RESUMEN
Cellular processes arise from the dynamic organization of proteins in networks of physical interactions. Mapping the interactome has therefore been a central objective of high-throughput biology. However, the dynamics of protein interactions across physiological contexts remain poorly understood. Here, we develop a quantitative proteomic approach combining protein correlation profiling with stable isotope labeling of mammals (PCP-SILAM) to map the interactomes of seven mouse tissues. The resulting maps provide a proteome-scale survey of interactome rewiring across mammalian tissues, revealing more than 125,000 unique interactions at a quality comparable to the highest-quality human screens. We identify systematic suppression of cross-talk between the evolutionarily ancient housekeeping interactome and younger, tissue-specific modules. Rewired proteins are tightly regulated by multiple cellular mechanisms and are implicated in disease. Our study opens up new avenues to uncover regulatory mechanisms that shape in vivo interactome responses to physiological and pathophysiological stimuli in mammalian systems.
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Especificidad de Órganos , Mapeo de Interacción de Proteínas , Animales , Marcaje Isotópico , Masculino , Mamíferos , Ratones Endogámicos C57BL , Reproducibilidad de los ResultadosRESUMEN
Plants are foundational for global ecological and economic systems, but most plant proteins remain uncharacterized. Protein interaction networks often suggest protein functions and open new avenues to characterize genes and proteins. We therefore systematically determined protein complexes from 13 plant species of scientific and agricultural importance, greatly expanding the known repertoire of stable protein complexes in plants. By using co-fractionation mass spectrometry, we recovered known complexes, confirmed complexes predicted to occur in plants, and identified previously unknown interactions conserved over 1.1 billion years of green plant evolution. Several novel complexes are involved in vernalization and pathogen defense, traits critical for agriculture. We also observed plant analogs of animal complexes with distinct molecular assemblies, including a megadalton-scale tRNA multi-synthetase complex. The resulting map offers a cross-species view of conserved, stable protein assemblies shared across plant cells and provides a mechanistic, biochemical framework for interpreting plant genetics and mutant phenotypes.
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Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Mapas de Interacción de Proteínas/fisiología , Espectrometría de Masas/métodos , Plantas/genética , Plantas/metabolismo , Mapeo de Interacción de Proteínas/métodos , Proteómica/métodosRESUMEN
Whole-genome duplications (WGDs) are widespread genomic events in eukaryotes that are hypothesized to contribute to the evolutionary success of many lineages, including flowering plants, Saccharomyces yeast, and vertebrates. WGDs generally can be classified into autopolyploids (ploidy increase descended from one species) or allopolyploids (ploidy increase descended from multiple species). Assignment of allopolyploid progenitor species (called subgenomes in the polyploid) is important to understanding the biology and evolution of polyploids, including the asymmetric subgenome evolution following hybridization (biased fractionation). Here, I review the different methodologies used to identify the ancestors of allopolyploid subgenomes, discuss the advantages and disadvantages of these methods, and outline the implications of how these methods affect the subsequent evolutionary analysis of these genomes.
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Evolución Molecular , Poliploidía , Filogenia , Animales , Genoma/genética , Genómica/métodos , Duplicación de Gen/genéticaRESUMEN
RNA localization in eukaryotes is a mechanism to regulate transcripts fate. Conversely, bacterial transcripts were not assumed to be specifically localized. We previously demonstrated that E. coli mRNAs may localize to where their products localize in a translation-independent manner, thus challenging the transcription-translation coupling extent. However, the scope of RNA localization in bacteria remained unknown. Here, we report the distribution of the E. coli transcriptome between the membrane, cytoplasm, and poles by combining cell fractionation with deep-sequencing (Rloc-seq). Our results reveal asymmetric RNA distribution on a transcriptome-wide scale, significantly correlating with proteome localization and prevalence of translation-independent RNA localization. The poles are enriched with stress-related mRNAs and small RNAs, the latter becoming further enriched upon stress in an Hfq-dependent manner. Genome organization may play a role in localizing membrane protein-encoding transcripts. Our results show an unexpected level of intricacy in bacterial transcriptome organization and highlight the poles as hubs for regulation.
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Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , ARN Bacteriano/genética , ARN Mensajero/genética , Transcriptoma , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Perfilación de la Expresión Génica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento , Proteína de Factor 1 del Huésped/genética , Proteína de Factor 1 del Huésped/metabolismo , Transporte de Proteínas , ARN Bacteriano/metabolismo , ARN Mensajero/metabolismo , Análisis de Secuencia de ARN , Estrés FisiológicoRESUMEN
Shifts in the hydrogen stable isotopic composition (2H/1H ratio) of lipids relative to water (lipid/water 2H-fractionation) at natural abundances reflect different sources of the central cellular reductant, NADPH, in bacteria. Here, we demonstrate that lipid/water 2H-fractionation (2εfattyacid/water) can also constrain the relative importance of key NADPH pathways in eukaryotes. We used the metabolically flexible yeast Saccharomyces cerevisiae, a microbial model for respiratory and fermentative metabolism in industry and medicine, to investigate 2εfattyacid/water. In chemostats, fatty acids from glycerol-respiring cells were >550 2H-enriched compared to those from cells aerobically fermenting sugars via overflow metabolism, a hallmark feature in cancer. Faster growth decreased 2H/1H ratios, particularly in glycerol-respiring cells by 200. Variations in the activities and kinetic isotope effects among NADP+-reducing enzymes indicate cytosolic NADPH supply as the primary control on 2εfattyacid/water. Contributions of cytosolic isocitrate dehydrogenase (cIDH) to NAPDH production drive large 2H-enrichments with substrate metabolism (cIDH is absent during fermentation but contributes up to 20 percent NAPDH during respiration) and slower growth on glycerol (11 percent more NADPH from cIDH). Shifts in NADPH demand associated with cellular lipid abundance explain smaller 2εfattyacid/water variations (<30) with growth rate during fermentation. Consistent with these results, tests of murine liver cells had 2H-enriched lipids from slower-growing, healthy respiring cells relative to fast-growing, fermenting hepatocellular carcinoma. Our findings point to the broad potential of lipid 2H/1H ratios as a passive natural tracker of eukaryotic metabolism with applications to distinguish health and disease, complementing studies that rely on complex isotope-tracer addition methods.
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Ácidos Grasos , Fermentación , NADP , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Ácidos Grasos/metabolismo , NADP/metabolismo , Aerobiosis , Deuterio/metabolismo , Humanos , Glicerol/metabolismo , Isocitrato Deshidrogenasa/metabolismoRESUMEN
Experimental results are presented showing the variation in the relationship between odd isotopes of tin (Sn) in mass-independent fractionation caused by the magnetic isotope effect (MIE), which has previously only been observed for mercury. These results are consistent with the trend predicted from the difference between the magnitudes of nuclear magnetic moments of odd isotopes with a nuclear spin. However, the correlation between odd isotopes in fractionation induced by the MIE for the reaction system used in this study (solvent extraction using a crown ether) was different from that reported for the photochemical reaction of methyltin. This difference between the two reaction systems is consistent with a theoretical prediction that the correlation between odd isotopes in fractionation induced by the MIE is controlled by the relationship between the spin conversion time and radical lifetime. The characteristic changes in the correlation between odd isotopes in fractionation induced by the MIE observed for Sn in this study provide a guideline for quantitatively determining fractionation patterns caused by the MIE for elements that have multiple isotopes with a nuclear spin. These results improve our understanding of the potential impact of the MIE on mass-independent fractionation observed in natural samples, such as meteorites, and analytical artifacts of high-precision isotope analysis for heavy elements.
RESUMEN
Hydrogen isotope ratios (δ2H) represent an important natural tracer of metabolic processes, but quantitative models of processes controlling H-fractionation in aquatic photosynthetic organisms are lacking. Here, we elucidate the underlying physiological controls of 2H/1H fractionation in algal lipids by systematically manipulating temperature, light, and CO2(aq) in continuous cultures of the haptophyte Gephyrocapsa oceanica. We analyze the hydrogen isotope fractionation in alkenones (αalkenone), a class of acyl lipids specific to this species and other haptophyte algae. We find a strong decrease in the αalkenone with increasing CO2(aq) and confirm αalkenone correlates with temperature and light. Based on the known biosynthesis pathways, we develop a cellular model of the δ2H of algal acyl lipids to evaluate processes contributing to these controls on fractionation. Simulations show that longer residence times of NADPH in the chloroplast favor a greater exchange of NADPH with 2H-richer intracellular water, increasing αalkenone. Higher chloroplast CO2(aq) and temperature shorten NADPH residence time by enhancing the carbon fixation and lipid synthesis rates. The inverse correlation of αalkenone to CO2(aq) in our cultures suggests that carbon concentrating mechanisms (CCM) do not achieve a constant saturation of CO2 at the Rubisco site, but rather that chloroplast CO2 varies with external CO2(aq). The pervasive inverse correlation of αalkenone with CO2(aq) in the modern and preindustrial ocean also suggests that natural populations may not attain a constant saturation of Rubisco with the CCM. Rather than reconstructing growth water, αalkenone may be a powerful tool to elucidate the carbon limitation of photosynthesis.
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Dióxido de Carbono , Haptophyta , Lípidos , Fotosíntesis , Dióxido de Carbono/metabolismo , Haptophyta/metabolismo , Lípidos/química , Hidrógeno/metabolismo , Cloroplastos/metabolismo , Deuterio/metabolismo , NADP/metabolismo , Temperatura , Fraccionamiento Químico/métodos , Metabolismo de los LípidosRESUMEN
Galactan polymer is a prominent component of the mycobacterial cell wall core. Its biogenesis starts at the cytoplasmic side of the plasma membrane by a build-up of the linker disaccharide [rhamnosyl (Rha) - N-acetyl-glucosaminyl (GlcNAc) phosphate] on the decaprenyl-phosphate carrier. This decaprenyl-P-P-GlcNAc-Rha intermediate is extended by two bifunctional galactosyl transferases, GlfT1 and GlfT2, and then it is translocated to the periplasmic space by an ABC transporter Wzm-Wzt. The cell wall core synthesis is finalized by the action of an array of arabinosyl transferases, mycolyl transferases, and ligases that catalyze an attachment of the arabinogalactan polymer to peptidoglycan through the linker region. Based on visualization of the GlfT2 enzyme fused with fluorescent tags it was proposed that galactan polymerization takes place in a specific compartment of the mycobacterial cell envelope, the intracellular membrane domain, representing pure plasma membrane free of cell wall components (previously denoted as the "PMf" domain), which localizes to the polar region of mycobacteria. In this work, we examined the activity of the galactan-producing cellular machine in the cell-wall containing cell envelope fraction and in the cell wall-free plasma membrane fraction prepared from Mycobacterium smegmatis by the enzyme assays using radioactively labeled substrate UDP-[14C]-galactose as a tracer. We found that despite a high abundance of GlfT2 in both of these fractions as confirmed by their thorough proteomic analyses, galactan is produced only in the reaction mixtures containing the cell wall components. Our findings open the discussion about the distribution of GlfT2 and the regulation of its activity in mycobacteria.
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Galactanos , Mycobacterium , Galactanos/biosíntesis , Polímeros/metabolismo , Proteómica , Transferasas/metabolismo , Mycobacterium/metabolismoRESUMEN
The allopolyploid okra (Abelmoschus esculentus) unveiled telomeric repeats flanking distal gene-rich regions and short interstitial TTTAGGG telomeric repeats, possibly representing hallmarks of chromosomal speciation. Ribosomal RNA (rRNA) genes organize into 5S clusters, distinct from the 18S-5.8S-28S units, indicating an S-type rRNA gene arrangement. The assembly, in line with cytogenetic and cytometry observations, identifies 65 chromosomes and a 1.45 Gb genome size estimate in a haploid sibling. The lack of aberrant meiotic configurations implies limited to no recombination among sub-genomes. k-mer distribution analysis reveals 75% has a diploid nature and 15% heterozygosity. The configurations of Benchmarking Universal Single-Copy Ortholog (BUSCO), k-mer, and repeat clustering point to the presence of at least two sub-genomes one with 30 and the other with 35 chromosomes, indicating the allopolyploid nature of the okra genome. Over 130 000 putative genes, derived from mapped IsoSeq data and transcriptome data from public okra accessions, exhibit a low genetic diversity of one single nucleotide polymorphisms per 2.1 kbp. The genes are predominantly located at the distal chromosome ends, declining toward central scaffold domains. Long terminal repeat retrotransposons prevail in central domains, consistent with the observed pericentromeric heterochromatin and distal euchromatin. Disparities in paralogous gene counts suggest potential sub-genome differentiation implying possible sub-genome dominance. Amino acid query sequences of putative genes facilitated phenol biosynthesis pathway annotation. Comparison with manually curated reference KEGG pathways from related Malvaceae species reveals the genetic basis for putative enzyme coding genes that likely enable metabolic reactions involved in the biosynthesis of dietary and therapeutic compounds in okra.
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Abelmoschus , Abelmoschus/genética , Abelmoschus/metabolismo , Genoma , Telómero , Diploidia , Variación GenéticaRESUMEN
Proteins are the molecular effectors of the information encoded in the genome. Proteomics aims at understanding the molecular functions of proteins in their biological context. In contrast to transcriptomics and genomics, the study of proteomes provides deeper insight into the dynamic regulatory layers encoded at the protein level, such as posttranslational modifications, subcellular localization, cell signaling, and protein-protein interactions. Currently, mass spectrometry (MS)-based proteomics is the technology of choice for studying proteomes at a system-wide scale, contributing to clinical biomarker discovery and fundamental molecular biology. MS technologies are continuously being developed to fulfill the requirements of speed, resolution, and quantitative accuracy, enabling the acquisition of comprehensive proteomes. In this review, we present how MS technology and acquisition methods have evolved to meet the requirements of cutting-edge proteomics research, which is describing the human proteome and its dynamic posttranslational modifications with unprecedented depth. Finally, we provide a perspective on studying proteomes at single-cell resolution.
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Proteoma , Proteómica , Genoma , Humanos , Espectrometría de Masas/métodos , Procesamiento Proteico-Postraduccional , Proteoma/análisis , Proteoma/química , Proteoma/metabolismo , Proteómica/métodosRESUMEN
Protein complexes are key functional units in cellular processes. High-throughput techniques, such as co-fractionation coupled with mass spectrometry (CF-MS), have advanced protein complex studies by enabling global interactome inference. However, dealing with complex fractionation characteristics to define true interactions is not a simple task, since CF-MS is prone to false positives due to the co-elution of non-interacting proteins by chance. Several computational methods have been designed to analyze CF-MS data and construct probabilistic protein-protein interaction (PPI) networks. Current methods usually first infer PPIs based on handcrafted CF-MS features, and then use clustering algorithms to form potential protein complexes. While powerful, these methods suffer from the potential bias of handcrafted features and severely imbalanced data distribution. However, the handcrafted features based on domain knowledge might introduce bias, and current methods also tend to overfit due to the severely imbalanced PPI data. To address these issues, we present a balanced end-to-end learning architecture, Software for Prediction of Interactome with Feature-extraction Free Elution Data (SPIFFED), to integrate feature representation from raw CF-MS data and interactome prediction by convolutional neural network. SPIFFED outperforms the state-of-the-art methods in predicting PPIs under the conventional imbalanced training. When trained with balanced data, SPIFFED had greatly improved sensitivity for true PPIs. Moreover, the ensemble SPIFFED model provides different voting schemes to integrate predicted PPIs from multiple CF-MS data. Using the clustering software (i.e. ClusterONE), SPIFFED allows users to infer high-confidence protein complexes depending on the CF-MS experimental designs. The source code of SPIFFED is freely available at: https://github.com/bio-it-station/SPIFFED.
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Mapeo de Interacción de Proteínas , Proteínas , Mapeo de Interacción de Proteínas/métodos , Proteínas/química , Algoritmos , Mapas de Interacción de Proteínas , Programas InformáticosRESUMEN
The variability of proteins at the sequence level creates an enormous potential for proteome complexity. Exploring the depths and limits of this complexity is an ongoing goal in biology. Here, we systematically survey human and plant high-throughput bottom-up native proteomics data for protein truncation variants, where substantial regions of the full-length protein are missing from an observed protein product. In humans, Arabidopsis, and the green alga Chlamydomonas, approximately one percent of observed proteins show a short form, which we can assign by comparison to RNA isoforms as either likely deriving from transcript-directed processes or limited proteolysis. While some detected protein fragments align with known splice forms and protein cleavage events, multiple examples are previously undescribed, such as our observation of fibrocystin proteolysis and nuclear translocation in a green alga. We find that truncations occur almost entirely between structured protein domains, even when short forms are derived from transcript variants. Intriguingly, multiple endogenous protein truncations of phase-separating translational proteins resemble cleaved proteoforms produced by enteroviruses during infection. Some truncated proteins are also observed in both humans and plants, suggesting that they date to the last eukaryotic common ancestor. Finally, we describe novel proteoform-specific protein complexes, where the loss of a domain may accompany complex formation.
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Arabidopsis , Proteómica , Arabidopsis/genética , Arabidopsis/metabolismo , Humanos , Proteómica/métodos , Chlamydomonas/metabolismo , Chlamydomonas/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteoma/genética , Proteolisis , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Empalme AlternativoRESUMEN
SignificanceSulfur isotopes confirm a key role for atmospheric sulfur gases in climatic cooling, mass extinction, and the demise of dinosaurs and other global biota after the Chicxulub bolide impact at the Cretaceous-Paleogene boundary. The sulfur isotope anomalies are confined to beds containing ejecta and, in the immediately overlying sediments, are temporally unrelated to known episodes of volcanism that also bracket this event, further addressing the controversial role of the Deccan Traps in the extinction.
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Dinosaurios , Extinción Biológica , Animales , Isótopos , AzufreRESUMEN
SignificanceThe permanent disappearance of mass-independent sulfur isotope fractionation (S-MIF) from the sedimentary record has become a widely accepted proxy for atmospheric oxygenation. This framework, however, neglects inheritance from oxidative weathering of pre-existing S-MIF-bearing sedimentary sulfide minerals (i.e., crustal memory), which has recently been invoked to explain apparent discrepancies within the sulfur isotope record. Herein, we demonstrate that such a crustal memory effect does not confound the Carletonville S-isotope record; rather, the pronounced Δ33S values identified within the Rooihoogte Formation represent the youngest known unequivocal oxygen-free photochemical products. Previously observed 33S-enrichments within the succeeding Timeball Hill Formation, however, contrasts with our record, revealing kilometer-scale heterogeneities that highlight significant uncertainties in our understanding of the dynamics of Earth's oxygenation.
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Post-transcriptional modification of RNA nucleosides has been implicated as a pivotal regulator of mRNA biology. In this issue of Genes & Development, Ke and colleagues (pp. 990-1006) provide insights into the temporal and spatial distribution of N6-methyladenosine (m6A) in RNA transcripts by analyzing different subcellular fractions. Using a recently developed biochemical approach for detecting m6A, the researchers show that m6A methylations are enriched in exons and are added to transcripts prior to splicing. Although m6A addition is widely thought to be readily reversible, they demonstrate in HeLa cells that once RNA is released from chromatin, the modifications are surprisingly static. This study integrates data from previous publications to clarify conflicting conclusions regarding the role of m6A in mRNA biogenesis and function. Ke and colleagues found that m6A methylation levels negatively correlate with transcript half-life but are not required for most pre-mRNA splicing events.
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Adenosina/metabolismo , Empalme del ARN/fisiología , ARN Mensajero/metabolismo , Adenosina/análisis , Animales , Exones/genética , Células HeLa , Humanos , Metilación , Metiltransferasas/metabolismoRESUMEN
Understanding the biologic role of N6-methyladenosine (m6A) RNA modifications in mRNA requires an understanding of when and where in the life of a pre-mRNA transcript the modifications are made. We found that HeLa cell chromatin-associated nascent pre-mRNA (CA-RNA) contains many unspliced introns and m6A in exons but very rarely in introns. The m6A methylation is essentially completed upon the release of mRNA into the nucleoplasm. Furthermore, the content and location of each m6A modification in steady-state cytoplasmic mRNA are largely indistinguishable from those in the newly synthesized CA-RNA or nucleoplasmic mRNA. This result suggests that quantitatively little methylation or demethylation occurs in cytoplasmic mRNA. In addition, only â¼10% of m6As in CA-RNA are within 50 nucleotides of 5' or 3' splice sites, and the vast majority of exons harboring m6A in wild-type mouse stem cells is spliced the same in cells lacking the major m6A methyltransferase Mettl3. Both HeLa and mouse embryonic stem cell mRNAs harboring m6As have shorter half-lives, and thousands of these mRNAs have increased half-lives (twofold or more) in Mettl3 knockout cells compared with wild type. In summary, m6A is added to exons before or soon after exon definition in nascent pre-mRNA, and while m6A is not required for most splicing, its addition in the nascent transcript is a determinant of cytoplasmic mRNA stability.
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Citoplasma/metabolismo , Precursores del ARN/metabolismo , Empalme del ARN , ARN Mensajero/metabolismo , Animales , Cromatina/metabolismo , Células Madre Embrionarias , Exones/genética , Técnicas de Inactivación de Genes , Células HeLa , Humanos , Intrones/genética , Metilación , Metiltransferasas/genética , Metiltransferasas/metabolismo , RatonesRESUMEN
In top-down (TD) proteomics, efficient proteoform separation is crucial to reduce the sample complexity and increase the depth of the analysis. Here, we developed a two-dimensional low pH/low pH reversed-phase liquid chromatography separation scheme for TD proteomics. The first dimension for offline fractionation was performed using a polymeric reversed-phase (PLRP-S) column with trifluoroacetic acid as ion-pairing reagent. The second dimension, a C4 nanocolumn with formic acid as ion-pairing reagent, was coupled online with a high-field asymmetric ion mobility spectrometry (FAIMS) Orbitrap Tribrid mass spectrometer. For both dimensions several parameters were optimized, such as the adaption of the LC gradients in the second dimension according to the elution time (i.e., fraction number) in the first dimension. Avoidance of elevated temperatures and prolonged exposure to acidic conditions minimized cleavage of acid labile aspartate-proline peptide bonds. Furthermore, a concatenation strategy was developed to reduce the total measurement time. We compared our low/low pH with a previously published high pH (C4, ammonium formate)/low pH strategy and found that both separation strategies led to complementary proteoform identifications, mainly below 20 kDa, with a higher number of proteoforms identified by the low/low pH separation. With the optimized separation scheme, more than 4900 proteoforms from 1250 protein groups were identified in Caco-2 cells.
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Cromatografía de Fase Inversa , Proteómica , Humanos , Cromatografía de Fase Inversa/métodos , Proteómica/métodos , Células CACO-2 , Cromatografía Líquida con Espectrometría de Masas , Concentración de Iones de HidrógenoRESUMEN
Fractionation of proteoforms is currently the most challenging topic in the field of proteoform analysis. The need for considering the existence of proteoforms in experimental approaches is not only important in Life Science research in general but especially in the manufacturing of therapeutic proteins (TPs) like recombinant therapeutic antibodies (mAbs). Some of the proteoforms of TPs have significantly decreased actions or even cause side effects. The identification and removal of proteoforms differing from the main species, having the desired action, is challenging because the difference in the composition of atoms is often very small and their concentration in comparison to the main proteoform can be low. In this study, we demonstrate that sample displacement batch chromatography (SDBC) is an easy-to-handle, economical, and efficient method for fractionating proteoforms. As a model sample a commercial ovalbumin fraction was used, containing many ovalbumin proteoforms. The most promising parameters for the SDBC were determined by a screening approach and applied for a 10-segment fractionation of ovalbumin with cation exchange chromatography resins. Mass spectrometry of intact proteoforms was used for characterizing the SDBC fractionation process. By SDBC, a significant separation of different proteoforms was obtained.
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Procesamiento Proteico-Postraduccional , Espectrometría de Masas en Tándem , Espectrometría de Masas en Tándem/métodos , Ovalbúmina/metabolismo , Cromatografía , Proteoma/análisisRESUMEN
Proteome coverage and accurate protein quantification are both important for evaluating biological systems; however, compromises between quantification, coverage, and mass spectrometry (MS) resources are often necessary. Consequently, experimental parameters that impact coverage and quantification must be adjusted, depending on experimental goals. Among these parameters is offline prefractionation, which is utilized in MS-based proteomics to decrease sample complexity resulting in higher overall proteome coverage upon MS analysis. Prefractionation leads to increases in required MS analysis time, although this is often mitigated by isobaric labeling using tandem-mass tags (TMT), which allow samples to be multiplexed. Here we evaluate common prefractionation schemes, TMT variants, and MS acquisition methods and their impact on protein quantification and coverage. Furthermore, we provide recommendations for experimental design depending on the experimental goals.
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Proteoma , Proteómica , Espectrometría de Masas en Tándem , Proteómica/métodos , Proteómica/normas , Espectrometría de Masas en Tándem/métodos , Proteoma/análisis , Humanos , Fraccionamiento Químico/métodos , Coloración y Etiquetado/métodosRESUMEN
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a widely employed technique in proteomics research for studying the proteome biology of various clinical samples. Hard tissues, such as bone and teeth, are routinely preserved using synthetic poly(methyl methacrylate) (PMMA) embedding resins that enable histological, immunohistochemical, and morphological examination. However, the suitability of PMMA-embedded hard tissues for large-scale proteomic analysis remained unexplored. This study is the first to report on the feasibility of PMMA-embedded bone samples for LC-MS/MS analysis. Conventional workflows yielded merely limited coverage of the bone proteome. Using advanced strategies of prefractionation by high-pH reversed-phase liquid chromatography in combination with isobaric tandem mass tag labeling resulted in proteome coverage exceeding 1000 protein identifications. The quantitative comparison with cryopreserved samples revealed that each sample preparation workflow had a distinct impact on the proteomic profile. However, workflow replicates exhibited a high reproducibility for PMMA-embedded samples. Our findings further demonstrate that decalcification prior to protein extraction, along with the analysis of solubilization fractions, is not preferred for PMMA-embedded bone. The biological applicability of the proposed workflow was demonstrated using samples of human PMMA-embedded alveolar bone and the iliac crest, which revealed anatomical site-specific proteomic profiles. Overall, these results establish a crucial foundation for large-scale proteomics studies contributing to our knowledge of bone biology.