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1.
Nature ; 624(7990): 192-200, 2023 Dec.
Article in English | MEDLINE | ID: mdl-37968396

ABSTRACT

Cellular functions are mediated by protein-protein interactions, and mapping the interactome provides fundamental insights into biological systems. Affinity purification coupled to mass spectrometry is an ideal tool for such mapping, but it has been difficult to identify low copy number complexes, membrane complexes and complexes that are disrupted by protein tagging. As a result, our current knowledge of the interactome is far from complete, and assessing the reliability of reported interactions is challenging. Here we develop a sensitive high-throughput method using highly reproducible affinity enrichment coupled to mass spectrometry combined with a quantitative two-dimensional analysis strategy to comprehensively map the interactome of Saccharomyces cerevisiae. Thousand-fold reduced volumes in 96-well format enabled replicate analysis of the endogenous GFP-tagged library covering the entire expressed yeast proteome1. The 4,159 pull-downs generated a highly structured network of 3,927 proteins connected by 31,004 interactions, doubling the number of proteins and tripling the number of reliable interactions compared with existing interactome maps2. This includes very-low-abundance epigenetic complexes, organellar membrane complexes and non-taggable complexes inferred by abundance correlation. This nearly saturated interactome reveals that the vast majority of yeast proteins are highly connected, with an average of 16 interactors. Similar to social networks between humans, the average shortest distance between proteins is 4.2 interactions. AlphaFold-Multimer provided novel insights into the functional roles of previously uncharacterized proteins in complexes. Our web portal ( www.yeast-interactome.org ) enables extensive exploration of the interactome dataset.


Subject(s)
Protein Interaction Mapping , Protein Interaction Maps , Proteome , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Mass Spectrometry , Protein Interaction Mapping/methods , Proteome/chemistry , Proteome/metabolism , Reproducibility of Results , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Epigenesis, Genetic , Databases, Factual
2.
Mol Cell Proteomics ; 23(2): 100713, 2024 Feb.
Article in English | MEDLINE | ID: mdl-38184013

ABSTRACT

Optimizing data-independent acquisition methods for proteomics applications often requires balancing spectral resolution and acquisition speed. Here, we describe a real-time full mass range implementation of the phase-constrained spectrum deconvolution method (ΦSDM) for Orbitrap mass spectrometry that increases mass resolving power without increasing scan time. Comparing its performance to the standard enhanced Fourier transformation signal processing revealed that the increased resolving power of ΦSDM is beneficial in areas of high peptide density and comes with a greater ability to resolve low-abundance signals. In a standard 2 h analysis of a 200 ng HeLa digest, this resulted in an increase of 16% in the number of quantified peptides. As the acquisition speed becomes even more important when using fast chromatographic gradients, we further applied ΦSDM methods to a range of shorter gradient lengths (21, 12, and 5 min). While ΦSDM improved identification rates and spectral quality in all tested gradients, it proved particularly advantageous for the 5 min gradient. Here, the number of identified protein groups and peptides increased by >15% in comparison to enhanced Fourier transformation processing. In conclusion, ΦSDM is an alternative signal processing algorithm for processing Orbitrap data that can improve spectral quality and benefit quantitative accuracy in typical proteomics experiments, especially when using short gradients.


Subject(s)
Proteome , Tandem Mass Spectrometry , Humans , Proteome/metabolism , Tandem Mass Spectrometry/methods , Peptides/analysis , HeLa Cells , Proteomics/methods
3.
Mol Syst Biol ; 20(8): 972-995, 2024 Aug.
Article in English | MEDLINE | ID: mdl-38907068

ABSTRACT

Mass spectrometry has revolutionized cell signaling research by vastly simplifying the analysis of many thousands of phosphorylation sites in the human proteome. Defining the cellular response to perturbations is crucial for further illuminating the functionality of the phosphoproteome. Here we describe µPhos ('microPhos'), an accessible phosphoproteomics platform that permits phosphopeptide enrichment from 96-well cell culture and small tissue amounts in <8 h total processing time. By greatly minimizing transfer steps and liquid volumes, we demonstrate increased sensitivity, >90% selectivity, and excellent quantitative reproducibility. Employing highly sensitive trapped ion mobility mass spectrometry, we quantify ~17,000 Class I phosphosites in a human cancer cell line using 20 µg starting material, and confidently localize ~6200 phosphosites from 1 µg. This depth covers key signaling pathways, rendering sample-limited applications and perturbation experiments with hundreds of samples viable. We employ µPhos to study drug- and time-dependent response signatures in a leukemia cell line, and by quantifying 30,000 Class I phosphosites in the mouse brain we reveal distinct spatial kinase activities in subregions of the hippocampal formation.


Subject(s)
Phosphopeptides , Phosphoproteins , Proteomics , Proteomics/methods , Humans , Animals , Mice , Phosphoproteins/metabolism , Phosphorylation , Cell Line, Tumor , Phosphopeptides/metabolism , Phosphopeptides/analysis , Mass Spectrometry/methods , Signal Transduction , Proteome/metabolism , Reproducibility of Results , Hippocampus/metabolism , Hippocampus/cytology
4.
Mol Cell Proteomics ; 22(2): 100489, 2023 02.
Article in English | MEDLINE | ID: mdl-36566012

ABSTRACT

Data-independent acquisition (DIA) methods have become increasingly popular in mass spectrometry-based proteomics because they enable continuous acquisition of fragment spectra for all precursors simultaneously. However, these advantages come with the challenge of correctly reconstructing the precursor-fragment relationships in these highly convoluted spectra for reliable identification and quantification. Here, we introduce a scan mode for the combination of trapped ion mobility spectrometry with parallel accumulation-serial fragmentation (PASEF) that seamlessly and continuously follows the natural shape of the ion cloud in ion mobility and peptide precursor mass dimensions. Termed synchro-PASEF, it increases the detected fragment ion current several-fold at sub-second cycle times. Consecutive quadrupole selection windows move synchronously through the mass and ion mobility range. In this process, the quadrupole slices through the peptide precursors, which separates fragment ion signals of each precursor into adjacent synchro-PASEF scans. This precisely defines precursor-fragment relationships in ion mobility and mass dimensions and effectively deconvolutes the DIA fragment space. Importantly, the partitioned parts of the fragment ion transitions provide a further dimension of specificity via a lock-and-key mechanism. This is also advantageous for quantification, where signals from interfering precursors in the DIA selection window do not affect all partitions of the fragment ion, allowing to retain only the specific parts for quantification. Overall, we establish the defining features of synchro-PASEF and explore its potential for proteomic analyses.


Subject(s)
Proteomics , Tandem Mass Spectrometry , Tandem Mass Spectrometry/methods , Proteomics/methods , Proteome/analysis , Peptides/analysis
5.
Mol Cell Proteomics ; 21(9): 100279, 2022 09.
Article in English | MEDLINE | ID: mdl-35944843

ABSTRACT

Data-independent acquisition (DIA) methods have become increasingly attractive in mass spectrometry-based proteomics because they enable high data completeness and a wide dynamic range. Recently, we combined DIA with parallel accumulation-serial fragmentation (dia-PASEF) on a Bruker trapped ion mobility (IM) separated quadrupole time-of-flight mass spectrometer. This requires alignment of the IM separation with the downstream mass selective quadrupole, leading to a more complex scheme for dia-PASEF window placement compared with DIA. To achieve high data completeness and deep proteome coverage, here we employ variable isolation windows that are placed optimally depending on precursor density in the m/z and IM plane. This is implemented in the freely available py_diAID (Python package for DIA with an automated isolation design) package. In combination with in-depth project-specific proteomics libraries and the Evosep liquid chromatography system, we reproducibly identified over 7700 proteins in a human cancer cell line in 44 min with quadruplicate single-shot injections at high sensitivity. Even at a throughput of 100 samples per day (11 min liquid chromatography gradients), we consistently quantified more than 6000 proteins in mammalian cell lysates by injecting four replicates. We found that optimal dia-PASEF window placement facilitates in-depth phosphoproteomics with very high sensitivity, quantifying more than 35,000 phosphosites in a human cancer cell line stimulated with an epidermal growth factor in triplicate 21 min runs. This covers a substantial part of the regulated phosphoproteome with high sensitivity, opening up for extensive systems-biological studies.


Subject(s)
Proteome , Tandem Mass Spectrometry , Animals , Chromatography, Liquid/methods , Epidermal Growth Factor , Humans , Mammals/metabolism , Proteome/metabolism , Proteomics/methods , Tandem Mass Spectrometry/methods
6.
Proteomics ; 23(7-8): e2200032, 2023 04.
Article in English | MEDLINE | ID: mdl-36300730

ABSTRACT

Mass spectrometry-based phosphoproteomics has identified >150,000 post-translational phosphorylation sites in the human proteome. To disentangle their functional relevance, complex experimental designs that require increased throughput are now coming into focus. Here, we apply dia-PASEF on a trapped ion mobility (TIMS) mass spectrometer to analyze the phosphoproteome of a human cancer cell line in short liquid chromatography gradients. At low sample amounts equivalent to ∼20 ug protein digest per analysis, we quantified over 13,000 phosphopeptides including ∼8700 class I phosphosites in 1 h without a spectral library. Decreasing the gradient time to 15 min yielded virtually identical coverage of the phosphoproteome, and with 7 min gradients we still quantified about 80% of the class I sites with a median coefficient of variation <10% in quadruplicates. We attribute this in part to the increased peak capacity, which effectively compensates for the higher peptide density per time unit in shorter gradients. Our data show a five-fold reduction in the number of co-isolated peptides with TIMS. In the most extreme case, these were positional isomers of nearby phosphosites that remained unresolved with fast liquid chromatography. In summary, our study demonstrates how key features of dia-PASEF translate to phosphoproteomics.


Subject(s)
Phosphopeptides , Proteome , Humans , Phosphopeptides/analysis , Chromatography, Liquid/methods , Mass Spectrometry/methods , Proteome/analysis , Cell Line
7.
Anal Chem ; 95(19): 7487-7494, 2023 05 16.
Article in English | MEDLINE | ID: mdl-37146101

ABSTRACT

We report an online analytical platform based on the coupling of asymmetrical flow field-flow fractionation (AF4) and native mass spectrometry (nMS) in parallel with UV-absorbance, multi-angle light scattering (MALS), and differential-refractive-index (UV-MALS-dRI) detectors to elucidate labile higher-order structures (HOS) of protein biotherapeutics. The technical aspects of coupling AF4 with nMS and the UV-MALS-dRI multi-detection system are discussed. The "slot-outlet" technique was used to reduce sample dilution and split the AF4 effluent between the MS and UV-MALS-dRI detectors. The stability, HOS, and dissociation pathways of the tetrameric biotherapeutic enzyme (anticancer agent) l-asparaginase (ASNase) were studied. ASNase is a 140 kDa homo-tetramer, but the presence of intact octamers and degradation products with lower molecular weights was indicated by AF4-MALS/nMS. Exposing ASNase to 10 mM NaOH disturbed the equilibrium between the different non-covalent species and led to HOS dissociation. Correlation of the information obtained by AF4-MALS (liquid phase) and AF4-nMS (gas phase) revealed the formation of monomeric, tetrameric, and pentameric species. High-resolution MS revealed deamidation of the main intact tetramer upon exposure of ASNase to high pH (NaOH and ammonium bicarbonate). The particular information retrieved from ASNase with the developed platform in a single run demonstrates that the newly developed platform can be highly useful for aggregation and stability studies of protein biopharmaceuticals.


Subject(s)
Fractionation, Field Flow , Proteins , Sodium Hydroxide , Mass Spectrometry , Refractometry , Asparaginase , Fractionation, Field Flow/methods
8.
Small ; 19(23): e2207207, 2023 06.
Article in English | MEDLINE | ID: mdl-36922728

ABSTRACT

In this study, a 96-well exposure system for safety assessment of nanomaterials is developed and characterized using an air-liquid interface lung epithelial model. This system is designed for sequential nebulization. Distribution studies verify the reproducible distribution over all 96 wells, with lower insert-to-insert variability compared to non-sequential application. With a first set of chemicals (TritonX), drugs (Bortezomib), and nanomaterials (silver nanoparticles and (non-)fluorescent crystalline nanocellulose), sequential exposure studies are performed with human lung epithelial cells followed by quantification of the deposited mass and of cell viability. The developed exposure system offers for the first time the possibility of exposing an air-liquid interface model in a 96-well format, resulting in high-throughput rates, combined with the feature for sequential dosing. This exposure system allows the possibility of creating dose-response curves resulting in the generation of more reliable cell-based assay data for many types of applications, such as safety analysis. In addition to chemicals and drugs, nanomaterials with spherical shapes, but also morphologically more complex nanostructures can be exposed sequentially with high efficiency. This allows new perspectives on in vivo-like and animal-free approaches for chemical and pharmaceutical safety assessment, in line with the 3R principle of replacing and reducing animal experiments.


Subject(s)
Metal Nanoparticles , Humans , Silver , Lung , Epithelial Cells , Bortezomib
9.
Nat Methods ; 17(12): 1229-1236, 2020 12.
Article in English | MEDLINE | ID: mdl-33257825

ABSTRACT

Data-independent acquisition modes isolate and concurrently fragment populations of different precursors by cycling through segments of a predefined precursor m/z range. Although these selection windows collectively cover the entire m/z range, overall, only a few per cent of all incoming ions are isolated for mass analysis. Here, we make use of the correlation of molecular weight and ion mobility in a trapped ion mobility device (timsTOF Pro) to devise a scan mode that samples up to 100% of the peptide precursor ion current in m/z and mobility windows. We extend an established targeted data extraction workflow by inclusion of the ion mobility dimension for both signal extraction and scoring and thereby increase the specificity for precursor identification. Data acquired from whole proteome digests and mixed organism samples demonstrate deep proteome coverage and a high degree of reproducibility as well as quantitative accuracy, even from 10 ng sample amounts.


Subject(s)
Data Science/methods , High-Throughput Screening Assays/methods , Ion Channels/metabolism , Ion Transport/physiology , Proteome/metabolism , Cell Line, Tumor , HeLa Cells , Humans , Ions/chemistry , Proteomics/methods , Reproducibility of Results , Tandem Mass Spectrometry/methods
10.
Mol Syst Biol ; 18(5): e10947, 2022 05.
Article in English | MEDLINE | ID: mdl-35579278

ABSTRACT

Deeper understanding of liver pathophysiology would benefit from a comprehensive quantitative proteome resource at cell type resolution to predict outcome and design therapy. Here, we quantify more than 150,000 sequence-unique peptides aggregated into 10,000 proteins across total liver, the major liver cell types, time course of primary cell cultures, and liver disease states. Bioinformatic analysis reveals that half of hepatocyte protein mass is comprised of enzymes and 23% of mitochondrial proteins, twice the proportion of other liver cell types. Using primary cell cultures, we capture dynamic proteome remodeling from tissue states to cell line states, providing useful information for biological or pharmaceutical research. Our extensive data serve as spectral library to characterize a human cohort of non-alcoholic steatohepatitis and cirrhosis. Dramatic proteome changes in liver tissue include signatures of hepatic stellate cell activation resembling liver cirrhosis and providing functional insights. We built a web-based dashboard application for the interactive exploration of our resource (www.liverproteome.org).


Subject(s)
Non-alcoholic Fatty Liver Disease , Proteome , Humans , Liver/metabolism , Liver Cirrhosis/metabolism , Non-alcoholic Fatty Liver Disease/metabolism , Proteome/metabolism , Proteomics
11.
Mol Syst Biol ; 18(3): e10798, 2022 03.
Article in English | MEDLINE | ID: mdl-35226415

ABSTRACT

Single-cell technologies are revolutionizing biology but are today mainly limited to imaging and deep sequencing. However, proteins are the main drivers of cellular function and in-depth characterization of individual cells by mass spectrometry (MS)-based proteomics would thus be highly valuable and complementary. Here, we develop a robust workflow combining miniaturized sample preparation, very low flow-rate chromatography, and a novel trapped ion mobility mass spectrometer, resulting in a more than 10-fold improved sensitivity. We precisely and robustly quantify proteomes and their changes in single, FACS-isolated cells. Arresting cells at defined stages of the cell cycle by drug treatment retrieves expected key regulators. Furthermore, it highlights potential novel ones and allows cell phase prediction. Comparing the variability in more than 430 single-cell proteomes to transcriptome data revealed a stable-core proteome despite perturbation, while the transcriptome appears stochastic. Our technology can readily be applied to ultra-high sensitivity analyses of tissue material, posttranslational modifications, and small molecule studies from small cell counts to gain unprecedented insights into cellular heterogeneity in health and disease.


Subject(s)
Proteome , Proteomics , Mass Spectrometry/methods , Protein Processing, Post-Translational , Proteome/metabolism , Proteomics/methods , Workflow
12.
Phys Rev Lett ; 131(22): 220201, 2023 Dec 01.
Article in English | MEDLINE | ID: mdl-38101372

ABSTRACT

From a thermodynamic point of view, all clocks are driven by irreversible processes. Additionally, one can use oscillatory systems to temporally modulate the thermodynamic flux towards equilibrium. Focusing on the most elementary thermalization events, this modulation can be thought of as a temporal probability concentration for these events. There are two fundamental factors limiting the performance of clocks: On the one level, the inevitable drifts of the oscillatory system, which are addressed by finding stable atomic or nuclear transitions that lead to astounding precision of today's clocks. On the other level, there is the intrinsically stochastic nature of the irreversible events upon which the clock's operation is based. This becomes relevant when seeking to maximize a clock's resolution at high accuracy, which is ultimately limited by the number of such stochastic events per reference time unit. We address this essential trade-off between clock accuracy and resolution, proving a universal bound for all clocks whose elementary thermalization events are memoryless.

13.
Phys Rev Lett ; 131(16): 160204, 2023 Oct 20.
Article in English | MEDLINE | ID: mdl-37925703

ABSTRACT

In order to unitarily evolve a quantum system, an agent requires knowledge of time, a parameter that no physical clock can ever perfectly characterize. In this Letter, we study how limitations on acquiring knowledge of time impact controlled quantum operations in different paradigms. We show that the quality of timekeeping an agent has access to limits the circuit complexity they are able to achieve within circuit-based quantum computation. We do this by deriving an upper bound on the average gate fidelity achievable under imperfect timekeeping for a general class of random circuits. Another area where quantum control is relevant is quantum thermodynamics. In that context, we show that cooling a qubit can be achieved using a timer of arbitrary quality for control: timekeeping error only impacts the rate of cooling and not the achievable temperature. Our analysis combines techniques from the study of autonomous quantum clocks and the theory of quantum channels to understand the effect of imperfect timekeeping on controlled quantum dynamics.

14.
Anal Bioanal Chem ; 415(27): 6633-6645, 2023 Nov.
Article in English | MEDLINE | ID: mdl-37758903

ABSTRACT

Recent advances have rekindled the interest in ion mobility as an additional dimension of separation in mass spectrometry (MS)-based proteomics. Ion mobility separates ions according to their size and shape in the gas phase. Here, we set out to investigate the effect of 22 different post-translational modifications (PTMs) on the collision cross section (CCS) of peptides. In total, we analyzed ~4300 pairs of matching modified and unmodified peptide ion species by trapped ion mobility spectrometry (TIMS). Linear alignment based on spike-in reference peptides resulted in highly reproducible CCS values with a median coefficient of variation of 0.26%. On a global level, we observed a redistribution in the m/z vs. ion mobility space for modified peptides upon changes in their charge state. Pairwise comparison between modified and unmodified peptides of the same charge state revealed median shifts in CCS between -1.4% (arginine citrullination) and +4.5% (O-GlcNAcylation). In general, increasing modified peptide masses were correlated with higher CCS values, in particular within homologous PTM series. However, investigating the ion populations in more detail, we found that the change in CCS can vary substantially for a given PTM and is partially correlated with the gas phase structure of its unmodified counterpart. In conclusion, our study shows PTM- and sequence-specific effects on the cross section of peptides, which could be further leveraged for proteome-wide PTM analysis.


Subject(s)
Peptides , Proteomics , Peptides/chemistry , Mass Spectrometry/methods , Proteome , Protein Processing, Post-Translational , Ions/chemistry
15.
Anal Bioanal Chem ; 415(15): 3007-3031, 2023 Jun.
Article in English | MEDLINE | ID: mdl-37106123

ABSTRACT

A comprehensive physicochemical characterization of heterogeneous nanoplastic (NPL) samples remains an analytical challenge requiring a combination of orthogonal measurement techniques to improve the accuracy and robustness of the results. Here, batch methods, including dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), tunable resistive pulse sensing (TRPS), transmission electron microscopy (TEM), and scanning electron microscopy (SEM), as well as separation/fractionation methods such as centrifugal liquid sedimentation (CLS) and field-flow fractionation (FFF)-multi-angle light scattering (MALS) combined with pyrolysis gas chromatography mass spectrometry (pyGC-MS) or Raman microspectroscopy (RM) were evaluated for NPL size, shape, and chemical composition measurements and for quantification. A set of representative/test particles of different chemical natures, including (i) polydisperse polyethylene (PE), (ii) (doped) polystyrene (PS) NPLs, (iii) titanium dioxide, and (iv) iron oxide nanoparticles (spherical and elongated), was used to assess the applicability and limitations of the selected methodologies. Particle sizes and number-based concentrations obtained by orthogonal batch methods (DLS, NTA, TRPS) were comparable for monodisperse spherical samples, while higher deviations were observed for polydisperse, agglomerated samples and for non-spherical particles, especially for light scattering methods. CLS and TRPS offer further insight with increased size resolution, while detailed morphological information can be derived by electron microscopy (EM)-based approaches. Combined techniques such as FFF coupled to MALS and RM can provide complementary information on physical and chemical properties by online measurements, while pyGC-MS analysis of FFF fractions can be used for the identification of polymer particles (vs. inorganic particles) and for their offline (semi)quantification. However, NPL analysis in complex samples will continue to present a serious challenge for the evaluated techniques without significant improvements in sample preparation.

16.
Mol Cell Proteomics ; 20: 100138, 2021.
Article in English | MEDLINE | ID: mdl-34416385

ABSTRACT

Recent advances in efficiency and ease of implementation have rekindled interest in ion mobility spectrometry, a technique that separates gas phase ions by their size and shape and that can be hybridized with conventional LC and MS. Here, we review the recent development of trapped ion mobility spectrometry (TIMS) coupled to TOF mass analysis. In particular, the parallel accumulation-serial fragmentation (PASEF) operation mode offers unique advantages in terms of sequencing speed and sensitivity. Its defining feature is that it synchronizes the release of ions from the TIMS device with the downstream selection of precursors for fragmentation in a TIMS quadrupole TOF configuration. As ions are compressed into narrow ion mobility peaks, the number of peptide fragment ion spectra obtained in data-dependent or targeted analyses can be increased by an order of magnitude without compromising sensitivity. Taking advantage of the correlation between ion mobility and mass, the PASEF principle also multiplies the efficiency of data-independent acquisition. This makes the technology well suited for rapid proteome profiling, an increasingly important attribute in clinical proteomics, as well as for ultrasensitive measurements down to single cells. The speed and accuracy of TIMS and PASEF also enable precise measurements of collisional cross section values at the scale of more than a million data points and the development of neural networks capable of predicting them based only on peptide sequences. Peptide collisional cross section values can differ for isobaric sequences or positional isomers of post-translational modifications. This additional information may be leveraged in real time to direct data acquisition or in postprocessing to increase confidence in peptide identifications. These developments make TIMS quadrupole TOF PASEF a powerful and expandable platform for proteomics and beyond.


Subject(s)
Proteomics/methods , Animals , Humans , Ion Mobility Spectrometry , Mass Spectrometry
17.
Z Rheumatol ; 82(4): 269-277, 2023 May.
Article in German | MEDLINE | ID: mdl-37099181

ABSTRACT

The major tasks of the immune system are protection against infectious agents, maintaining homeostasis by recognizing and neutralizing noxious substances from the environment, and monitoring pathological, e.g. neoplastic tissue changes. It accomplishes these tasks through complex interactions of cellular and humoral components of the innate and adaptive immune system. This review article focuses on a central problem of self versus non-self discrimination in the development of B and T lymphocytes as carriers of adaptive immunity. During maturation of the lymphocytes in the bone marrow, large repertoires of lymphocyte receptors are randomly generated by somatic recombination, which as a whole have the capability of recognizing any foreign antigen. In order to reduce the implicit risk of autoaggressive immunity that might arise from evolutionary conserved structural motifs in self and foreign antigens, the adaptive immune system must provide redundant mechanisms (clonal deletion, anergy, quiescence and suppression) to eliminate or inactivate lymphocytes expressing highly avid receptors for autoantigens. Thus, the provision of costimulatory signals resulting in a reduced activation threshold of potentially autoreactive anergic T cells through infection, molecular mimicry, disrupted apoptosis regulation, altered "self" by post-translational modification, genetic changes in transcription factors with critical importance for thymic tolerance induction or signaling components of apoptosis can lead to a disruption of self-tolerance and the induction of pathogenic autoimmunity.


Subject(s)
Autoimmune Diseases , Rheumatic Diseases , Humans , Immune Tolerance , T-Lymphocytes , Self Tolerance , Autoimmunity , Autoantigens
18.
Anal Chem ; 94(23): 8146-8155, 2022 06 14.
Article in English | MEDLINE | ID: mdl-35621336

ABSTRACT

Top-down proteomics provides a straightforward approach to the level of proteoforms but remains technologically challenging. Using ion mobility spectrometry/mass spectrometry (IMS/MS) to separate top-down fragment ions improves signal/noise and dynamic range. Such applications, however, do not yet leverage the primary information obtained from IMS/MS, which is the characterization of the fragment ion structure by the measured momentum transfer cross sections. Here, we perform top-down analysis of intact proteins and assemblies using our tandem trapped ion mobility spectrometer/mass spectrometer (tTIMS/MS) and compile over 1400 cross section values of fragment ions. Our analysis reveals that most fragment ions exhibit multiple, stable conformations similar to those of intact polypeptides and proteins. The data further indicate that the conformational heterogeneity is strongly influenced by the amino acid sequences of the fragment ions. Moreover, time-resolved tTIMS/MS experiments reveal that conformations of top-down fragment ions can be metastable on the timescale of ion mobility measurements. Taken together, our analysis indicates that top-down fragment ions undergo a folding process in the gas phase and that this folding process can lead to kinetic trapping of intermediate states in ion mobility measurements. Hence, because the folding free energy surface of a polypeptide ion is encoded by its amino acid sequence and charge state, our analysis suggests that cross sections can be exploited as sequence-specific determinants of top-down fragment ions.


Subject(s)
Ion Mobility Spectrometry , Proteins , Ion Mobility Spectrometry/methods , Ions/chemistry , Peptides , Proteins/chemistry , Proteomics/methods , Tandem Mass Spectrometry/methods
19.
Molecules ; 27(3)2022 Feb 01.
Article in English | MEDLINE | ID: mdl-35164246

ABSTRACT

Whereas the characterization of nanomaterials using different analytical techniques is often highly automated and standardized, the sample preparation that precedes it causes a bottleneck in nanomaterial analysis as it is performed manually. Usually, this pretreatment depends on the skills and experience of the analysts. Furthermore, adequate reporting of the sample preparation is often missing. In this overview, some solutions for techniques widely used in nano-analytics to overcome this problem are discussed. Two examples of sample preparation optimization by automation are presented, which demonstrate that this approach is leading to increased analytical confidence. Our first example is motivated by the need to exclude human bias and focuses on the development of automation in sample introduction. To this end, a robotic system has been developed, which can prepare stable and homogeneous nanomaterial suspensions amenable to a variety of well-established analytical methods, such as dynamic light scattering (DLS), small-angle X-ray scattering (SAXS), field-flow fractionation (FFF) or single-particle inductively coupled mass spectrometry (sp-ICP-MS). Our second example addresses biological samples, such as cells exposed to nanomaterials, which are still challenging for reliable analysis. An air-liquid interface has been developed for the exposure of biological samples to nanomaterial-containing aerosols. The system exposes transmission electron microscopy (TEM) grids under reproducible conditions, whilst also allowing characterization of aerosol composition with mass spectrometry. Such an approach enables correlative measurements combining biological with physicochemical analysis. These case studies demonstrate that standardization and automation of sample preparation setups, combined with appropriate measurement processes and data reduction are crucial steps towards more reliable and reproducible data.

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