2020
Johnson, A. ; Stadlmeier, M. ; Wühr, M. TMTPro Complementary Ion Quantification Increases Plexing and Sensitivity for Accurate Multiplexed Proteomics at the MS2 Level.
bioRxiv 2020.
AbstractMultiplexed proteomics is a powerful tool to assay cell states in health and disease, but accurate quantification of relative protein changes is impaired by interference from co-isolated peptides. Interference can be reduced by using MS3-based quantification, but this reduces sensitivity and requires specialized instrumentation. An alternative approach is quantification by complementary ions, which allows accurate and precise multiplexed quantification at the MS2 level and is compatible with the most widely distributed instruments. However, complementary ions of the popular TMT tag form inefficiently and multiplexing is limited to five channels. Here, we evaluate and optimize complementary ion quantification for the recently released TMTPro tag, which increases plexing capacity to eight channels (TMTProC). We find that the beneficial fragmentation properties of TMTPro increase quantification signal five-fold compared to TMT. This increased sensitivity results in ~65% more proteins quantified compared to TMTPro-MS3 and even slightly outperforms TMTPro-MS2. Furthermore, TMTProC quantification is more accurate than TMTPro-MS2 and even superior to TMTPro-MS3. To demonstrate the power of TMTProC, we analyzed a human and yeast interference sample and were able to quantify 13,290 proteins in 24 fractions. Thus, TMTProC advances multiplexed proteomics data quality and widens access to accurate multiplexed proteomics beyond laboratories with MS3-capable instrumentation.Competing Interest StatementThe authors have declared no competing interest.
2020_johnson_wuhr_biorxiv.pdf Crapse, J. ; Pappireddi, N. ; Gupta, M. ; Shvartsman, S. Y. ; Wieschaus, E. ; Wühr, M. Evaluating the Simple Arrhenius Equation for the Temperature Dependence of Complex Developmental Processes.
bioRxiv 2020.
Publisher's VersionAbstractThe famous Arrhenius equation is well motivated to describe the temperature dependence of chemical reactions but has also been used for complicated biological processes. Here, we evaluate how well the simple Arrhenius equation predicts complex multistep biological processes, using frog and fruit fly embryogenesis as two canonical models. We find the Arrhenius equation provides a good approximation for the temperature dependence of embryogenesis, even though individual developmental stages scale differently with temperature. At low and high temperatures, however, we observed significant departures from idealized Arrhenius Law behavior. When we model multistep reactions of idealized chemical networks we are unable to generate comparable deviations from linearity. In contrast, we find the single enzyme GAPDH shows non-linearity in the Arrhenius plot similar to our observations of embryonic development. Thus, we find that complex embryonic development can be well approximated by the simple Arrhenius Law and propose that the observed departure from this law results primarily from non-idealized individual steps rather than the complexity of the system.Competing Interest StatementThe authors have declared no competing interest.
2020_crapse_wuhr_biorxiv.pdf Hart, E. M. ; Gupta, M. ; Wühr, M. ; Silhavy, T. J. The gain-of-function allele bamAE470K bypasses the essential requirement for BamD in β-barrel outer membrane protein assembly.
Proceedings of the National Academy of Sciences 2020.
Publisher's VersionAbstractThe assembly of β-barrel outer membrane proteins (OMPs) is broadly conserved in diderm bacteria as well as in mitochondria and chloroplasts. The β-barrel assembly machine (BAM), which assembles OMPs into the outer membrane of gram-negative microbes, contains two essential proteins, BamA and BamD. Here we identify a genetic background in which BamD is nonessential, indicating that BamD does not function in OMP catalysis, but rather plays a regulatory role in OMP assembly. BamD is not conserved in the complexes that assemble OMPs in chloroplasts and mitochondria, likely because these organelles, unlike bacteria, inhabit a carefully controlled cytoplasmic environment.The outer membrane (OM) of gram-negative bacteria confers innate resistance to toxins and antibiotics. Integral β-barrel outer membrane proteins (OMPs) function to establish and maintain the selective permeability of the OM. OMPs are assembled into the OM by the β-barrel assembly machine (BAM), which is composed of one OMP—BamA—and four lipoproteins—BamB, C, D, and E. BamB, C, and E can be removed individually with only minor effects on barrier function; however, depletion of either BamA or BamD causes a global defect in OMP assembly and results in cell death. We have identified a gain-of-function mutation, bamAE470K, that bypasses the requirement for BamD. Although bamD::kan bamAE470K cells exhibit growth and OM barrier defects, they assemble OMPs with surprising robustness. Our results demonstrate that BamD does not play a catalytic role in OMP assembly, but rather functions to regulate the activity of BamA.
2020_hart_silhavy_pnas.pdf Cao, W. X. ; Kabelitz, S. ; Gupta, M. ; Yeung, E. ; Lin, S. ; Rammelt, C. ; Ihling, C. ; Pekovic, F. ; Low, T. C. H. ; Siddiqui, N. U. ; et al. Precise Temporal Regulation of Post-transcriptional Repressors Is Required for an Orderly Drosophila Maternal-to-Zygotic Transition.
Cell Reports 2020.
Publisher's VersionAbstractIn animal embryos, the maternal-to-zygotic transition (MZT) hands developmental control from maternal to zygotic gene products. We show that the maternal proteome represents more than half of the protein-coding capacity of Drosophila melanogaster’s genome, and that 2% of this proteome is rapidly degraded during the MZT. Cleared proteins include the post-transcriptional repressors Cup, Trailer hitch (TRAL), Maternal expression at 31B (ME31B), and Smaug (SMG). Although the ubiquitin-proteasome system is necessary for clearance of these repressors, distinct E3 ligase complexes target them: the C-terminal to Lis1 Homology (CTLH) complex targets Cup, TRAL, and ME31B for degradation early in the MZT and the Skp/Cullin/F-box-containing (SCF) complex targets SMG at the end of the MZT. Deleting the C-terminal 233 amino acids of SMG abrogates F-box protein interaction and confers immunity to degradation. Persistent SMG downregulates zygotic re-expression of mRNAs whose maternal contribution is degraded by SMG. Thus, clearance of SMG permits an orderly MZT.
2020_cao_lipshitz_cell_reports.pdf Li, A. ; Mao, D. ; Yoshimura, A. ; Rosen, P. C. ; Martin, W. L. ; Gallant, É. ; Wühr, M. ; Seyedsayamdost, M. R. Multi-Omic Analyses Provide Links between Low-Dose Antibiotic Treatment and Induction of Secondary Metabolism in Burkholderia thailandensis.
mBio 2020,
11, e03210-19.
Publisher's VersionAbstractLow doses of antibiotics can trigger secondary metabolite biosynthesis in bacteria, but the underlying mechanisms are generally unknown. We sought to better understand this phenomenon by studying how the antibiotic trimethoprim activates the synthesis of the virulence factor malleilactone in Burkholderia thailandensis. Using transcriptomics, quantitative multiplexed proteomics, and primary metabolomics, we systematically mapped the changes induced by trimethoprim. Surprisingly, even subinhibitory doses of the antibiotic resulted in broad transcriptional and translational alterations, with ∼8.5% of the transcriptome and ∼5% of the proteome up- or downregulated >4-fold. Follow-up studies with genetic-biochemical experiments showed that the induction of malleilactone synthesis can be sufficiently explained by the accumulation of methionine biosynthetic precursors, notably homoserine, as a result of inhibition of the folate pathway. Homoserine activated the malleilactone gene cluster via the transcriptional regulator MalR and gave rise to a secondary metabolome which was very similar to that generated by trimethoprim. Our work highlights the expansive changes that low-dose trimethoprim induces on bacterial physiology and provides insights into its stimulatory effect on secondary metabolism.IMPORTANCE The discovery of antibiotics ranks among the most significant accomplishments of the last century. Although the targets of nearly all clinical antibiotics are known, our understanding regarding their natural functions and the effects of subinhibitory concentrations is in its infancy. Stimulatory rather than inhibitory functions have been attributed to low-dose antibiotics. Among these, we previously found that antibiotics activate silent biosynthetic genes and thereby enhance the metabolic output of bacteria. The regulatory circuits underlying this phenomenon are unknown. We take a first step toward elucidating these circuits and show that low doses of trimethoprim (Tmp) have cell-wide effects on the saprophyte Burkholderia thailandensis. Most importantly, inhibition of one-carbon metabolic processes by Tmp leads to an accumulation of homoserine, which induces the production of an otherwise silent cytotoxin via a LuxR-type transcriptional regulator. These results provide a starting point for uncovering the molecular basis of the hormetic effects of antibiotics.
2020_li_seyedsayamdost_mbio.pdf Yeung, E. ; McFann, S. ; Marsh, L. ; Dufresne, E. ; Filippi, S. ; Harrington, H. A. ; Shvartsman, S. Y. ; Wühr, M. Inference of Multisite Phosphorylation Rate Constants and Their Modulation by Pathogenic Mutations.
Current Biology 2020.
Publisher's VersionAbstractSummary Multisite protein phosphorylation plays a critical role in cell regulation [1, 2, 3]. It is widely appreciated that the functional capabilities of multisite phosphorylation depend on the order and kinetics of phosphorylation steps, but kinetic aspects of multisite phosphorylation remain poorly understood [4, 5, 6]. Here, we focus on what appears to be the simplest scenario, when a protein is phosphorylated on only two sites in a strict, well-defined order. This scenario describes the activation of ERK, a highly conserved cell-signaling enzyme. We use Bayesian parameter inference in a structurally identifiable kinetic model to dissect dual phosphorylation of ERK by MEK, a kinase that is mutated in a large number of human diseases [7, 8, 9, 10, 11, 12]. Our results reveal how enzyme processivity and efficiencies of individual phosphorylation steps are altered by pathogenic mutations. The presented approach, which connects specific mutations to kinetic parameters of multisite phosphorylation mechanisms, provides a systematic framework for closing the gap between studies with purified enzymes and their effects in the living organism.
2020_yeung_wuhr_currbiol.pdf Stadlmeier, M. ; Runtsch, L. S. ; Streshnev, F. ; Wühr, M. ; Carell, T. A Click-Chemistry-Based Enrichable Crosslinker for Structural and Protein Interaction Analysis by Mass Spectrometry.
ChemBioChem 2020,
21, 103-107.
Publisher's VersionAbstractAbstract Mass spectrometry is the method of choice for the characterisation of proteomes. Most proteins operate in protein complexes, in which their close association modulates their function. However, with standard MS analysis, information on protein–protein interactions is lost and no structural information is retained. To gain structural and interactome data, new crosslinking reagents are needed that freeze inter- and intramolecular interactions. Herein, the development of a new reagent, which has several features that enable highly sensitive crosslinking MS, is reported. The reagent enables enrichment of crosslinked peptides from the majority of background peptides to facilitate efficient detection of low-abundant crosslinked peptides. Due to the special cleavable properties, the reagent can be used for MS2 and potentially for MS3 experiments. Thus, the new crosslinking reagent, in combination with high-end MS, should enable sensitive analysis of interactomes, which will help researchers to obtain important insights into cellular states in health and diseases.
2019_stadlmeier_carell_chembiochem.pdf 2019
Cao, W. X. ; Kabelitz, S. ; Gupta, M. ; Yeung, E. ; Lin, S. ; Rammelt, C. ; Ihling, C. ; Pekovic, F. ; Low, T. C. H. ; Siddiqui, N. U. ; et al. Precise temporal regulation of post-transcriptional repressors is required for an orderly Drosophila maternal-to-zygotic transition.
bioRxiv 2019, 862490.
Publisher's VersionAbstractIn animal embryos the maternal-to-zygotic transition (MZT) hands developmental control from maternal to zygotic gene products. We show that the maternal proteome represents over half of the protein coding capacity of the Drosophila melanogaster genome and that 2% of this proteome is rapidly degraded during the MZT. Cleared proteins include the post-transcriptional repressors Cup, Trailer hitch (TRAL), Maternal expression at 31B (ME31B), and Smaug (SMG). While the ubiquitin-proteasome system is necessary for clearance of all four repressors, distinct E3 ligase complexes target them: the C-terminal to Lis1 Homology (CTLH) complex targets Cup, TRAL and ME31B for degradation early in the MZT; the Skp/Cullin/F-box-containing (SCF) complex targets SMG at the end of the MZT. Deleting the C-terminal 233 amino acids of SMG makes the protein immune to degradation. We show that artificially persistent SMG downregulates the zygotic re-expression of mRNAs whose maternal contribution is cleared by SMG. Thus, clearance of SMG permits an orderly MZT.
2019_cao_lipshitz_biorxiv.pdf Peshkin, L. *; Gupta, M. *; Ryazanova, L. ; Wühr, M. Bayesian Confidence Intervals for Multiplexed Proteomics Integrate Ion-Statistics with Peptide Quantification Concordance.
Molecular & Cellular Proteomics 2019.
Publisher's VersionAbstractMultiplexed proteomics has emerged as a powerful tool to measure relative protein expression levels across multiple conditions. The relative protein abundances are inferred by comparing the signals generated by isobaric tags, which encode the samples’ origins. Intuitively, the trust associated with a protein measurement depends on the similarity of ratios from the protein’s peptides and the signal-strength of these measurements. However, typically the average peptide ratio is reported as the estimate of relative protein abundance, which is only the most likely ratio with a very naive model. Moreover, there is no sense on the confidence in these measurements. Here, we present a mathematically rigorous approach that integrates peptide signal strengths and peptidemeasurement agreement into an estimation of the true protein ratio and the associated confidence (BACIQ). The main advantages of BACIQ are: 1) It removes the need to threshold reported peptide signal based on an arbitrary cut-off, thereby reporting more measurements from a given experiment; 2) Confidence can be assigned without replicates; 3) For repeated experiments BACIQ provides confidence intervals for the union, not the intersection, of quantified proteins; 4) For repeated experiments, BACIQ confidence intervals are more predictive than confidence intervals based on protein measurement agreement. To demonstrate the power of BACIQ we reanalyzed previously published data on subcellular protein movement upon treatment with an Exportin-1 inhibiting drug. We detect ~2x more highly significant movers, down to subcellular localization changes of ~1% . Thus, our method drastically increases the value obtainable from quantitative proteomics experiments helping researchers to interpret their data and prioritize resources. To make our approach easily accessible we distribute it via a Python/Stan package
2019_peshkin_wuhr_mcp.pdf Hart, E. M. ; Gupta, M. ; Wühr, M. ; Silhavy, T. J. The Synthetic Phenotype of ΔbamB ΔbamE Double Mutants Results from a Lethal Jamming of the Bam Complex by the Lipoprotein RcsF.
mBio 2019,
10, e00662-19.
Publisher's VersionAbstractThe selective permeability of the Gram-negative outer membrane (OM)
is maintained by integral -barrel outer membrane proteins (OMPs). The heteropentomeric
-barrel assembly machine (Bam) folds and inserts OMPs into the OM. Coordination
of the essential proteins BamA and BamD is critical for OMP assembly and
therefore the viability of the cell. The role of the nonessential lipoproteins BamBCE
has yet to be characterized; however, genetic evidence suggests that they have nonoverlapping
roles in OMP assembly. In this work, we quantify changes of the proteome
in the conditional lethal ΔbamB ΔbamE double mutant. We show that cells
lacking BamB and BamE have a global OMP defect that is a result of a lethal obstruction
of an assembly-competent Bam complex by the lipoprotein RcsF. RcsF is a
stress-sensing lipoprotein that is threaded through the lumen of abundant -barrel
OMPs by the Bam complex to expose the amino terminus on the cell surface. We
demonstrate that simply removing this lipoprotein corrects the severe OMP assembly
defect of the double mutant nearly as efficiently as a previously isolated suppressor
mutation in bamA. We propose that BamB and BamE play crucial, nonoverlapping
roles to coordinate the activities of BamA and BamD during OMP
biogenesis.
2019_hart_silhavy_mbio.pdf Peshkin, L. ; Lukyanov, A. ; Kalocsay, M. ; Gage, R. M. ; Wang, D. Z. ; Pells, T. J. ; Karimi, K. ; Vize, P. D. ; Wühr, M. ; Kirschner, M. W. The protein repertoire in early vertebrate embryogenesis.
bioRxiv 2019.
Publisher's VersionAbstractWe present an unprecedentedly comprehensive characterization of protein
dynamics across early development in Xenopus laevis, available immediately via a convenient
Web portal. This resource allows interrogation of the protein expression data in conjunction with
other data modalities such as genome wide mRNA expression. This study provides detailed
data for absolute levels of ~14K unique Xenopus proteins representing homologues of ~9K
unique human genes -- a rich resource for developmental biologists. The purpose of this
manuscript is limited to presenting and releasing the data browser.
2019_peshkin_kirschner_biorxiv.pdf Tye, B. W. ; Commins, N. ; Ryazanova, L. V. ; Wühr, M. ; Springer, M. ; Pincus, D. ; Churchman, S. L. Proteotoxicity from aberrant ribosome biogenesis compromises cell fitness.
eLife 2019,
8:e43002.
Publisher's VersionAbstractTo achieve maximal growth, cells must manage a massive economy of ribosomal proteins (r-proteins) and RNAs (rRNAs) to produce thousands of ribosomes every minute. Although ribosomes are essential in all cells, natural disruptions to ribosome biogenesis lead to heterogeneous phenotypes. Here, we model these perturbations in Saccharomyces cerevisiae and show that challenges to ribosome biogenesis result in acute loss of proteostasis. Imbalances in the synthesis of r-proteins and rRNAs lead to the rapid aggregation of newly synthesized orphan r-proteins and compromise essential cellular processes, which cells alleviate by activating proteostasis genes. Exogenously bolstering the proteostasis network increases cellular fitness in the face of challenges to ribosome assembly, demonstrating the direct contribution of orphan r-proteins to cellular phenotypes. We propose that ribosome assembly is a key vulnerability of proteostasis maintenance in proliferating cells that may be compromised by diverse genetic, environmental, and xenobiotic perturbations that generate orphan r-proteins.
2019_tye_churchman_elife.pdf Nguyen, T. ; Mitchison, T. J. ; Wühr, M. Immunofluorescence of Microtubule Assemblies in Amphibian Oocytes and Early Embryos.
Methods Mol Biol. 2019,
1920, 17-32.
Publisher's VersionAbstractAmphibian oocytes and embryos are classical models to study cellular and developmental processes. For these studies, it is often advantageous to visualize protein organization. However, the large size and yolk distribution make imaging of deep structures in amphibian zygotes challenging. Here we describe in detail immunofluorescence (IF) protocols for imaging microtubule assemblies in early amphibian development. We developed these protocols to elucidate how the cell division machinery adapts to drastic changes in embryonic cell sizes. We describe how to image mitotic spindles, microtubule asters, chromosomes, and nuclei in whole-mount embryos, even when they are hundreds of micrometers removed from the embryo’s surface. Though the described methods were optimized for microtubule assemblies, they have also proven useful for the visualization of other proteins.
2019_nguyen_wuhr_methodsmolbio.pdf Pappireddi, N. ; Martin, L. ; Wühr, M. A Review on Quantitative Multiplexed Proteomics.
ChemBioChem 2019.
Publisher's VersionAbstractOver the last few decades, mass spectrometry‐based proteomics has become an increasingly powerful tool that is now able to routinely detect and quantify thousands of proteins. A major advance for global protein quantification was the introduction of isobaric tags, which in a single experiment enable the global quantification of proteins across multiple samples. In this review, we refer to these methods as multiplexed proteomics. We discuss the principles, advantages, and drawbacks of various multiplexed proteomics techniques, and compare them to alternative approaches. We discuss how the emerging combination of multiplexing with targeted proteomics might enable the reliable and high‐quality quantification of very low‐abundance proteins across multiple conditions. Lastly, we suggest that fusing multiplexed proteomics with data‐independent acquisition approaches might enable the comparison of hundreds of different samples without missing values while maintaining the superb measurement precision and accuracy obtainable with isobaric tag quantification.
2019_pappireddi_wuhr_chembiochem.pdf Nguyen, T. ; Pappireddi, N. ; Wühr, M. Proteomics of nucleocytoplasmic partitioning.
Current Opinion in Chemical Biology 2019,
2019, 55–63.
Publisher's VersionAbstract
The partitioning of the proteome between nucleus and cytoplasm affects nearly every aspect of eukaryotic biology. Despite this central role, we still have a poor understanding of which proteins localize in the nucleus and how this varies in different cell types and conditions. Recent advances in quantitative proteomics and high-throughput imaging are starting to close this knowledge gap. Studies on protein interaction are beginning to reveal the spectrum of cargos of nuclear import and export receptors.
We anticipate that it will soon be possible to predict each protein’s nucleocytoplasmic localization based on its importin/exportin interactions and its estimated diffusion rate through the nuclear pore. This insight is likely to provide us with a fundamental understanding of how cells use nucleocytoplasmic partitioning to encode and relay information.
2018_nguyen_wuhr_curropinchembio.pdf 2018
Gupta, M. ; Sonnett, M. ; Ryazanova, L. ; Presler, M. ; Wühr, M. Quantitative Proteomics of Xenopus Embryos I, Sample Preparation.
Methods in molecular biology 2018, 175–194.
Publisher's VersionAbstractXenopus oocytes and embryos are model systems optimally suited for quantitative proteomics. This is due to the availability of large amount of protein material and the ease of physical manipulation. Furthermore, facile in vitro fertilization provides superbly synchronized embryos for cell cycle and developmental stages. Here, we detail protocols developed over the last few years for sample preparation of multiplexed proteomics with TMT-tags followed by quantitative mass spectrometry analysis using the MultiNotch MS3 approach. In this approach, each condition is barcoded with an isobaric tag at the peptide level. After barcoding, samples are combined and the relative abundance of \textasciitilde100,000 peptides is quantified on a mass spectrometer. High reproducibility of the sample preparation process prior to peptides being tagged and combined is of upmost importance for obtaining unbiased data. Otherwise, differences in sample handling can inadvertently appear as biological changes. We detail and exemplify the application of our sample workflow on an embryonic time-series of ten developmental stages of Xenopus laevis embryos ranging from the egg to stage 35 (just before hatching). Our accompanying paper (Chapter 14 ) details a bioinformatics pipeline to analyze the quality of the given sample preparation and strategies to convert spectra of X. laevis peptides into biologically interpretable data.
2018_gupta_wuhr_methods_molbio.pdf Sonnett, M. ; Gupta, M. ; Nguyen, T. ; Wühr, M. Quantitative Proteomics for Xenopus Embryos II, Data Analysis.
Methods in molecular biology 2018,
1865, 195-215.
Publisher's VersionAbstractThe oocytes, embryos, and cell-free lysates of the frog Xenopus laevis have emerged as powerful models for quantitative proteomic experiments. In the accompanying paper (Chapter 13) we describe how to prepare samples and acquire multiplexed proteomics spectra from those. As an illustrative example we use a 10-stage developmental time series from the egg to stage 35 (just before hatching). Here, we outline how to convert the ~700,000 acquired mass spectra from this time series into protein expression dynamics for ~9000 proteins. We first outline a preliminary quality-control analysis to discover any errors that occurred during sample preparation. We discuss how peptide and protein identification error rates are controlled, and how peptide and protein species are quantified. Our analysis relies on the freely available MaxQuant proteomics pipeline. Finally, we demonstrate how to start interpreting this large dataset by clustering and gene-set enrichment analysis.
2018_sonnett_wuhr_methods_molbio.pdf Sonnett, M. ; Yeung, E. ; Wühr, M. Accurate, Sensitive, and Precise Multiplexed Proteomics Using the Complement Reporter Ion Cluster.
Analytical Chemistry 2018.
Publisher's VersionAbstractQuantitative analysis of proteomes across multiple time points, organelles, and
perturbations is essential for understanding both fundamental biology and disease
states. The development of isobaric tags (e.g. TMT) have enabled the simultaneous
measurement of peptide abundances across several different conditions. These
multiplexed approaches are promising in principle because of advantages in throughput
and measurement quality. However, in practice existing multiplexing approaches suffer
from key limitations. In its simple implementation (TMT-MS2), measurements are
distorted by chemical noise leading to poor measurement accuracy. The current state-ofthe-
art (TMT-MS3) addresses this, but requires specialized quadrupole-iontrap-Orbitrap
instrumentation. The complement reporter ion approach (TMTc) produces high accuracy
measurements and is compatible with many more instruments, like quadrupole-
Orbitraps. However, the required deconvolution of the TMTc cluster leads to poor
measurement precision. Here, we introduce TMTc+, which adds the modeling of the MS2-
isolation step into the deconvolution algorithm. The resulting measurements are
comparable in precision to TMT-MS3/MS2. The improved duty cycle, and lower filtering
requirements make TMTc+ more sensitive than TMT-MS3 and comparable with TMT-MS2.
At the same time, unlike TMT-MS2, TMTc+ is exquisitely able to distinguish signal from
chemical noise even outperforming TMT-MS3. Lastly, we compare TMTc+ to quantitative
label-free proteomics of total HeLa lysate and find that TMTc+ quantifies 7.8k versus 3.9k
proteins in a 5-plex sample. At the same time the median coefficient of variation
improves from 13% to 4%. Thus, TMTc+ advances quantitative proteomics by enabling
accurate, sensitive, and precise multiplexed experiments on more commonly used
instruments.
2018_sonnett_wuhr_analytical_chemistry.pdf Stadlmeier, M. ; Bogena, J. ; Wallner, M. ; Wühr, M. ; Carell, T. A sulfoxide-based isobaric labelling reagent for accurate quantitative mass spectrometry.
Angewandte Chemie 2018.
AbstractModern 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. Current generation reagents require a high fragmentation energy for cleavage, leading to incomplete fragmentation and hence loss of signal intensity. 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.
2018_stadlmeier_carell_angewandte.pdf