; for the present project, higher signal intensities were seen for samples collected using VAMS compared to DSSs.

A protocol is presented for the determination of low-abundance biomarkers from dried serum samples exemplified with the biomarker progastrin-releasing peptide (ProGRP). Antibody-coated magnetic beads are used for the selective cleanup and enrichment of a proteotypic ProGRP peptide. The captured peptide is subsequently analyzed by liquid chromatography-tandem mass spectrometry.

Düşük Bolluktaki Proteinlerin Hedeflenen Kütle Spektrometrisine Dayalı Protein Analizinde Mikro Örnekleme

This paper presents a protocol with detailed descriptions for efficient sample cleanup of low-abundance proteins from dried samples. This is performed ...

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1 An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2 In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4 and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated format.

Proteinler Peptid immunoaffiniti Zenginleştirme kullanarak Niceleme Kütle Spektrometresi ile birleştiğinde

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in ...

Biyoloji

Comprehensive Workflow of Mass Spectrometry-based Shotgun Proteomics of Tissue Samples

Recent advances in mass spectrometry have resulted in deep proteomic&#160;analysis along with the generation of&#160;robust and&#160;reproducible datasets. However, despite the considerable technical advancements, sample preparation from biospecimens such as patient blood, CSF, and tissue still poses considerable challenges. For identifying biomarkers, tissue proteomics often provides an attractive sample source to translate the research findings from the bench to the clinic. It can reveal potential candidate biomarkers for early diagnosis of cancer and neurodegenerative diseases such as Alzheimer&#39;s disease, Parkinson&#39;s disease, etc. Tissue proteomics also yields a wealth of systemic information based on the abundance of proteins and helps to address interesting biological questions.
Quantitative proteomics analysis can be grouped into two broad categories: a label-based and a label-free approach. In the label-based approach, proteins or peptides are labeled using stable isotopes such as SILAC (stable isotope labeling with amino acids in cell culture) or by chemical tags such as ICAT (isotope-coded affinity tags), TMT (tandem mass tag) or iTRAQ (isobaric tag for relative and absolute quantitation). Label-based approaches have the advantage of more accurate quantitation of proteins and using isobaric labels, multiple samples can be analyzed in a single experiment. The label-free approach provides a cost-effective alternative to label-based approaches. Hundreds of patient samples belonging to a particular cohort can be analyzed and compared with other cohorts based on clinical features. Here, we have described an optimized quantitative proteomics workflow for tissue samples using label-free and label-based proteome profiling methods, which is crucial for applications in life sciences, especially biomarker discovery-based projects.

The described protocol provides an optimized quantitative proteomics analysis of tissue samples using two approaches: label-based and label free quantitation. Label-based approaches have the advantage of more accurate quantitation of proteins, while a label-free approach is more cost-effective and used to analyze hundreds of samples of a cohort.

Doku Örneklerinin Kütle Spektrometresi Bazlı Av Tüfeği Proteomik Kapsamlı İş Akışı

Recent advances in mass spectrometry have resulted in deep proteomic&#160;analysis along with the generation of&#160;robust and&#160;reproducible ...

Quantitative Proteomics Workflow using Multiple Reaction Monitoring Based Detection of Proteins from Human Brain Tissue

The proteomic analysis of the human brain tissue over the last decade has greatly enhanced our understanding of the brain. However, brain related disorders continue to be a major contributor of deaths around the world, necessitating the need for even greater understanding of their pathobiology. Traditional antibody-based techniques like western blotting or immunohistochemistry suffer from being low-throughput besides being labor-intensive and qualitative or semi-quantitative. Even conventional mass spectrometry-based shotgun approaches fail to provide conclusive evidence to support a certain hypothesis. Targeted proteomics approaches are largely hypothesis driven and differ from the conventional shotgun proteomics approaches that have been long in use. Multiple reaction monitoring is one such targeted approach that requires the use of a special mass spectrometer called the tandem quadrupole mass spectrometer or triple quadrupole mass spectrometer. In the current study, we have systematically highlighted the major steps involved in performing a successful tandem quadrupole mass spectrometry-based proteomics workflow using human brain tissue with an aim to introduce this workflow to a broader research community.

The protocol aims to introduce the use of a triple quadrupole mass spectrometer for Multiple Reaction Monitoring (MRM) of proteins from clinical samples. We have provided a systematic workflow starting from sample preparation to data analysis for clinical samples with all the necessary precautions to be taken.