RESUMEN
Peptidomics is the detection and identification of the peptides present in a sample, and quantitative peptidomics provides additional information about the amounts of these peptides. It is possible to perform absolute quantitation of peptide levels in which the biological sample is compared to synthetic standards of each peptide. More commonly, relative quantitation is performed to compare peptide levels between two or more samples. Relative quantitation can measure differences between all peptides that are detectable, which can exceed 1000 peptides in a complex sample. In this chapter, various techniques used for quantitative peptidomics are described along with discussion of the advantages and disadvantages of each approach. A guide to selecting the optimal quantitative approach is provided, based on the goals of the experiment and the resources that are available.
Asunto(s)
Proteómica , Espectrometría de Masas en Tándem , Espectrometría de Masas en Tándem/métodos , Proteómica/métodos , Péptidos , Estándares de ReferenciaRESUMEN
Neuropeptides are key signaling molecules in many pathways and can serve as potential biomarkers or therapeutics. Mass spectrometry has emerged as a powerful tool for studying neuropeptides with high sensitivity and accuracy. Isobaric tagging can further enhance this method by improving throughput and reducing sampling needs. In this chapter, we discuss the benefits and limitations of using isobaric tags to analyze neuropeptides. Methods for optimizing the data acquisition are also presented to enable a greater number of neuropeptides to be identified and quantified when using isobaric tags, specifically N,N-dimethyl leucine (DiLeu).
Asunto(s)
Neuropéptidos , Espectrometría de Masas en Tándem , Leucina/análogos & derivados , Leucina/química , Neuropéptidos/metabolismo , Proteómica/métodos , Espectrometría de Masas en Tándem/métodosRESUMEN
Restenosis, re-narrowing of arterial lumen following intervention for cardiovascular disease, remains a major issue limiting the long-term therapeutic efficacy of treatment. The signaling molecules, TGFß (transforming growth factor-beta) and Smad3, play important roles in vascular restenosis, but very little is yet known about the down-stream dynamics in global protein expression and phosphorylation. Here, we develop a highly multiplexed quantitative proteomic and phosphoproteomic strategy employing 12-plex N,N-dimethyl leucine (DiLeu) isobaric tags and The DiLeu Tool software to globally assess protein expression and phosphorylation changes in smooth muscle cells (SMCs) treated with TGFß/Smad3 and/or SDF-1α (stromal cell-derived factor). A total of 4086 proteins were quantified in the combined dataset of proteome and phosphoproteome across 12-plex DiLeu-labeled SMC samples. 2317 localized phosphorylation sites were quantified, corresponding to 1193 phosphoproteins. TGFß/Smad3 induced up-regulation of 40 phosphosites and down-regulation of 50 phosphosites, and TGFß/Smad3-specific SDF-1α exclusively facilitated up-regulation of 27 phosphosites and down-regulation of 47 phosphosites. TGFß/Smad3 inhibited the expression of contractile-associated proteins including smooth muscle myosin heavy chain, calponin, cardiac muscle alpha-actin, and smooth muscle protein 22α. Gene ontology and pathway enrichment analysis revealed that elevated TGFß/Smad3 activated cell proliferation and TGFß signaling pathway, sequentially stimulating phosphorylation of CXCR4 (C-X-C chemokine receptor 4). SDF-1α/CXCR4 activated extracellular signal-regulating kinase signaling pathway and facilitated the expression of synthetic marker, osteopontin, which was validated through targeted analysis. These findings provide new insights into the mechanisms of TGFß regulated SMC dedifferentiation, as well as new avenues for designing effective therapeutics for vascular disease.
Asunto(s)
Desdiferenciación Celular , Músculo Liso Vascular , Miocitos del Músculo Liso , Proteómica , Factor de Crecimiento Transformador betaRESUMEN
The use of mass spectrometry for protein identification and quantification in cerebrospinal fluid (CSF) is at the forefront of research efforts to identify and explore biomarkers for the early diagnosis and prognosis of neurologic disorders. Here we implemented a 4-plex N,N-dimethyl leucine (DiLeu) isobaric labeling strategy in a longitudinal study aiming to investigate protein dynamics in children with B-cell acute lymphoblastic leukemia (B-cell ALL) undergoing chemotherapy. The temporal profile of CSF proteome during chemotherapy treatment at weeks 5, 10-14, and 24-28 highlighted many differentially expressed proteins, such as neural cell adhesion molecule, neuronal growth regulator 1, and secretogranin-3, all of which play important roles in neurodegenerative diseases. A total of 63 proteins were significantly altered across all of the time points investigated. The most over-represented biological processes from gene ontology analysis included platelet degranulation, complement activation, cell adhesion, fibrinolysis, neuron projection, regeneration, and regulation of neuron death. We expect that results from this and future studies will provide a means to monitor neurotoxicity and develop strategies to prevent central nervous system injury in response to chemotherapy in children.
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Leucemia-Linfoma Linfoblástico de Células Precursoras , Proteómica , Linfocitos B , Niño , Humanos , Leucina , Estudios Longitudinales , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Espectrometría de Masas en TándemRESUMEN
Mass spectrometry-based stable isotope labeling provides the advantages of multiplexing capability and accurate quantification but requires tailored bioinformatics tools for data analysis. Despite the rapid advancements in analytical methodology, it is often challenging to analyze stable isotope labeling-based metabolomics data, particularly for isobaric labeling using MS/MS reporter ions for quantification. We report Metandem, a novel online software tool for isobaric labeling-based metabolomics, freely available at http://metandem.com/web/. Metandem provides a comprehensive data analysis pipeline integrating feature extraction, metabolite quantification, metabolite identification, batch processing of multiple data files, online parameter optimization for custom datasets, data normalization, and statistical analysis. Systematic evaluation of the Metandem tool was demonstrated on UPLC-MS/MS, nanoLC-MS/MS, CE-MS/MS and MALDI-MS platforms, via duplex, 4-plex, 10-plex, and 12-plex isobaric labeling experiments and the application to various biological samples.
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Metabolómica/métodos , Programas Informáticos , Internet , Marcaje Isotópico , Espectrometría de Masas en Tándem , Interfaz Usuario-ComputadorRESUMEN
Extracellular matrix (ECM) is an important component of the pancreatic microenvironment which regulates ß cell proliferation, differentiation, and insulin secretion. Protocols have recently been developed for the decellularization of the human pancreas to generate functional scaffolds and hydrogels. In this work, we characterized human pancreatic ECM composition before and after decellularization using isobaric dimethylated leucine (DiLeu) labeling for relative quantification of ECM proteins. A novel correction factor was employed in the study to eliminate the bias introduced during sample preparation. In comparison to the commonly employed sample preparation methods (urea and FASP) for proteomic analysis, a recently developed surfactant and chaotropic agent assisted sequential extraction/on pellet digestion (SCAD) protocol has provided an improved strategy for ECM protein extraction of human pancreatic ECM matrix. The quantitative proteomic results revealed the preservation of matrisome proteins while most of the cellular proteins were removed. This method was compared with a well-established label-free quantification (LFQ) approach which rendered similar expressions of different categories of proteins (collagens, ECM glycoproteins, proteoglycans, etc.). The distinct expression of ECM proteins was quantified comparing adult and fetal pancreas ECM, shedding light on the correlation between matrix composition and postnatal ß cell maturation. Despite the distinct profiles of different subcategories in the native pancreas, the distribution of matrisome proteins exhibited similar trends after the decellularization process. Our method generated a large data set of matrisome proteins from a single tissue type. These results provide valuable insight into the possibilities of constructing a bioengineered pancreas. It may also facilitate better understanding of the potential roles that matrisome proteins play in postnatal ß cell maturation.
Asunto(s)
Proteínas de la Matriz Extracelular/genética , Páncreas/metabolismo , Proteoglicanos/genética , Proteómica , Colágeno/genética , Colágeno/metabolismo , Proteínas de la Matriz Extracelular/aislamiento & purificación , Humanos , Hidrogeles/química , Proteoglicanos/aislamiento & purificaciónRESUMEN
Peptidomics is the detection and identification of the peptides present in a sample, while quantitative peptidomics provides additional information about the amounts of these peptides. Comparison of peptide levels among two or more samples is termed relative quantitation. It is also possible to perform absolute quantitation of peptide levels in which the biological sample is compared to synthetic standards, which requires a separate standard for each peptide. In contrast, relative quantitation can compare levels of all peptides that are detectable in a sample, which can exceed 1000 peptides in a complex sample. In this chapter, various techniques used for quantitative peptidomics are described along with discussion of the advantages and disadvantages of each approach. A guide to selecting the optimal quantitative approach is provided, based on the goals of the experiment and the resources that are available.
Asunto(s)
Carboxipeptidasa H/fisiología , Cromatografía Liquida/métodos , Fragmentos de Péptidos/análisis , Fragmentos de Péptidos/metabolismo , Hipófisis/metabolismo , Proteómica/métodos , Espectrometría de Masas en Tándem/métodos , Animales , Ratones , Ratones NoqueadosRESUMEN
Mass spectrometry (MS)-based isobaric labeling has undergone rapid development in recent years due to its capability for high throughput quantitation. Apart from its originally designed use with collision-induced dissociation (CID) and higher-energy collisional dissociation (HCD), isobaric tagging technique could also work with electron-transfer dissociation (ETD), which provides complementarity to CID and is preferred in sequencing peptides with post-translational modifications (PTMs). However, ETD suffers from long reaction time, reduced duty cycle and bias against peptides with lower charge states. In addition, common fragmentation mechanism in ETD results in altered reporter ion production, decreased multiplexing capability, and even loss of quantitation capability for some of the isobaric tags, including custom-designed dimethyl leucine (DiLeu) tags. Here, we demonstrate a novel electron-transfer/higher-energy collision dissociation (EThcD) approach that preserves original reporter ion channels, mitigates bias against lower charge states, improves sensitivity, and significantly improves data quality for quantitative proteomics and proteome-wide PTM studies. Systematic optimization was performed to achieve a balance between data quality and sensitivity. We provide direct comparison of EThcD with ETD and HCD for DiLeu- and TMT-labeled HEK cell lysate and IMAC enriched phosphopeptides. Results demonstrate improved data quality and phosphorylation localization accuracy while preserving sufficient reporter ion production. Biological studies were performed to investigate phosphorylation changes in a mouse vascular smooth muscle cell line treated with four different conditions. Overall, EThcD exhibits superior performance compared to conventional ETD and offers distinct advantages compared to HCD in isobaric labeling based quantitative proteomics and quantitative PTM studies.
Asunto(s)
Exactitud de los Datos , Procesamiento Proteico-Postraduccional , Proteómica , Animales , Línea Celular , Iones , Espectrometría de Masas , Ratones , Miocitos del Músculo Liso , ProteomaRESUMEN
Isobaric labeling has become a popular technique for high-throughput, mass spectrometry (MS)-based relative quantification of peptides and proteins. However, widespread use of the approach for large-scale proteomics applications has been limited by the high cost of commercial isobaric tags. To address this, we have developed our own N,N-dimethyl leucine (DiLeu) multiplex isobaric tags as a cost-effective alternative that can be synthesized with ease using readily available isotopic reagents. When paired with high-resolution tandem mass (MS(n)) acquisition, mass defect-based DiLeu isobaric tags allow relative quantification of up to twelve samples in a single liquid chromatography (LC)-MS(2) experiment. Herein, we present detailed methods for synthesis of 12-plex DiLeu isobaric tags, labeling of complex protein digest samples, analysis by high-resolution nanoLC-MS(n), and processing of acquired data.
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Proteómica/métodos , Cromatografía Liquida , Leucina/químicaRESUMEN
Quantitative proteomics studies require an absolute quantification step to accurately measure changes in protein concentration. Absolute quantification using liquid chromatography-mass spectrometry (LC-MS) traditionally combines triple quadrupole instrumentation with stable isotope-labeled standards to measure protein concentrations via their enzymatically produced peptides. Chemical modification of peptides using labels like mass differential tags for relative and absolute quantification (mTRAQ) provides another route to determine protein quantities. This chapter describes a cost-effective and high-throughput chemical labeling method that utilizes five amine-reactive, isotopic N,N-dimethyl leucine (iDiLeu) reagents. These tags enable generation of four-point calibration curves in one LC-MS run to determine protein concentrations from labeled peptides. In particular, we provide a detailed workflow for protein quantification using the iDiLeu reagent that includes important considerations like labeling conditions and isotopic interference correction.