RESUMO
Gut microbiota can regulate host brain functions and influence various physiological and pathological processes through the brain-gut axis. To systematically elucidate the intervention of different gut environments on different brain regions, we implemented an integrated approach that combines 11-plex DiLeu isobaric tags with a "BRIDGE" normalization strategy to comparatively analyze the proteome of six brain regions in germ-free (GF)- and conventionally raised (ConvR)-mice. A total of 5945 proteins were identified and 5656 were quantifiable, while 1906 of them were significantly changed between GF- and ConvR-mice; 281 proteins were filtered with FC greater than 1.2 in at least one brain region, of which heatmap analysis showed clear protein profile disparities, both between brain regions and gut microbiome conditions. Gut microbiome impact is most overt in the hypothalamus and the least in the thalamus region. Collectively, this approach allows an in-depth investigation of the induced protein changes by multiple gut microbiome environments in a brain region-specific manner. This comprehensive proteomic work improves the understanding of the brain region protein association networks impacted by the gut microbiome and highlights the critical roles of the brain-gut axis.
Assuntos
Microbioma Gastrointestinal , Camundongos , Animais , Proteômica , Encéfalo , ProteomaRESUMO
Top-down proteomics, the tandem mass spectrometric analysis of intact proteoforms, is the dominant method for proteoform characterization in complex mixtures. While this strategy produces detailed molecular information, it also requires extensive instrument time per mass spectrum obtained and thus compromises the depth of proteoform coverage that is accessible on liquid chromatography time scales. Such a top-down analysis is necessary for making original proteoform identifications, but once a proteoform has been confidently identified, the extensive characterization it provides may no longer be required for a subsequent identification of the same proteoform. We present a strategy to identify proteoforms in tissue samples on the basis of the combination of an intact mass determination with a measured count of the number of cysteine residues present in each proteoform. We developed and characterized a cysteine tagging chemistry suitable for the efficient and specific labeling of cysteine residues within intact proteoforms and for providing a count of the cysteine amino acids present. On simple protein mixtures, the tagging chemistry yields greater than 98% labeling of all cysteine residues, with a labeling specificity of greater than 95%. Similar results are observed on more complex samples. In a proof-of-principle study, proteoforms present in a human prostate tumor biopsy were characterized. Observed proteoforms, each characterized by an intact mass and a cysteine count, were grouped into proteoform families (groups of proteoforms originating from the same gene). We observed 2190 unique experimental proteoforms, 703 of which were grouped into 275 proteoform families.
Assuntos
Cisteína , Espectrometria de Massas em Tandem , Humanos , Cisteína/metabolismo , Espectrometria de Massas em Tandem/métodos , Proteínas/metabolismo , Cromatografia Líquida , Proteômica/métodos , Proteoma/análise , Processamento de Proteína Pós-TraducionalRESUMO
Gut microbiota can regulate host physiological and pathological status through gut-brain communications or pathways. However, the impact of the gut microbiome on neuropeptides and proteins involved in regulating brain functions and behaviors is still not clearly understood. To address the problem, integrated label-free and 10-plex DiLeu isobaric tag-based quantitative methods were implemented to compare the profiling of neuropeptides and proteins in the hypothalamus of germ-free (GF)- vs conventionally raised (ConvR)-mice. A total of 2943 endogenous peptides from 63 neuropeptide precursors and 3971 proteins in the mouse hypothalamus were identified. Among these 368 significantly changed peptides (fold changes over 1.5 and a p-value of <0.05), 73.6% of the peptides showed higher levels in GF-mice than in ConvR-mice, and 26.4% of the peptides had higher levels in ConvR-mice than in GF-mice. These peptides were mainly from secretogranin-2, phosphatidylethanolamine-binding protein-1, ProSAAS, and proenkephalin-A. A quantitative proteomic analysis employing DiLeu isobaric tags revealed that 282 proteins were significantly up- or down-regulated (fold changes over 1.2 and a p-value of <0.05) among the 3277 quantified proteins. These neuropeptides and proteins were mainly involved in regulating behaviors, transmitter release, signaling pathways, and synapses. Interestingly, pathways including long-term potentiation, long-term depression, and circadian entrainment were involved. In the present study, a combined label-free and 10-plex DiLeu-based quantitative method enabled a comprehensive profiling of gut microbiome-induced dynamic changes of neuropeptides and proteins in the hypothalamus, suggesting that the gut microbiome might mediate a range of behavioral changes, brain development, and learning and memory through these neuropeptides and proteins.
Assuntos
Microbioma Gastrointestinal/fisiologia , Hipotálamo/metabolismo , Leucina/análogos & derivados , Leucina/química , Neuropeptídeos/metabolismo , Proteoma/metabolismo , Aminas/química , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Proteômica , Espectrometria de Massas em TandemRESUMO
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.
Assuntos
Desdiferenciação Celular , Músculo Liso Vascular , Miócitos de Músculo Liso , Proteômica , Fator de Crescimento Transformador betaRESUMO
The unbiased selection of peptide precursors makes data-independent acquisition (DIA) an advantageous alternative to data-dependent acquisition (DDA) for discovery proteomics, but traditional multiplexed quantification approaches employing mass difference labeling or isobaric tagging are incompatible with DIA. Here, we describe a strategy that permits multiplexed quantification by DIA using mass defect-based N,N-dimethyl leucine (mdDiLeu) tags and high-resolution tandem mass spectrometry (MS2) analysis. Millidalton mass differences between mdDiLeu isotopologues produce fragment ion multiplet peaks separated in mass by as little as 5.8 mDa, enabling up to 4-plex quantification in DIA MS2 spectra. Quantitative analysis of yeast samples displayed comparable accuracy and precision for MS2-based DIA and MS1-based DDA methods. Multiplexed DIA analysis of cerebrospinal fluid revealed the dynamic proteome changes in Alzheimer's disease, demonstrating its utility for discovery of potential clinical biomarkers. We show that the mdDiLeu tagging approach for multiplexed DIA is a viable methodology for investigating proteome changes, particularly for low-abundance proteins, in different biological matrices.
Assuntos
Leucina/análogos & derivados , Proteoma/análise , Proteômica/métodos , Proteínas de Saccharomyces cerevisiae/análise , Doença de Alzheimer/líquido cefalorraquidiano , Sequência de Aminoácidos , Biomarcadores/líquido cefalorraquidiano , Biomarcadores/química , Proteínas do Líquido Cefalorraquidiano/líquido cefalorraquidiano , Proteínas do Líquido Cefalorraquidiano/química , Humanos , Pessoa de Meia-Idade , Estudo de Prova de Conceito , Proteoma/química , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/química , Espectrometria de Massas em TandemRESUMO
Isobaric tags enable multiplexed quantitative analysis of many biological samples in a single LC-MS/MS experiment. As a cost-effective alternative to expensive commercial isobaric tagging reagents, we developed our own custom N,N-dimethylleucine "DiLeu" isobaric tags for quantitative proteomics. Here, we present a new generation of DiLeu tags that achieves 21-plex quantification in high-resolution HCD MS/MS spectra via distinct reporter ions that differ in mass from each other by a minimum of 3 mDa. The 21-plex set retains the compact tag structure and existing isotopologues of the 12-plex set but includes nine new reporter variants formulated with unique configurations of 13C, 15N, and 2H stable isotopes, each synthesized in-house via a stepwise N-monomethylation synthesis strategy using readily available reagents. Thus, multiplexing capacity is expanded significantly, while preserving the performance and low cost of the previous implementation. We show that 21-plex DiLeu tags generate strong reporter ions following HCD fragmentation of labeled peptides acquired on Orbitrap platforms at a minimum of 60,000 resolving power (at 400 m/z), and we demonstrate accurate 21-plex quantification of labeled K562 human cell line protein digests via single-shot nanoLC-MS/MS analysis on a Q Exactive HF system.
Assuntos
Leucina/química , Proteínas de Neoplasias/análise , Proteômica , Cromatografia Líquida , Ensaios de Triagem em Larga Escala , Humanos , Células K562 , Leucina/análogos & derivados , Leucina/síntese química , Estrutura Molecular , Espectrometria de Massas em TandemRESUMO
Yeast display immunoprecipitation is a combinatorial library screening platform for the discovery and engineering of antibodies against membrane proteins using detergent-solubilized membrane fractions or cell lysates as antigen sources. Here, we present the extension of this method for the screening of antibodies that bind to membrane protein complexes, enabling discovery of antibodies that target antigens involved in a functional protein-protein interaction of interest. For this proof-of-concept study, we focused on the receptor-mediated endocytosis machinery at the blood-brain barrier, and adaptin 2 (AP-2) was chosen as the functional interaction hub. The goal of this study was to identify antibodies that bound to blood-brain barrier (BBB) membrane protein complexes containing AP-2. Screening of a nonimmune yeast display antibody library was carried out using detergent-solubilized BBB plasma membranes as an antigen pool, and antibodies that could interact with protein complexes containing AP-2 were identified. Downstream characterization of isolated antibodies confirmed targeting of proteins known to play important roles in membrane trafficking. This functional yeast display immunoprecipitation screen may be applied to other systems where antibodies against other functional classes of protein complexes are sought.
Assuntos
Antígenos/imunologia , Barreira Hematoencefálica/imunologia , Proteínas de Membrana/imunologia , Complexos Multiproteicos/imunologia , Saccharomyces cerevisiae , Anticorpos de Cadeia Única , Animais , Células HEK293 , Humanos , Masculino , Ratos , Ratos Sprague-Dawley , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/imunologia , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologiaRESUMO
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.
Assuntos
Metabolômica/métodos , Software , Internet , Marcação por Isótopo , Espectrometria de Massas em Tandem , Interface Usuário-ComputadorRESUMO
A mass defect-based labeling strategy provides high accuracy as an MS1-centric quantification method, avoiding the ratio compression that affects isobaric label-based reporter ion quantification. We have developed cost-effective 5-plex mass defect N, N-dimethyl leucine (mdDiLeu) tags for quantification of various biological samples with increased multiplexing at a given resolving power afforded by the addition of mass difference isotopologues. The combination of mass difference and mass defect produces two labeled peak clusters separated by 5 Da in MS1 spectra that are detected as five isotopic peaks at high resolution with mass differences of 15, 17, and 18 mDa per tag. Synthesis of each of the 5-plex mdDiLeu tags is accomplished by a single straightforward reaction step, making it accessible to any lab. To demonstrate 5-plex mdDiLeu for quantitative proteomics, we perform proof-of-principle experiments of mdDiLeu-labeled Saccharomyces cerevisiae lysate digest on an Orbitrap Fusion Lumos mass spectrometer.
Assuntos
Leucina/análogos & derivados , Proteínas de Saccharomyces cerevisiae/análise , Saccharomyces cerevisiae/química , Espectrometria de Massas em Tandem/métodos , Metilação , Proteômica/métodosRESUMO
Absolute quantification in targeted proteomics is challenging due to a variety of factors, including low specificity in complex backgrounds, limited analytical throughput, and wide dynamic range. To address these problems, we developed a hybrid offset-triggered multiplex absolute quantification (HOTMAQ) strategy that combines cost-effective mass difference and isobaric tags to enable simultaneous construction of an internal standard curve in the MS1 precursor scan, real-time identification of peptides at the MS2 level, and mass offset-triggered accurate quantification of target proteins in synchronous precursor selection (SPS)-MS3 spectra. This approach increases the analytical throughput of targeted quantitative proteomics by up to 12-fold. The HOTMAQ strategy was employed to verify candidate protein biomarkers in preclinical Alzheimer's disease with high accuracy. The greatly enhanced throughput and quantitative performance, paired with sample flexibility, makes HOTMAQ broadly applicable to targeted peptidomics, proteomics, and phosphoproteomics.
Assuntos
Doença de Alzheimer/diagnóstico , Proteínas/análise , Proteômica , Doença de Alzheimer/líquido cefalorraquidiano , Biomarcadores/análise , Biomarcadores/líquido cefalorraquidiano , Cromatografia Líquida , Humanos , Espectrometria de MassasRESUMO
Glycans are highly complex entities with multiple building units and different degrees of branched polymerization. Intensive research efforts have been directed to mass spectrometry (MS)-based qualitative and quantitative glycomic analysis due to the important functions of glycans. Among various strategies, isobaric labeling has become popular because of its higher multiplexing capacity. Over the past few years, several isobaric chemical tags have been developed for quantitative glycomics. However, caveats also exist for these tags, such as relatively low reporter ion yield for aminoxyTMT-labeled complex glycans. To overcome the limitations of existing isobaric chemical tags, we designed a class of novel isobaric multiplex reagents for carbonyl-containing compound (SUGAR) tags that can be used to label glycans for quantitative glycomic analysis. The quantitative performance including labeling efficiency, quantification accuracy, and dynamic range of these SUGAR tags has been evaluated, showing promising results. Finally, the 4-plex SUGAR tags have been utilized to investigate N-glycan changes of B-cell acute lymphoblastic leukemia (ALL) pediatric patients before and after chemotherapy.
Assuntos
Acetonitrilas/química , Proteínas Sanguíneas/química , Glicômica , Indicadores e Reagentes/química , Polissacarídeos/análise , Leucemia-Linfoma Linfoblástico de Células Precursoras B/sangue , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras B/diagnósticoRESUMO
Multiplex isobaric tags have become valuable tools for high-throughput quantitative analysis of complex biological samples in discovery-based proteomics studies. Hybrid labeling strategies that pair stable isotope mass difference labeling with multiplex isobaric tag-based quantification further facilitate these studies by greatly increasing multiplexing capability. In this work, we present a cost-effective chemical labeling approach that couples duplex stable isotope dimethyl labeling with our custom 12-plex N,N-dimethyl leucine (DiLeu) isobaric tags in a combined precursor isotopic labeling and isobaric tagging (cPILOT) strategy that is compatible with a wide variety of biological samples and permits 24-plex quantification in a single LC-MS/MS experiment. We demonstrate the utility of the DiLeu cPILOT approach by labeling yeast digests and performing proof-of-principle quantification experiments on the Orbitrap Fusion Lumos.
Assuntos
Marcação por Isótopo , Leucina/análogos & derivados , Sequência de Aminoácidos , Cromatografia Líquida/métodos , Análise de Fourier , Estudo de Prova de Conceito , Proteômica , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/química , Espectrometria de Massas em Tandem/métodosRESUMO
We recently developed a novel amine-reactive mass-defect-based chemical tag, dimethyl pyrimidinyl ornithine (DiPyrO), for quantitative proteomic analysis at the MS1 level. In this work, we further extend the application of the DiPyrO tag, which provides amine group reactivity, optical detection capability, and improved electrospray sensitivity, to quantify N-linked glycans enzymatically released from glycoproteins in the glycosylamine form. Duplex DiPyrO tags that differ in mass by 45.3 mDa were used to label the glycosylamine moieties of freshly released N-glycosylamines from glycoprotein standards and human serum proteins. We demonstrate that both MALDI-LTQ-Orbitrap and nano-HILIC LC/MS/MS Fusion Lumos Orbitrap platforms are capable of resolving the singly or multiply charged N-glycans labeled with mass-defect DiPyrO tags. Dynamic range of quantification, based on MS1 peak intensities, was evaluated across 2 orders of magnitude. With optimized N-glycan release conditions, glycosylamine labeling conditions, and MS acquisition parameters, the N-glycan profiles and abundances in human serum proteins of cancer patients before and after chemotherapy were compared. Moreover, this study also opens a door for using well-developed amine-reactive tags for relative quantification of glycans, which could be widely applied.
Assuntos
Glicômica/métodos , Espectrometria de Massas/métodos , Ornitina/química , Polissacarídeos/química , Polissacarídeos/metabolismo , Antineoplásicos/uso terapêutico , Criança , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismoRESUMO
We have developed a novel amine-reactive mass defect-based chemical tag, dimethyl pyrimidinyl ornithine (DiPyrO), that is compact in size, is suitable for various biological samples, and enables highly multiplexed quantification of peptides at the MS1 level without increasing mass spectral complexity. The DiPyrO tag structure incorporates heavy isotopes in a variety of configurations to impart as much as 45.3 mDa or as little as 5.8 mDa per tag between labeled peptides. Notably, peptides containing lysine are labeled with two tags, doubling the imparted mass defect to up to 90.6 mDa for the duplex tags and effectively reducing the resolving power requirement compared to previously reported mass defect-based quantification approaches. This permits current and previous generation LTQ-Orbitrap platforms to perform confident quantitative analyses of two DiPyrO-labeled samples at 100K resolving power, whereas 3-plex and 6-plex quantifications are possible at 240K and 480K resolving powers, respectively. In this work, we discuss the design and synthesis of the DiPyrO tag, characterize its effect on labeled proteome analysis by nanoLC-MS2, and demonstrate proof-of-principle applications of the duplex and triplex tags for quantitative proteomics using high-resolution MS acquisition on the Orbitrap Elite and Orbitrap Fusion Lumos.
Assuntos
Ornitina/química , Peptídeos/análise , Proteômica/métodos , Aminas/química , Cromatografia Líquida de Alta Pressão , Nanotecnologia , Peptídeos/química , Proteoma/análise , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/análise , Proteínas de Saccharomyces cerevisiae/metabolismo , Espectrometria de Massas em TandemRESUMO
Mass spectrometry-based stable isotope labeling has become a key technology for protein and small-molecule analyses. We developed a multiplexed quantification method for simultaneous proteomics and amine metabolomics analyses via nano reversed-phase liquid chromatography-tandem mass spectrometry (nanoRPLC-MS/MS), called mass defect-based N,N-dimethyl leucine (mdDiLeu) labeling. The duplex mdDiLeu reagents were custom-synthesized with a mass difference of 20.5 mDa, arising from the subtle variation in nuclear binding energy between the two DiLeu isotopologues. Optimal MS resolving powers were determined to be 240K for labeled peptides and 120K for labeled metabolites on the Orbitrap Fusion Lumos instrument. The mdDiLeu labeling does not suffer from precursor interference and dynamic range compression, providing excellent accuracy for MS1-centric quantification. Quantitative information is only revealed at high MS resolution without increasing spectrum complexity and overlapping isotope distribution. Chromatographic performance of polar metabolites was dramatically improved by mdDiLeu labeling with modified hydrophobicity, enhanced ionization efficiency, and picomole levels of detection limits. Paralleled proteomics and amine metabolomics analyses using mdDiLeu were systematically evaluated and then applied to pancreatic cancer cells.
Assuntos
Aminas/metabolismo , Leucina/análogos & derivados , Metabolômica/métodos , Neoplasias Pancreáticas/metabolismo , Proteínas/metabolismo , Proteômica/métodos , Aminas/análise , Linhagem Celular Tumoral , Cromatografia de Fase Reversa/métodos , Humanos , Leucina/análise , Leucina/metabolismo , Metilação , Proteínas/análise , Espectrometria de Massas em Tandem/métodosRESUMO
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.
Assuntos
Proteômica/métodos , Cromatografia Líquida , Leucina/químicaRESUMO
RATIONALE: Relative quantification of proteins via their enzymatically digested peptide products determines disease biomarker candidate lists in discovery studies. Isobaric label-based strategies using TMT and iTRAQ allow for up to 10 samples to be multiplexed in one experiment, but their expense limits their use. The demand for cost-effective tagging reagents capable of multiplexing many samples led us to develop an 8-plex version of our isobaric labeling reagent, DiLeu. METHODS: The original 4-plex DiLeu reagent was extended to an 8-plex set by coupling isotopic variants of dimethylated leucine to an alanine balance group designed to offset the increasing mass of the label's reporter group. Tryptic peptides from a single protein digest, a protein mixture digest, and Saccharomyces cerevisiae lysate digest were labeled with 8-plex DiLeu and analyzed via nanospray liquid chromatography/tandem mass spectrometry (nanoLC/MS(2) ) on a Q-Exactive Orbitrap mass spectrometer. Characteristics of 8-plex DiLeu-labeled peptides, including quantitative accuracy and fragmentation, were examined. RESULTS: An 8-plex set of DiLeu reagents with 1 Da spaced reporters was synthesized at a yield of 36%. The average cost to label eight 100 µg peptide samples was calculated to be approximately $15. Normalized collision energy tests on the Q-Exactive revealed that a higher-energy collisional dissociation value of 27 generated the optimum number of high-quality spectral matches. Relative quantification of DiLeu-labeled peptides yielded normalized median ratios accurate to within 12% of their expected values. CONCLUSIONS: Cost-effective 8-plex DiLeu reagents can be synthesized and applied to relative peptide and protein quantification. These labels increase the multiplexing capacity of our previous 4-plex implementation without requiring high-resolution instrumentation to resolve reporter ion signals.
Assuntos
Leucina/análogos & derivados , Peptídeos/química , Proteômica/métodos , Cromatografia Líquida/métodos , Deutério/química , Leucina/química , Peptídeos/análise , Peptídeos/síntese química , Saccharomyces cerevisiae/química , Espectrometria de Massas em Tandem/instrumentação , Espectrometria de Massas em Tandem/métodosRESUMO
In the foreign body response, infiltrating PMNs exocytose granule subsets to influence subsequent downstream inflammatory and wound healing events. In previous studies, we found that PMNs cultured on poly(ethylene glycol) (PEG)-containing hydrogels (i.e., PEG and gelatin + PEG hydrogels) had enhanced primary granule release, yet similar tertiary granule release compared with PMNs cultured on polydimethylsiloxane or tissue culture polystyrene. PMN primary granules contain microbicidal proteins and proteases, which can potentially injure bystander cells, degrade the extracellular matrix, and promote inflammation. Here, we sought to understand the mechanism of the enhanced primary granule release from PMNs on PEG hydrogels. We found that primary granule release from PMNs on PEG hydrogels was adhesion mediated and involved Src family kinases and PI3K-γ. The addition of gelatin to PEG hydrogels did not further enhance PMN primary granule release. Using stable-isotope dimethyl labeling-based shotgun proteomics, we identified many serum proteins - including Ig gamma constant chain region proteins and alpha-1-acid glycoprotein 1 - that were absorbed/adsorbed in higher quantities on PEG hydrogels than on TCPS, and may be involved in mediating PMN primary granule release. Ultimately, this mechanistic knowledge can be used to direct inflammation and wound healing following biomaterial implantation to promote a more favorable healing response.
Assuntos
Materiais Biocompatíveis/farmacologia , Grânulos Citoplasmáticos/metabolismo , Neutrófilos/enzimologia , Fosfatidilinositol 3-Quinases/metabolismo , Quinases da Família src/metabolismo , Adsorção , Adulto , Animais , Proteínas Sanguíneas/metabolismo , Bovinos , Degranulação Celular/efeitos dos fármacos , Células Cultivadas , Grânulos Citoplasmáticos/efeitos dos fármacos , Humanos , Hidrogéis/farmacologia , Metaloproteinase 9 da Matriz/metabolismo , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/efeitos dos fármacos , Peroxidase/metabolismo , Polietilenoglicóis/farmacologia , Poliestirenos/farmacologia , Receptores de Formil Peptídeo/metabolismoRESUMO
Multiplex isobaric tags (e.g., tandem mass tags (TMT) and isobaric tags for relative and absolute quantification (iTRAQ)) are a valuable tool for high-throughput mass spectrometry based quantitative proteomics. We have developed our own multiplex isobaric tags, DiLeu, that feature quantitative performance on par with commercial offerings but can be readily synthesized in-house as a cost-effective alternative. In this work, we achieve a 3-fold increase in the multiplexing capacity of the DiLeu reagent without increasing structural complexity by exploiting mass defects that arise from selective incorporation of (13)C, (15)N, and (2)H stable isotopes in the reporter group. The inclusion of eight new reporter isotopologues that differ in mass from the existing four reporters by intervals of 6 mDa yields a 12-plex isobaric set that preserves the synthetic simplicity and quantitative performance of the original implementation. We show that the new reporter variants can be baseline-resolved in high-resolution higher-energy C-trap dissociation (HCD) spectra, and we demonstrate accurate 12-plex quantitation of a DiLeu-labeled Saccharomyces cerevisiae lysate digest via high-resolution nano liquid chromatography-tandem mass spectrometry (nanoLC-MS(2)) analysis on an Orbitrap Elite mass spectrometer.
Assuntos
Cromatografia Líquida/métodos , Leucina/química , Proteômica/métodos , Proteínas de Saccharomyces cerevisiae/metabolismo , Espectrometria de Massas em Tandem/métodos , Marcação por Isótopo , Saccharomyces cerevisiae/metabolismoRESUMO
Protein glycosylation plays fundamental roles in many biological processes as one of the most common, and the most complex, posttranslational modification. Alterations in glycosylation profile are now known to be associated with many diseases. As a result, the discovery and detailed characterization of glycoprotein disease biomarkers is a primary interest of biomedical research. Advances in mass spectrometry (MS)-based glycoproteomics and glycomics are increasingly enabling qualitative and quantitative approaches for site-specific structural analysis of protein glycosylation. While the complexity presented by glycan heterogeneity and the wide dynamic range of clinically relevant samples like plasma, serum, cerebrospinal fluid, and tissue make comprehensive analyses of the glycoproteome a challenging task, the ongoing efforts into the development of glycoprotein enrichment, enzymatic digestion, and separation strategies combined with novel quantitative MS methodologies have greatly improved analytical sensitivity, specificity, and throughput. This review summarizes current MS-based glycoproteomics approaches and highlights recent advances in its application to cancer biomarker and neurodegenerative disease research.