RESUMEN
We report the identification of a photocleavable anionic surfactant, 4-hexylphenylazosulfonate (Azo), which can be rapidly degraded by ultraviolet irradiation, for top-down proteomics. Azo can effectively solubilize proteins with performance comparable to that of sodium dodecyl sulfate (SDS) and is compatible with mass spectrometry. Azo-aided top-down proteomics enables the solubilization of membrane proteins for comprehensive characterization of post-translational modifications. Moreover, Azo is simple to synthesize and can be used as a general SDS replacement in SDS-polyacrylamide gel electrophoresis.
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Compuestos Azo/química , Electroforesis en Gel de Poliacrilamida/métodos , Proteómica/métodos , Dodecil Sulfato de Sodio/química , Tensoactivos/química , Compuestos Azo/efectos de la radiación , Interacciones Hidrofóbicas e Hidrofílicas , Espectrometría de Masas , Proteínas de la Membrana/análisis , Fotólisis , Dodecil Sulfato de Sodio/efectos de la radiación , Solubilidad , Tensoactivos/efectos de la radiación , Rayos UltravioletaRESUMEN
One gene can give rise to many functionally distinct proteoforms, each of which has a characteristic molecular mass. Top-down mass spectrometry enables the analysis of intact proteins and proteoforms. Here members of the Consortium for Top-Down Proteomics provide a decision tree that guides researchers to robust protocols for mass analysis of intact proteins (antibodies, membrane proteins and others) from mixtures of varying complexity. We also present cross-platform analytical benchmarks using a protein standard sample, to allow users to gauge their proficiency.
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Benchmarking , Espectrometría de Masas/métodos , Proteínas/química , Desnaturalización Proteica , Procesamiento Proteico-Postraduccional , ProteómicaRESUMEN
RATIONALE: Human pluripotent stem cell (hPSC)-derived cardiomyocytes exhibit the properties of fetal cardiomyocytes, which limits their applications. Various methods have been used to promote maturation of hPSC-cardiomyocytes; however, there is a lack of an unbiased and comprehensive method for accurate assessment of the maturity of hPSC-cardiomyocytes. OBJECTIVE: We aim to develop an unbiased proteomics strategy integrating high-throughput top-down targeted proteomics and bottom-up global proteomics for the accurate and comprehensive assessment of hPSC-cardiomyocyte maturation. METHODS AND RESULTS: Utilizing hPSC-cardiomyocytes from early- and late-stage 2-dimensional monolayer culture and 3-dimensional engineered cardiac tissue, we demonstrated the high reproducibility and reliability of a top-down proteomics method, which enabled simultaneous quantification of contractile protein isoform expression and associated post-translational modifications. This method allowed for the detection of known maturation-associated contractile protein alterations and, for the first time, identified contractile protein post-translational modifications as promising new markers of hPSC-cardiomyocytes maturation. Most notably, decreased phosphorylation of α-tropomyosin was found to be associated with hPSC-cardiomyocyte maturation. By employing a bottom-up global proteomics strategy, we identified candidate maturation-associated markers important for sarcomere organization, cardiac excitability, and Ca2+ homeostasis. In particular, upregulation of myomesin 1 and transmembrane 65 was associated with hPSC-cardiomyocyte maturation and validated in cardiac development, making these promising markers for assessing maturity of hPSC-cardiomyocytes. We have further validated α-actinin isoforms, phospholamban, dystrophin, αB-crystallin, and calsequestrin 2 as novel maturation-associated markers, in the developing mouse cardiac ventricles. CONCLUSIONS: We established an unbiased proteomics method that can provide accurate and specific assessment of the maturity of hPSC-cardiomyocytes and identified new markers of maturation. Furthermore, this integrated proteomics strategy laid a strong foundation for uncovering the molecular pathways involved in cardiac development and disease using hPSC-cardiomyocytes.
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Diferenciación Celular , Cromatografía Liquida , Células Madre Pluripotentes Inducidas/metabolismo , Miocitos Cardíacos/metabolismo , Proteínas/metabolismo , Proteómica/métodos , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en Tándem , Sesgo , Técnicas de Cultivo de Célula , Línea Celular , Ensayos Analíticos de Alto Rendimiento , Humanos , Fenotipo , Reproducibilidad de los Resultados , Factores de TiempoRESUMEN
Understanding the relationship between the acceptor dopant size and proton conductivity in barium zirconate, BaZrO3, is important for maximizing efficiency in this promising fuel cell material. While proton conduction pathways with larger YZr ' and smaller AlZr ' defects have been explored, proton pathways with ScZr ', a defect of comparable size to the replaced ion, have not been investigated using centrality measures, periodic pathway searches, and kinetic Monte Carlo (KMC). Centrality measures in BaSc0.125Zr0.875O3 highlight a trapping region by ScZr ' and scattered high centrality regions on undoped planes. Connected long-range high centrality regions are found mainly in undoped planes for BaAl0.125Zr0.875O3 and in the dopant planes for BaY0.125Zr0.875O3. The best long-range proton conduction periodic pathways in AlZr ' and ScZr ' systems travel between dopant planes, while those for yttrium-doped BaZrO3 remained on dopant planes. KMC trajectories at 1000 K show long-range proton conduction barriers of 0.86 eV, 0.52 eV, and 0.25 eV for AlZr ', ScZr ', and YZr ' systems, respectively. Long-range periodic conduction highway limiting barrier averages correlate well with the connectivity of the most central regions in each system but ignore diffusion around the dopant and through other high centrality regions. BaSc0.125Zr0.875O3 shows an intermediate overall conduction barrier limited by trapping, which earlier experiments and simulations suggest that it can be mitigated with increased oxygen vacancy concentration.
RESUMEN
Determining changes in protein expression and post-translational modifications (PTMs) is crucial for elucidating cellular signal transduction and disease mechanisms. Conventional antibody-based approaches have inherent problems such as the limited availability of high-quality antibodies and batch-to-batch variation. Top-down mass spectrometry (MS)-based proteomics has emerged as the most powerful method for characterization and quantification of protein modifications. Nevertheless, robust methods to simultaneously determine changes in protein expression and PTMs remain lacking. Herein, we have developed a straightforward and robust top-down liquid chromatography (LC)/MS-based targeted proteomics platform for simultaneous quantification of protein expression and PTMs with high throughput and high reproducibility. We employed this method to analyze the sarcomeric subproteome from various muscle types of different species, which successfully revealed skeletal muscle heterogeneity and cardiac developmental changes in sarcomeric protein isoform expression and PTMs. As demonstrated, this targeted top-down proteomics platform offers an excellent 'antibody-independent' alternative for the accurate quantification of sarcomeric protein expression and PTMs concurrently in complex mixtures, which is generally applicable to different species and various tissue types.
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Corazón/crecimiento & desarrollo , Músculo Esquelético/crecimiento & desarrollo , Proteómica/métodos , Sarcómeros/metabolismo , Animales , Cromatografía Liquida , Regulación del Desarrollo de la Expresión Génica , Masculino , Músculo Esquelético/metabolismo , Miocardio/metabolismo , Procesamiento Proteico-Postraduccional , Ratas , Ovinos , Espectrometría de Masas en TándemRESUMEN
Heterogeneity in skeletal muscle contraction time, peak power output, and resistance to fatigue, among others, is necessary to accommodate the wide range of functional demands imposed on the body. Underlying this functional heterogeneity are a myriad of differences in the myofilament protein isoform expression and post-translational modifications; yet, characterizing this heterogeneity remains challenging. Herein, we have utilized top-down liquid chromatography (LC)-mass spectrometry (MS)-based proteomics to characterize myofilament proteoform heterogeneity in seven rat skeletal muscle tissues including vastus lateralis, vastus medialis, vastus intermedius, rectus femoris, soleus, gastrocnemius, and plantaris. Top-down proteomics revealed that myofilament proteoforms varied greatly across the seven different rat skeletal muscle tissues. Subsequently, we quantified and characterized myofilament proteoforms using online LC-MS. We have comprehensively characterized the fast and slow skeletal troponin I isoforms, which demonstrates the ability of top-down MS to decipher isoforms with high sequence homology. Taken together, we have shown that top-down proteomics can be used as a robust and high-throughput method to characterize the molecular heterogeneity of myofilament proteoforms from various skeletal muscle tissues.
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Proteínas Musculares/análisis , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Miofibrillas/metabolismo , Animales , Cromatografía Liquida/métodos , Electroforesis en Gel de Poliacrilamida , Masculino , Proteómica/métodos , Ratas Endogámicas F344 , Espectrometría de Masas en Tándem , Troponina T/análisis , Troponina T/metabolismoRESUMEN
Sarcopenia, the age-related loss of skeletal muscle mass and strength, is a significant cause of morbidity in the elderly and is a major burden on health care systems. Unfortunately, the underlying molecular mechanisms in sarcopenia remain poorly understood. Herein, we utilized top-down proteomics to elucidate sarcopenia-related changes in the fast- and slow-twitch skeletal muscles of aging rats with a focus on the sarcomeric proteome, which includes both myofilament and Z-disc proteins-the proteins that constitute the contractile apparatuses. Top-down quantitative proteomics identified significant changes in the post-translational modifications (PTMs) of critical myofilament proteins in the fast-twitch skeletal muscles of aging rats, in accordance with the vulnerability of fast-twitch muscles to sarcopenia. Surprisingly, age-related alterations in the phosphorylation of Cypher isoforms, proteins that localize to the Z-discs in striated muscles, were also noted in the fast-twitch skeletal muscle of aging rats. This represents the first report of changes in the phosphorylation of Z-disc proteins in skeletal muscle during aging. In addition, increased glutathionylation of slow skeletal troponin I, a novel modification that may help protect against oxidative damage, was observed in slow-twitch skeletal muscles. Furthermore, we have identified and characterized novel muscle type-specific proteoforms of myofilament proteins and Z-disc proteins, including a novel isoform of the Z-disc protein Enigma. The finding that the phosphorylation of Z-disc proteins is altered in response to aging in the fast-twitch skeletal muscles of aging rats opens new avenues for the investigation of the role of Z-discs in age-related muscle dysfunction.
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Músculo Esquelético/metabolismo , Sarcómeros/metabolismo , Sarcopenia/metabolismo , Envejecimiento/metabolismo , Animales , Masculino , Procesamiento Proteico-Postraduccional , Proteómica , RatasRESUMEN
Cellular metabolites act as important signaling cues, but are subject to complex unknown chemistry. Kynurenine is a tryptophan metabolite that plays a crucial role in cancer and the immune system. Despite its atypical, non-ligand-like, highly polar structure, kynurenine activates the aryl hydrocarbon receptor (AHR), a PER, ARNT, SIM (PAS) family transcription factor that responds to diverse environmental and cellular ligands. The activity of kynurenine is increased 100-1000-fold by incubation or long-term storage and relies on the hydrophobic ligand-binding pocket of AHR, with identical structural signatures for AHR induction before and after activation. We purified trace-active derivatives of kynurenine and identified two novel, closely related condensation products, named trace-extended aromatic condensation products (TEACOPs), which are active at low picomolar levels. The synthesized compound for one of the predicted structures matched the purified compound in both chemical structure and AHR pharmacology. Our study provides evidence that kynurenine acts as an AHR pro-ligand, which requires novel chemical conversions to act as a receptor agonist.
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Quinurenina/química , Quinurenina/metabolismo , Receptores de Hidrocarburo de Aril/metabolismo , Animales , Sitios de Unión , Cinética , Ligandos , Ratones , Estructura Molecular , Receptores de Hidrocarburo de Aril/química , Receptores de Hidrocarburo de Aril/genéticaRESUMEN
The transition of mass spectrometry for clinical analysis is highly desirable, and major progress has been made with direct sampling ionization for operation simplification. High-precision quantitation, however, remains a major challenge in this transition. Herein, a novel method was developed for direct quantitation of biofluid samples, using an extremely simplified procedure for incorporation of internal standards selected against the traditional rules. Slug flow microextraction was used for the development, with conditions predicted by a theoretical model, viz., using internal standards of partition coefficients very different from the analytes and large sample-to-extraction solvent volume ratios. Direct quantitation of drug compounds in urine and blood samples was demonstrated. This development enabled an extremely simplified protocol that is expected to have a significant impact on on-site or clinical analysis.
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Líquidos Corporales , Preparaciones Farmacéuticas/sangre , Preparaciones Farmacéuticas/orina , Espectrometría de Masa por Ionización de Electrospray/métodos , Amitriptilina/sangre , Animales , Bovinos , Humanos , Límite de Detección , Lincomicina/sangre , Lincomicina/farmacocinética , Microextracción en Fase Líquida/métodos , Morfina/orina , Espectrometría de Masa por Ionización de Electrospray/instrumentaciónRESUMEN
Mass spectrometry (MS) based top-down proteomics provides rich information about proteoforms arising from combinatorial amino acid sequence variations and post-translational modifications (PTMs). Fourier transform ion cyclotron resonance (FT-ICR) MS affords ultrahigh resolving power and provides high-accuracy mass measurements, presenting a powerful tool for top-down MS characterization of proteoforms. However, the detection and characterization of large proteins from complex mixtures remain challenging due to the exponential decrease in S: N with increasing molecular weight (MW) and coeluting low-MW proteins; thus, size-based fractionation of complex protein mixtures prior to MS analysis is necessary. Here, we directly combine MS-compatible serial size exclusion chromatography (sSEC) fractionation with 12 T FT-ICR MS for targeted top-down characterization of proteins from complex mixtures extracted from human and swine heart tissue. Benefiting from the ultrahigh resolving power of FT-ICR, we isotopically resolved 31 distinct proteoforms (30-50 kDa) simultaneously in a single mass spectrum within a 100 m/ z window. Notably, within a 5 m/ z window, we obtained baseline isotopic resolution for 6 distinct large proteoforms (30-50 kDa). The ultrahigh resolving power of FT-ICR MS combined with sSEC fractionation enabled targeted top-down analysis of large proteoforms (>30 kDa) from the human heart proteome without extensive chromatographic separation or protein purification. Further separation of proteoforms inside the mass spectrometer (in-MS) allowed for isolation of individual proteoforms and targeted electron capture dissociation (ECD), yielding high sequence coverage. sSEC/FT-ICR ECD facilitated the identification and sequence characterization of important metabolic enzymes. This platform, which facilitates deep interrogation of proteoform primary structure, is highly tunable, allows for adjustment of MS and MS/MS parameters in real time, and can be utilized for a variety of complex protein mixtures.
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Cromatografía en Gel/instrumentación , Ciclotrones , Análisis de Fourier , Espectrometría de Masas/instrumentación , Proteómica/instrumentación , Humanos , Miocardio/metabolismoRESUMEN
Antibody-drug conjugates (ADCs) are designed to combine the target specificity of monoclonal antibodies and potent cytotoxin drugs to achieve better therapeutic outcomes. Comprehensive evaluation of the quality attributes of ADCs is critical for drug development but remains challenging due to heterogeneity of the construct. Currently, peptide mapping with reversed-phase liquid chromatography (RPLC) coupled to mass spectrometry (MS) is the predominant approach to characterize ADCs. However, it is suboptimal for sequence characterization and quantification of ADCs because it lacks a comprehensive view of coexisting variants and suffers from varying ionization effects of drug-conjugated peptides compared to unconjugated counterparts. Here, we present the first middle-down RPLC-MS analysis of both cysteine (Adcetris; BV) and lysine (Kadcyla; T-DM1) conjugated ADCs at the subunit level (â¼25 kDa) with electron transfer dissociation (ETD). We successfully achieved high-resolution separation of subunit isomers arising from different drug conjugation and subsequently localized the conjugation sites. Moreover, we obtained a comprehensive overview of the microvariants associated with each subunits and characterized them such as oxidized variants with different sites. Furthermore, we observed relatively high levels of conjugation near complementarity-determining regions (CDRs) from the heavy chain but no drug conjugation near CDRs of light chain (Lc) from lysine conjugated T-DM1. Based on the extracted ion chromatograms, we accurately measured average drug to antibody ratio (DAR) values and relative occupancy of drug-conjugated subunits. Overall, the middle-down MS approach enables the evaluation of multiple quality attributes including DAR, positional isomers, conjugation sites, occupancy, and microvariants, which potentially opens up a new avenue to characterize ADCs.
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Ado-Trastuzumab Emtansina/química , Brentuximab Vedotina/química , Inmunoconjugados/análisis , Inmunoconjugados/química , Ado-Trastuzumab Emtansina/análisis , Brentuximab Vedotina/análisis , Cromatografía de Fase Inversa , Cisteína/química , Transporte de Electrón , Lisina/química , Espectrometría de Masas en Tándem/métodosRESUMEN
O-linked ß-N-acetylglucosamine (O-GlcNAc) transferase (OGT) is an essential human glycosyltransferase that adds O-GlcNAc modifications to numerous proteins. However, little is known about the mechanism with which OGT recognizes various protein substrates. Here we report on GlcNAc electrophilic probes (GEPs) to expedite the characterization of OGT-substrate recognition. Data from mass spectrometry, X-ray crystallization, and biochemical and radiolabeled kinetic assays support the application of GEPs to rapidly report the impacts of OGT mutations on protein substrate or sugar binding and to discover OGT residues crucial for protein recognition. Interestingly, we found that the same residues on the inner surface of the N-terminal domain contribute to OGT interactions with different protein substrates. By tuning reaction conditions, a GEP enables crosslinking of OGT with acceptor substrates in situ, affording a unique method to discover genuine substrates that weakly or transiently interact with OGT. Hence, GEPs provide new strategies to dissect OGT-substrate binding and recognition.
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Sondas Moleculares/metabolismo , N-Acetilglucosaminiltransferasas/metabolismo , Cristalografía por Rayos X , Humanos , Cinética , Espectrometría de Masas , Modelos Moleculares , Conformación Molecular , Sondas Moleculares/síntesis química , Sondas Moleculares/química , Mutación , N-Acetilglucosaminiltransferasas/química , N-Acetilglucosaminiltransferasas/genética , Especificidad por SustratoRESUMEN
There is currently no large population data-based data set in Kashgar Prefecture Uyghur. The allele frequencies of 18 autosomal short tandem repeat (STR) loci included in the DNATyper™ 19 kit were evaluated in 2600 Uyghur individuals living in Kashgar Prefecture, Northwest China. The values of combined power of discrimination (CPD) and combined probability of exclusion (CPE) of all 18 autosomal STR loci were 0.99999999999999999998235 and 0.99999998670, respectively. Phylogenetic analyses revealed that the Uyghur population has a closer relationship with the Xinjiang-Kazakh, Inner Mongolia-Mongolian, and other three Uyghur populations. In addition, our results are consistent with the hypothesis that Uyghur population is an admixture of Eastern Asian and European populations.
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Etnicidad/genética , Frecuencia de los Genes , Genética de Población , Repeticiones de Microsatélite , China , Dermatoglifia del ADN , Humanos , Filogenia , Reacción en Cadena de la Polimerasa , Análisis de Componente PrincipalRESUMEN
Ili is located in northernmost Xinjiang, China. The Uyghur population only accounts for 15.90% of the total population in the nation. There is currently no large population data-based data set in Ili Uyghur. In this study, we investigated the genetic diversities of 18 autosomal short tandem repeat (STR) loci in 1129 Uyghur individuals living in Ili. The values of combined power of discrimination (CPD) and combined probability of exclusion (CPE) were 0.99999999999999999999990244 and 0.99999995645, respectively. Furthermore, we explored the genetic relationships between the Ili Uyghur population and 32 previously published populations. The results indicated that the Ili Uyghur population was more closely related to the Xinjiang Kazakh population. In addition, It was worth noting that significant differences were observed between Ili the Uyghur population and the Uyghur1 and Uyghur2 populations at the shared 15 loci, with significant differences at 7 and 11 loci after Bonferroni adjustment (p = 0.05/495 ≈ 0.00010).
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Etnicidad/genética , Variación Genética , Genética de Población , Repeticiones de Microsatélite , Filogenia , China , Dermatoglifia del ADN , Frecuencia de los Genes , HumanosRESUMEN
Despite advancements in symptom management for heart failure (HF), this devastating clinical syndrome remains the leading cause of death in the developed world. Studies using animal models have greatly advanced our understanding of the molecular mechanisms underlying HF; however, differences in cardiac physiology and the manifestation of HF between animals, particularly rodents, and humans necessitates the direct interrogation of human heart tissue samples. Nevertheless, an ever-present concern when examining human heart tissue samples is the potential for artefactual changes related to temperature changes during tissue shipment or sample processing. Herein, we examined the effects of temperature on the post-translational modifications (PTMs) of sarcomeric proteins, the proteins responsible for muscle contraction, under conditions mimicking those that might occur during tissue shipment or sample processing. Using a powerful top-down proteomics method, we found that sarcomeric protein PTMs were differentially affected by temperature. Specifically, cardiac troponin I and enigma homolog isoform 2 showed robust increases in phosphorylation when tissue was incubated at either 4⯰C or 22⯰C. The observed increase is likely due to increased cyclic AMP levels and activation of protein kinase A in the tissue. On the contrary, cardiac troponin T and myosin regulatory light chain phosphorylation decreased when tissue was incubated at 4⯰C or 22⯰C. Furthermore, significant protein degradation was also observed after incubation at 4⯰C or 22⯰C. Overall, these results indicate that temperature exerts various effects on sarcomeric protein PTMs and careful tissue handling is critical for studies involving human heart samples. Moreover, these findings highlight the power of top-down proteomics for examining the integrity of cardiac tissue samples.
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Miocardio/metabolismo , Procesamiento Proteico-Postraduccional , Proteómica/métodos , Sarcómeros/metabolismo , Temperatura , Proteínas Adaptadoras Transductoras de Señales , Análisis de Varianza , Cromatografía de Fase Inversa , AMP Cíclico/análisis , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Insuficiencia Cardíaca/metabolismo , Humanos , Proteínas con Dominio LIM , Cadenas Ligeras de Miosina/metabolismo , Fosforilación , Isoformas de Proteínas/metabolismo , Proteolisis , Manejo de Especímenes/efectos adversos , Espectrometría de Masas en Tándem , Troponina I/metabolismo , Troponina T/metabolismoRESUMEN
Therapeutic monoclonal antibodies (mAbs) are an important class of drugs for a wide spectrum of human diseases. Liquid chromatography (LC) coupled to mass spectrometry (MS) is one of the techniques in the forefront for comprehensive characterization of analytical attributes of mAbs. Among various protein chromatography modes, hydrophobic interaction chromatography (HIC) is a popular offline nondenaturing separation technique utilized to purify and analyze mAbs, typically with the use of non-MS-compatible mobile phases. Herein we demonstrate for the first time, the application of direct HIC-MS and HIC-tandem MS (MS/MS) with electron capture dissociation (ECD) for analyzing intact mAbs on quadrupole-time-of-flight (Q-TOF) and Fourier transform ion cyclotron resonance (FTICR) mass spectrometers, respectively. Our method allows for rapid determination of relative hydrophobicity, intact masses, and glycosylation profiles of mAbs as well as sequence and structural characterization of the complementarity-determining regions in an online configuration.
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Anticuerpos Monoclonales/análisis , Interacciones Hidrofóbicas e Hidrofílicas , Cromatografía Liquida , Humanos , Espectrometría de MasasRESUMEN
Protein phosphorylation is a ubiquitous and critical post-translational modification (PTM) involved in numerous cellular processes. Mass spectrometry (MS)-based proteomics has emerged as the preferred technology for protein identification, characterization, and quantification. Whereas ionization/detection efficiency of peptides in electrospray ionization (ESI)-MS are markedly influenced by the presence of phosphorylation, the physicochemical properties of intact proteins are assumed not to vary significantly due to the relatively smaller modification on large intact proteins. Thus, the ionization/detection efficiency of intact phosphoprotein is hypothesized not to alter appreciably for subsequent MS quantification. However, this hypothesis has never been rigorously tested. Herein, we systematically investigated the impact of phosphorylation on ESI-MS quantification of mono- and multiply phosphorylated proteins. We verified that a single phosphorylation did not appreciably affect the ESI-MS quantification of phosphoproteins as demonstrated in the enigma homolog isoform 2 (28 kDa) with monophosphorylation. Moreover, different ionization and desolvation parameters did not impact phosphoprotein quantification. In contrast to monophosphorylation, multiphosphorylation noticeably affected ESI-MS quantification of phosphoproteins likely due to differential ionization/detection efficiency between unphosphorylated and phosphorylated proteoforms as shown in the pentakis-phosphorylated ß-casein (24 kDa).
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Fosfoproteínas/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Proteínas Adaptadoras Transductoras de Señales/análisis , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Caseínas/análisis , Caseínas/metabolismo , Cromatografía Líquida de Alta Presión , Humanos , Proteínas con Dominio LIM/análisis , Proteínas con Dominio LIM/metabolismo , Fosfopéptidos/análisis , Fosfoproteínas/metabolismo , Fosforilación , ProteómicaRESUMEN
Short tandem repeats (STRs) play a vitally important role in forensics. Population data is needed to improve the field. There is currently no large population data-based data set in Chamdo Tibetan. In our study, the allele frequencies and forensic statistical parameters of 18 autosomal STR loci (D5S818, D21S11, D7S820, CSF1PO, D2S1338, D3S1358, VWA, D8S1179, D16S539, PentaE, TPOX, TH01, D19S433, D18S51, FGA, D6S1043, D13S317, and D12S391) included in the DNATyper™19 kit were investigated in 2249 healthy, unrelated Tibetan subjects living in Tibet Chamdo, Southwest China. The combined power of discrimination and the combined probability of exclusion of all 18 loci were 0.9999999999999999999998174 and 0.99999994704, respectively. Furthermore, the genetic relationship between our Tibetan group and 33 previously published populations was also investigated. Phylogenetic analyses revealed that the Chamdo Tibetan population is more closely related genetically with the Lhasa Tibetan group. Our results suggest that these autosomal STR loci are highly polymorphic in the Tibetan population living in Tibet Chamdo and can be used as a powerful tool in forensics, linguistics, and population genetic analyses.
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Etnicidad/genética , Genética de Población , Polimorfismo Genético , China , Dermatoglifia del ADN , Frecuencia de los Genes , Humanos , Repeticiones de Microsatélite , Filogenia , Tibet/etnologíaRESUMEN
Myosin is the principal component of the thick filaments that, through interactions with the actin thin filaments, mediates force production during muscle contraction. Myosin is a hexamer, consisting of two heavy chains, each associated with an essential (ELC) and a regulatory (RLC) light chain, which bind the lever-arm of the heavy chain and play important modulatory roles in striated muscle contraction. Nevertheless, a comprehensive assessment of the sequences of the ELC and RLC isoforms, as well as their post-translational modifications, in the heart remains lacking. Herein, utilizing top-down high-resolution mass spectrometry (MS), we have comprehensively characterized the sequences and N-terminal modifications of the atrial and ventricular isoforms of the myosin light chains from human and swine hearts, as well as the sites of phosphorylation in the swine proteins. In addition to the correction of disparities in the database sequences of the swine proteins, we show for the first time that, whereas the ventricular isoforms of the ELC and RLC are methylated at their N-termini, which is consistent with previous studies, the atrial isoforms of the ELC and RLC from both human and swine are Nα-methylated and Nα-acetylated, respectively. Furthermore, top-down MS with electron capture dissociation enabled localization of the sites of phosphorylation in swine RLC isoforms from the ventricles and atria to Ser14 and Ser22, respectively. Collectively, these results provide new insights into the sequences and modifications of myosin light chain isoforms in the human and swine hearts, which will pave the way for a better understanding of their functional roles in cardiac physiology and pathophysiology.
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Atrios Cardíacos/metabolismo , Miocardio/metabolismo , Cadenas Ligeras de Miosina/genética , Isoformas de Proteínas/genética , Animales , Ventrículos Cardíacos/metabolismo , Humanos , Miocardio/patología , Cadenas Ligeras de Miosina/metabolismo , Fosforilación , Isoformas de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional/genética , Sarcómeros/metabolismo , PorcinosRESUMEN
Myosin heavy chain (MHC), the major component of the myosin motor molecule, plays an essential role in force production during muscle contraction. However, a comprehensive analysis of MHC proteoforms arising from sequence variations and post-translational modifications (PTMs) remains challenging due to the difficulties in purifying MHC (â¼223 kDa) and achieving complete sequence coverage. Herein, we have established a strategy to effectively purify and comprehensively characterize MHC from heart tissue by combining size-exclusion chromatography (SEC) and middle-down mass spectrometry (MS). First, we have developed a MS-compatible SEC method for purifying MHC from heart tissue with high efficiency. Next, we have optimized the Glu-C, Asp-N, and trypsin limited digestion conditions for middle-down MS. Subsequently, we have applied this strategy with optimized conditions to comprehensively characterize human MHC and identified ß-MHC as the predominant isoform in human left ventricular tissue. Full sequence coverage based on highly accurate mass measurements has been achieved using middle-down MS combining 1 Glu-C, 1 Asp-N, and 1 trypsin digestion. Three different PTMs: acetylation, methylation, and trimethylation were identified in human ß-MHC and the corresponding sites were localized to the N-terminal Gly, Lys34, and Lys129, respectively, by electron capture dissociation (ECD). Taken together, we have demonstrated this strategy is highly efficient for purification and characterization of MHC, which can be further applied to studies of the role of MHC proteoforms in muscle-related diseases. We also envision that this integrated SEC/middle-down MS strategy can be extended for the characterization of other large proteins over 200 kDa.