Isobaric Reagents Research Articles

Multiplex Quantitative Glycomics Enabled by Periodate Oxidation and Triplex Mass Defect Isobaric Multiplex Reagents for Carbonyl-Containing Compound Tags.

Glycosylation is one of the most important post-translational modifications (PTMs) with essential physiological functions, including protein folding, cell signaling, and immune response. Thus, various qualitative and quantitative glycomics analysis strategies have been developed. Recently, the isobaric multiplex reagents for carbonyl-containing compound (SUGAR) tag was developed for quantitative glycomics with multiplexing capacity and increased reporter ion yield. To further improve quantification efficiency and enable quantifying low-abundance species, the mass defect based triplex SUGAR (mdSUGAR) tag has been designed. In addition, we also introduce additional reaction sites for mdSUGAR at the terminal sialic acid by periodate oxidation of the polyhydroxy chain to extend the mass difference and lower the requirement for resolving power. As a result, mdSUGAR tags show complete labeling efficiency, improved fragmentation pattern, and accurate quantification. Moreover, the quantitative performance of the mdSUGAR tags in a complex system has been systematically evaluated and demonstrated reliable results.

Exosome Proteomics and Machine Learning Identify Novel Biomarkers of Primary Graft Dysfunction

Purpose The etiology of primary graft dysfunction (PGD) is unclear and prediction of PGD remains an area of active research. Exosomes are secreted microvesicles found in numerous body fluids and have protein and RNA content with biomarker potential. Here we investigate pre-transplant serum exosomes using proteomics to identify novel recipient biomarkers of PGD prior to heart transplant. Methods Pre-transplant serum samples were collected prospectively at Columbia University from June 2014 to December 2015 (n=16). Pre-transplant serum samples were assembled retrospectively from HLA laboratories of Cedars Sinai Medical Center (n=44) and Pitie Salpetriere (n=29). Exosomes were purified with the Total Exosome Isolation Kit (Life Technologies) and proteins were labelled with TMT10plex isobaric reagent. We performed LC-MS/MS with an Orbitrap Fusion Tribrid mass spectrometer. Proteome Discoverer software was used to search the acquired MS/MS data against Uniprot human protein database and generate TMT ratios. Conditional logistic regressions with bootstrapping modeled the association of each protein to PGD status controlling for LVAD status and batch. Logistic Regression (LR), Random Forest (RF), Support Vector Machines (SVM), and Gradient Boosting Classifier (GBC) models, with regularization, and 10-fold cross validated recursive feature elimination (RFE) were used to select the most predictive proteins in the dataset. Results 131 proteins were found in the prospective cohort showing a differential protein expression signature (FDR Conclusion Exosome proteomics of serum samples from both prospective and retrospective cohorts followed by machine learning based analysis identified potential biomarkers for PGD within the recipient prior to transplant.

Trends in the Design of New Isobaric Labeling Reagents for Quantitative Proteomics.

Modern mass spectrometry is one of the most frequently used methods of quantitative proteomics, enabling determination of the amount of peptides in a sample. Although mass spectrometry is not inherently a quantitative method due to differences in the ionization efficiency of various analytes, the application of isotope-coded labeling allows relative quantification of proteins and proteins. Over the past decade, a new method for derivatization of tryptic peptides using isobaric labels has been proposed. The labels consist of reporter and balanced groups. They have the same molecular weights and chemical properties, but differ in the distribution of stable heavy isotopes. These tags are designed in such a way that during high energy collision induced dissociation (CID) by tandem mass spectrometry, the isobaric tag is fragmented in the specific linker region, yielding reporter ions with different masses. The mass shifts among the reporter groups are compensated by the balancing groups so that the overall mass is the same for all forms of the reagent. Samples of peptides are labeled with the isobaric mass tags in parallel and combined for analysis. Quantification of individual peptides is achieved by comparing the intensity of reporter ions in the tandem mass (MS/MS) spectra. Isobaric markers have found a wide range of potential applications in proteomics. However, the currently available isobaric labeling reagents have some drawbacks, such as high cost of production, insufficient selectivity of the derivatization, and relatively limited enhancement of sensitivity of the analysis. Therefore, efforts have been devoted to the development of new isobaric markers with increased usability. The search for new isobaric markers is focused on developing a more selective method of introducing a tag into a peptide molecule, increasing the multiplexicity of markers, lowering the cost of synthesis, and increasing the sensitivity of measurement by using ionization tags containing quaternary ammonium salts. Here, the trends in the design of new isobaric labeling reagents for quantitative proteomics isobaric derivatization strategies in proteomics are reviewed, with a particular emphasis on isobaric ionization tags. The presented review focused on different types of isobaric reagents used in quantitative proteomics, their chemistry, and advantages offer by their application.

Active Instrument Engagement Combined with a Real-Time Database Search for Improved Performance of Sample Multiplexing Workflows.

Quantitative proteomics employing isobaric reagents has been established as a powerful tool for biological discovery. Current workflows often utilize a dedicated quantitative spectrum to improve quantitative accuracy and precision. A consequence of this approach is a dramatic reduction in the spectral acquisition rate, which necessitates the use of additional instrument time to achieve comprehensive proteomic depth. This work assesses the performance and benefits of online and real-time spectral identification in quantitative multiplexed workflows. A Real-Time Search (RTS) algorithm was implemented to identify fragment spectra within milliseconds as they are acquired using a probabilistic score and to trigger quantitative spectra only upon confident peptide identification. The RTS-MS3 was benchmarked against standard workflows using a complex two-proteome model of interference and a targeted 10-plex comparison of kinase abundance profiles. Applying the RTS-MS3 method provided the comprehensive characterization of a 10-plex proteome in 50% less acquisition time. These data indicate that the RTS-MS3 approach provides dramatic performance improvements for quantitative multiplexed experiments.

Isobaric Multiplex Labeling Reagents for Carbonyl-Containing Compound (SUGAR) Tags: A Probe for Quantitative Glycomic Analysis.

Glycans are highly complex entities with multiple building units and different degrees of branched polymerization. Intensive research efforts have been directed to mass spectrometry (MS)-based qualitative and quantitative glycomic analysis due to the important functions of glycans. Among various strategies, isobaric labeling has become popular because of its higher multiplexing capacity. Over the past few years, several isobaric chemical tags have been developed for quantitative glycomics. However, caveats also exist for these tags, such as relatively low reporter ion yield for aminoxyTMT-labeled complex glycans. To overcome the limitations of existing isobaric chemical tags, we designed a class of novel isobaric multiplex reagents for carbonyl-containing compound (SUGAR) tags that can be used to label glycans for quantitative glycomic analysis. The quantitative performance including labeling efficiency, quantification accuracy, and dynamic range of these SUGAR tags has been evaluated, showing promising results. Finally, the 4-plex SUGAR tags have been utilized to investigate N-glycan changes of B-cell acute lymphoblastic leukemia (ALL) pediatric patients before and after chemotherapy.

Multiplexed quantitative phosphoproteomics of cell line and tissue samples.

Post-translational modifications (PTMs) of proteins increase a biological system's repertoire of regulatory tools to control cellular mechanisms. Protein phosphorylation is the most studied PTM and known to be dysregulated in many diseases, including cancer, and protein kinases are among the most important drug targets. Many proteins across the eukaryotic proteome are phosphorylated, and more than 50,000 unique protein phosphorylation sites have been identified in a single human cell line. Understanding the vast biological networks directed by protein phosphorylation requires deep quantitative mapping of the phosphoproteome across many samples. Multiplexed proteomics using isobaric labeling reagents to barcode proteome samples for simultaneous quantification has greatly increased the throughput of mass spectrometry-based proteomics and enabled the number of analyses required to understand complex biological systems. We are presenting a detailed protocol to use multiplexed proteomics for mapping phosphoproteomes in samples from cell culture experiments and in tissue samples. The protocol includes phosphopeptide enrichment with TiO2 and phosphotyrosine antibody technology. We are using tandem mass tag (TMT) reagents for barcoding the samples allowing parallel quantification of up to 11 samples. The mass spectrometry method is based on the MultiNotch MS3 method to generate quantitative data of high accuracy and reproducibility. Tandem mass spectrometry (MS2) based on regular collision-induced dissociation (CID) and higher-energy collisional dissociation (HCD) is used to maximize the number of quantified phosphopeptides. The protocol typically enables the quantification of more than 20,000 unique phosphoforms (unique patterns of peptide phosphorylations) from proteome samples of human origin requiring less than 8h of mass spectrometry time per sample.

Abstract 5660: Proteomic comparative assessment of flash-frozen and OCT embedded breast cancer tissues for utilization in precision oncology discovery studies

Abstract Proteomic analysis is an important tool in classifying molecular architecture of tissue samples for subtyping and precision oncology applications. Tissues are commonly either flash frozen (FF) or embedded in optimal cutting temperature (OCT) compound for long-term storage. OCT provides a convenient specimen matrix for cryostat sectioning but contains glycols and resins which are incompatible with mass spectrometric analysis of proteins. In contrast, flash frozen preservation of tissue is historically considered to be more suitable and compatible with mass spectrometry applications. As a pilot study, we evaluated different tissue preservation methods, sample processing methods, and protein extraction methods, and their impact on quantitation and coverage of the proteome. In this pilot study, 30 breast cancer cases with matching flash frozen and OCT-embedded samples were used for proteomic analysis. Tissues were procured from the Clinical Breast Care Project (CBCP) in partnership with the Chan Soon-Shiong Institute of Molecular Medicine at Windber, and the Walter Reed National Military Medical Center (WRNMMC). Breast cancer subtypes were determined by clinical and expanded immunohistochemical panels. These subtypes were Her2, Luminal A (LA), Luminal B (LB) and Triple Negative (TN). Flash frozen tissues were cryosectioned to isolate 60-100 mg total tissue per case, and proteins were extracted using an 8M urea lysis buffer. The OCT specimens were laser microdissected to isolate primarily tumor cells from the samples. Proteins were extracted using the illustra TriplePrep kit. Digested proteins were then multiplexed using Tandem-Mass-Tag (TMT) 10plex isobaric labeling reagents, and chromatographically resolved using a Waters 2D nanoAcquity liquid chromatographer (LC) coupled to a Thermo Q Exactive Plus mass spectrometer (MS). Data was then analyzed using Proteome Discoverer. In total, 6130 proteins were identified using a 1% peptide FDR confidence filter in the 30 samples analyzed. Unsupervised hierarchical clustering of proteins across the samples resulted in two primary clusters, OCT and FF. Within the primary clustering, there were sub-clusters of Luminal, Her2, and Triple Negatives. At 2-fold threshold, 246 proteins were differentially expressed between OCT and FF tissues. However, the differential proteins between ER+ (LA and LB) and ER- (Her2 and TN) tumors correlate irrespective of the cryopreservation and processing methods. In conclusion, proteome analysis of samples stored under two different conditions provide subtyping information; however, there is differential expression of proteins between the two types of sample handling techniques which can impact biological interpretation of breast cancer precision oncology studies. Citation Format: Kiki Panagopoulos, Punit Shah, Albert Kovatich, Michael A. Kiebish, Jeffrey Hooke, Leigh Campbell, Mary Lou Cutler, Audrey Kovatich, Hai Hu, Brenda Deyarmin, Lori Sturtz, Praveen Kumar Raj Kumar, Stella Somiari, Emily Y. Chen, Elder Granger, Viatcheslav Akmaev, Rangaprasad Sarangarajan, Niven Narain, Craig D. Shriver. Proteomic comparative assessment of flash-frozen and OCT embedded breast cancer tissues for utilization in precision oncology discovery studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5660.

EASI-tag enables accurate multiplexed and interference-free MS2-based proteome quantification

We developed EASI-tag (easily abstractable sulfoxide-based isobaric-tag), a new type of amine-derivatizing and sulfoxide-containing isobaric labeling reagents for highly accurate quantitative proteomics analysis using mass spectrometry. We observed that EASI-tag labels dissociate at low collision energy and generate peptide-coupled, interference-free reporter ions with high yield. Efficient isolation of 12C precursors and quantification at the MS2 level allowed accurate determination of quantitative differences between up to six multiplexed samples.

A Sulfoxide-Based Isobaric Labelling Reagent for Accurate Quantitative Mass Spectrometry.

Modern proteomics requires reagents for exact quantification of peptides in complex mixtures. Peptide labelling is most typically achieved with isobaric tags that consist of a balancer and a reporter part that separate in the gas phase. An ingenious distribution of stable isotopes provides multiple reagents with identical molecular weight but a different mass of the reporter groups, allowing relative quantification of multiple samples in one measurement. Here we report a new isobaric labelling reagent, where the balancer and the reporter are linked by a sulfoxide group, which, based on the sulfoxide pyrolysis, leads to easy and asymmetric cleavage at low fragmentation energy. The fragmentation of our new design is significantly improved, yielding more intense complementary ion signals, allowing complementary ion cluster analysis as well.

Towards a 44-plex cPILOT

Event Abstract Back to Event Towards a 44-plex cPILOT David A. Vassallo Rodríguez1 and Renā A. Robinson1* 1 Vanderbilt University, United States Our laboratory created a novel method, “combined precursor isotopic labeling and isobaric tagging (cPILOT)” to increase the multiplexing capability of quantitative proteomics. This scheme takes advantage of the pka difference between the N-terminal and Lysine primary amines to selectively incorporate chemical tags through manipulation of buffer pH. The use of heavy isotopic tags and isobaric reagents such as tandem mass tags (TMT) has made it possible to multiplex 24 samples in a single experiment to date. The current work aims to develop novel chemical strategies to expand the multiplexing capability of cPILOT. Specifically, we are evaluating if other amine chemistries, such as diethylation and propionylation, can be incorporated into the cPILOT workflow. For this work, we are using mouse brain tissue to generate complex tryptic peptide mixtures that we can independently diethylate or propionylate. Diethylation and propionylation increases peptide masses by 56.0626 and 56.0262 Da, respectively. Experimental variables such as buffer composition, buffer pH, reaction time and temperature are optimized in order to increase the specificity and labelling efficiency for N-terminal primary amines. For diethylation, peptides are resuspended in a coupling buffer, to which acetaldehyde is later added and this final solution is incubated at 37°C for 25 minutes. For propionylation, propionic anhydride is added to the peptides, pH adjusted, the solution is incubated at 37°C for 15 minutes, and these steps are performed twice more for high labelling efficiency. Samples are then TMT tagged and taken through a cPILOT workflow on a liquid chromatography coupled tandem mass spectrometry system. This poster will present our preliminary data on the incorporation of diethylation and propionylation in to the cPILOT workflow. Keywords: multiplexing, cPILOT, Enhanced multiplexing, Proteomics, reductive amination Conference: National Organization for the Professional Advancement of Black Chemists and Chemical Engineers (NOBCChE) 45th Annual Conference , Orlando, Florida, United States, 17 Sep - 20 Sep, 2018. Presentation Type: Poster Presentation Topic: Analytical Chemsitry Citation: Vassallo Rodríguez DA and Robinson RA (2019). Towards a 44-plex cPILOT. Front. Chem. Conference Abstract: National Organization for the Professional Advancement of Black Chemists and Chemical Engineers (NOBCChE) 45th Annual Conference . doi: 10.3389/conf.fchem.2018.01.00059 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 20 Oct 2018; Published Online: 17 Jan 2019. * Correspondence: Prof. Renā A Robinson, Vanderbilt University, Nashville, United States, rena.as.robinson@vanderbilt.edu Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers David A Vassallo Rodríguez Renā A Robinson Google David A Vassallo Rodríguez Renā A Robinson Google Scholar David A Vassallo Rodríguez Renā A Robinson PubMed David A Vassallo Rodríguez Renā A Robinson Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Enhanced Sample Multiplexing of Tissues Using Combined Precursor Isotopic Labeling and Isobaric Tagging (cPILOT).

There is an increasing demand to analyze many biological samples for disease understanding and biomarker discovery. Quantitative proteomics strategies that allow simultaneous measurement of multiple samples have become widespread and greatly reduce experimental costs and times. Our laboratory developed a technique called combined precursor isotopic labeling and isobaric tagging (cPILOT), which enhances sample multiplexing of traditional isotopic labeling or isobaric tagging approaches. Global cPILOT can be applied to samples originating from cells, tissues, bodily fluids, or whole organisms and gives information on relative protein abundances across different sample conditions. cPILOT works by 1) using low pH buffer conditions to selectively dimethylate peptide N-termini and 2) using high pH buffer conditions to label primary amines of lysine residues with commercially-available isobaric reagents (see Table of Materials/Reagents). The degree of sample multiplexing available is dependent on the number of precursor labels used and the isobaric tagging reagent. Here, we present a 12-plex analysis using light and heavy dimethylation combined with six-plex isobaric reagents to analyze 12 samples from mouse tissues in a single analysis. Enhanced multiplexing is helpful for reducing experimental time and cost and more importantly, allowing comparison across many sample conditions (biological replicates, disease stage, drug treatments, genotypes, or longitudinal time-points) with less experimental bias and error. In this work, the global cPILOT approach is used to analyze brain, heart, and liver tissues across biological replicates from an Alzheimer's disease mouse model and wild-type controls. Global cPILOT can be applied to study other biological processes and adapted to increase sample multiplexing to greater than 20 samples.

Improvement of Quantitative Measurements in Multiplex Proteomics Using High-Field Asymmetric Waveform Spectrometry.

Quantitative proteomics using isobaric reagent tandem mass tags (TMT) or isobaric tags for relative and absolute quantitation (iTRAQ) provides a convenient approach to compare changes in protein abundance across multiple samples. However, the analysis of complex protein digests by isobaric labeling can be undermined by the relative large proportion of co-selected peptide ions that lead to distorted reporter ion ratios and affect the accuracy and precision of quantitative measurements. Here, we investigated the use of high-field asymmetric waveform ion mobility spectrometry (FAIMS) in proteomic experiments to reduce sample complexity and improve protein quantification using TMT isobaric labeling. LC-FAIMS-MS/MS analyses of human and yeast protein digests led to significant reductions in interfering ions, which increased the number of quantifiable peptides by up to 68% while significantly improving the accuracy of abundance measurements compared to that with conventional LC-MS/MS. The improvement in quantitative measurements using FAIMS is further demonstrated for the temporal profiling of protein abundance of HEK293 cells following heat shock treatment.

Developing a Multiplexed Quantitative Cross-Linking Mass Spectrometry Platform for Comparative Structural Analysis of Protein Complexes.

Cross-linking mass spectrometry (XL-MS) represents a recently popularized hybrid methodology for defining protein-protein interactions (PPIs) and analyzing structures of large protein assemblies. In particular, XL-MS strategies have been demonstrated to be effective in elucidating molecular details of PPIs at the peptide resolution, providing a complementary set of structural data that can be utilized to refine existing complex structures or direct de novo modeling of unknown protein structures. To study structural and interaction dynamics of protein complexes, quantitative cross-linking mass spectrometry (QXL-MS) strategies based on isotope-labeled cross-linkers have been developed. Although successful, these approaches are mostly limited to pairwise comparisons. In order to establish a robust workflow enabling comparative analysis of multiple cross-linked samples simultaneously, we have developed a multiplexed QXL-MS strategy, namely, QMIX (Quantitation of Multiplexed, Isobaric-labeled cross (X)-linked peptides) by integrating MS-cleavable cross-linkers with isobaric labeling reagents. This study has established a new analytical platform for quantitative analysis of cross-linked peptides, which can be directly applied for multiplexed comparisons of the conformational dynamics of protein complexes and PPIs at the proteome scale in future studies.

BioSpotlight / Citations

BioTechniquesVol. 60, No. 4 BioSpotlight / CitationsOpen AccessBioSpotlight / CitationsPatrick C.H. Lo & Nathan S. BlowPatrick C.H. LoSearch for more papers by this author & Nathan S. BlowSearch for more papers by this authorPublished Online:16 Mar 2018https://doi.org/10.2144/000114398AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInReddit Tagging along with carbonylated proteinsCarbonylation of proteins is a biomarker for oxidative stress that is traditionally studied using two complementary techniques: immunoblotting and tandem mass spectroscopy. For both of these methods, tagging of the carbonylated protein using probes that form a covalent bond with the reactive carbonyl plays an important role. In this month's issue, Timothy J. Griffin and colleagues from the University of Minnesota, along with researchers at Thermo Fisher, introduce a novel multifunctional tagging reagent for analysis of carbonylated proteins. This derivative of the tandem mass tag (TMT) isobaric labeling reagents, called aminoxyTMT, contains a carbonyl-reactive aminooxy group. The authors demonstrate that, in combination with the anti-TMT antibody, aminoxyTMT labeling of carbonylated proteins allows for their visualization in immunoblots and their enrichment from complex mixtures. When used for tandem mass spectroscopy, aminoxyTMT enables the identification of carbonylated proteins and the sites of their modification as well as the relative quantification of the carbonylation state.See “AminoxyTMT: A novel multi-functional reagent for characterization of protein carbonylation”dPCR samples can't stand the heatDigital PCR (dPCR) has greatly facilitated the accurate quantification of DNA mutations in tissue samples. Fragmentation of intact genomic DNA samples is required prior to dPCR, but there has been no study of the effects of different fragmentation methods on the performance of dPCR point mutation assays. In this issue of BioTechniques, Muneesh Tewari and her group at the University of Michigan examined three methods of fragmentation: heating, sonication, and restriction digestion. Wild-type genomic DNA treated by each method was analyzed by droplet dPCR to determine the wild-type and mutant allele counts for the KRAS and BRAF genes. While heating to 95°C is the easiest fragmentation method, it resulted in a high background of mutation detection for particular KRAS mutations compared to the other two methods. This effect appeared to be due to heat-induced mutations and specifically affected dPCR assays detecting guanine to adenine (G>A) mutations. Fragmentation by heating also overestimated gene copy number. These results indicate that researchers should be careful about which DNA fragmentation method they select for their dPCR-based mutation detection assays.See “Mutant DNA quantification by digital PCR can be confounded by heating during sample preparation”Measuring gene regulationRelating transcription factor binding to specific patterns of gene expression has proven to be difficult, particularly at the single-cell level. But now, Sepulveda et al. describe a workflow for measuring gene regulation in individual cells. Using the lysogeny maintenance promoter of phage lambda as a model system, the authors first simultaneously measured concentrations of the lambda repressor CI in single cells, as well as numbers of mRNA transcripts from the promoter. To measure CI concentrations, they used an immunofluorescence assay, while single-molecule fluorescence in situ hybridization (FISH) provided measurements of promoter activity. By then modeling the CI concentration and transcript data, the researchers were able to define activity patterns that corresponded to specific CI binding configurations and uncover unique insights into promoter state switching. While applied initially to the lysogeny maintenance promoter, this approach should be adaptable to other genetic systems for promoter binding and gene expression studies.Sepulveda et al. 2016. Measurement of gene regulation in individual cells reveals rapid switching between promoter states. Science. 351:1218-1222.Tissue bioprinting gets thickThree-dimensional bioprinting advances have made it possible to create complex tissue architectures in the lab. But more development is needed to fully realize the potential of this technique and use it to faithfully mimic the structure and longevity of human tissue. Now, Kolesky et al. report a new technique for 3-D bioprinting that allows long-term perfusion of thicker tissues on chips. To generate vascularized tissues within perfusion chips, they first printed silicone ink on the glass surface. After this, a cell-laden ink along with ink composed of a triblock copolymer was printed on the chip. The result was a network of interconnected channels lined with cells, which could be perfused over an extended period using external pumps. The authors validated their printing method by creating a simple tissue construct composed of two parallel channels embedded in a fibroblast matrix and then a more complex, heterogenous tissue microenvironment >1 cm thick. This new bioprinting approach is another step towards creating more complex tissue architectures in the lab that can be used for ex vivo and, eventually, in vivo applications.Kolesky, D. et al. 2016. Three-dimensional bioprinting of thick vascularized tissues. Proc. Natl. Acad. Sci. 113: 3179-3184.FiguresReferencesRelatedDetails Vol. 60, No. 4 Follow us on social media for the latest updates Metrics History Published online 16 March 2018 Published in print April 2016 Information© 2016 Author(s)PDF download

AminoxyTMT: A novel multi-functional reagent for characterization of protein carbonylation.

Protein carbonylation is a common oxidative stress (OS)-driven post-translational modification (PTM). Proteome-wide carbonylation events can best be characterized using a combination of analytical approaches. Immunoblotting of carbonylated proteins provides data on the extent of modifications within complex samples, as well as a broad comparison of carbonylation profiles between different biological states (e.g., disease versus control), while mass spectrometry (MS)-based analysis provides information on proteins susceptible to carbonylation, as well as the potential for quantitative characterization of specific sites of amino acid modification. Here, we present a novel use for aminoxyTMT, a derivative of the Tandem Mass Tag (TMT) isobaric labeling reagent, which utilizes an aminooxy functional group for covalent labeling of reactive carbonyls in proteins. When coupled with anti-TMT antibody, we demonstrate the use of aminoxyTMT for immunoblot profiling of protein carbonylation in complex mixtures, as well as enrichment of modified peptides from these mixtures. Proof-of-principle experiments also show the amenability of aminoxyTMT-labeled carbonylated peptides enriched from complex mixtures to identification using tandem MS (MS/MS) and database searching, as well as quantitative analysis using TMT-based reporter ion intensity measurements.

Generation of multiple reporter ions from a single isobaric reagent increases multiplexing capacity for quantitative proteomics.

Isobaric labeling strategies for mass spectrometry-based proteomics enable multiplexed simultaneous quantification of samples and therefore substantially increase the sample throughput in proteomics. However, despite these benefits, current limits to multiplexing capacity are prohibitive for large sample sizes and impose limitations on experimental design. Here, we introduce a novel mechanism for increasing the multiplexing density of isobaric reagents. We present Combinatorial Isobaric Mass Tags (CMTs), an isobaric labeling architecture with the unique ability to generate multiple series of reporter ions simultaneously. We demonstrate that utilization of multiple reporter ion series improves multiplexing capacity of CMT with respect to a commercially available isobaric labeling reagent with preserved quantitative accuracy and depth of coverage in complex mixtures. We provide a blueprint for the realization of 16-plex reagents with 1 Da spacing between reporter ions and up to 28-plex at 6 mDa spacing using only 5 heavy isotopes per reagent. We anticipate that this improvement in multiplexing capacity will further advance the application of quantitative proteomics, particularly in high-throughput screening assays.

Development and characterization of novel 8-plex DiLeu isobaric labels for quantitative proteomics and peptidomics.

Relative quantification of proteins via their enzymatically digested peptide products determines disease biomarker candidate lists in discovery studies. Isobaric label-based strategies using TMT and iTRAQ allow for up to 10 samples to be multiplexed in one experiment, but their expense limits their use. The demand for cost-effective tagging reagents capable of multiplexing many samples led us to develop an 8-plex version of our isobaric labeling reagent, DiLeu. The original 4-plex DiLeu reagent was extended to an 8-plex set by coupling isotopic variants of dimethylated leucine to an alanine balance group designed to offset the increasing mass of the label's reporter group. Tryptic peptides from a single protein digest, a protein mixture digest, and Saccharomyces cerevisiae lysate digest were labeled with 8-plex DiLeu and analyzed via nanospray liquid chromatography/tandem mass spectrometry (nanoLC/MS(2) ) on a Q-Exactive Orbitrap mass spectrometer. Characteristics of 8-plex DiLeu-labeled peptides, including quantitative accuracy and fragmentation, were examined. An 8-plex set of DiLeu reagents with 1 Da spaced reporters was synthesized at a yield of 36%. The average cost to label eight 100 µg peptide samples was calculated to be approximately $15. Normalized collision energy tests on the Q-Exactive revealed that a higher-energy collisional dissociation value of 27 generated the optimum number of high-quality spectral matches. Relative quantification of DiLeu-labeled peptides yielded normalized median ratios accurate to within 12% of their expected values. Cost-effective 8-plex DiLeu reagents can be synthesized and applied to relative peptide and protein quantification. These labels increase the multiplexing capacity of our previous 4-plex implementation without requiring high-resolution instrumentation to resolve reporter ion signals.

Isobaric labeling-based relative quantification in shotgun proteomics.

Mass spectrometry plays a key role in relative quantitative comparisons of proteins in order to understand their functional role in biological systems upon perturbation. In this review, we review studies that examine different aspects of isobaric labeling-based relative quantification for shotgun proteomic analysis. In particular, we focus on different types of isobaric reagents and their reaction chemistry (e.g., amine-, carbonyl-, and sulfhydryl-reactive). Various factors, such as ratio compression, reporter ion dynamic range, and others, cause an underestimation of changes in relative abundance of proteins across samples, undermining the ability of the isobaric labeling approach to be truly quantitative. These factors that affect quantification and the suggested combinations of experimental design and optimal data acquisition methods to increase the precision and accuracy of the measurements will be discussed. Finally, the extended application of isobaric labeling-based approach in hyperplexing strategy, targeted quantification, and phosphopeptide analysis are also examined.

Proteomic quantification and site-mapping of S-nitrosylated proteins using isobaric iodoTMT reagents.

S-Nitrosylation is a redox-based protein post-translational modification in response to nitric oxide signaling and is involved in a wide range of biological processes. Detection and quantification of protein S-nitrosylation have been challenging tasks due to instability and low abundance of the modification. Many studies have used mass spectrometry (MS)-based methods with different thiol-reactive reagents to label and identify proteins with S-nitrosylated cysteine (SNO-Cys). In this study, we developed a novel iodoTMT switch assay (ISA) using an isobaric set of thiol-reactive iodoTMTsixplex reagents to specifically detect and quantify protein S-nitrosylation. Irreversible labeling of SNO-Cys with the iodoTMTsixplex reagents enables immune-affinity detection of S-nitrosylated proteins, enrichment of iodoTMT-labeled peptides by anti-TMT resin, and importantly, unambiguous modification site-mapping and multiplex quantification by liquid chromatography–tandem MS. Additionally, we significantly improved anti-TMT peptide enrichment efficiency by competitive elution. Using ISA, we identified a set of SNO-Cys sites responding to lipopolysaccharide (LPS) stimulation in murine BV-2 microglial cells and revealed effects of S-allyl cysteine from garlic on LPS-induced protein S-nitrosylation in antioxidative signaling and mitochondrial metabolic pathways. ISA proved to be an effective proteomic approach for quantitative analysis of S-nitrosylation in complex samples and will facilitate the elucidation of molecular mechanisms of nitrosative stress in disease.

ITRAQ Analysis with Paul Ion Trap–Obstacle Solved

Mass spectrometers equipped with ion trap analyzers have been significantly improved due to their high performance and wide application area accompanying the low costs of purchase. Despite several advantages, such as reasonable resolution at low cost, high sensitivity, and capability for multistage analysis, ion traps have an important drawback: low mass cutoff during tandem mass spectrometry analysis MS(n). Although the low mass cutoff associated with the ion trap does not seriously obstruct peptide identification, it may cause a serious problem in identification of small molecules (posttranslational modifications, e.g., glycan structures) and quantification of peptides with multiplexed isobaric tag reagents. The presented approach offers the possibility to use isobaric tags for relative and absolute quantification labeling (iTRAQ) for quantitative, proteomic analysis using typical, widely available ion trap devices and manufacturer's software. We have performed series of analyses of standard protein labeled with isobaric tags in various concentration ratios to prove quantitative capabilities of this approach.