February 2024
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20 Reads
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6 Citations
Journal of the American Society for Mass Spectrometry
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February 2024
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20 Reads
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6 Citations
Journal of the American Society for Mass Spectrometry
February 2024
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15 Reads
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2 Citations
Journal of Proteome Research
November 2023
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35 Reads
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2 Citations
Journal of Proteome Research
August 2023
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35 Reads
The extracellular matrix (ECM) is a complex assembly of proteins that provide interstitial scaffolding and elastic recoil to human lungs. The pulmonary extracellular matrix (ECM) is increasingly recognized as an independent bioactive entity by creating biochemical and mechanical signals that influence disease pathogenesis, making it an attractive therapeutic target. However, the pulmonary ECM proteome (matrisome) remains challenging to analyze by mass spectrometry due to its inherent biophysical properties and relatively low abundance. Here, we introduce a strategy designed for rapid and efficient characterization of the human pulmonary ECM using the photocleavable surfactant Azo. We coupled this approach with trapped ion mobility MS with diaPASEF to maximize depth of matrisome coverage. Using this strategy, we identify nearly 400 unique matrisome proteins with excellent reproducibility that are known to be important in lung biology, including key insoluble ECM proteins.
July 2023
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90 Reads
Sarcopenia is a progressive disorder characterized by age-related loss of skeletal muscle mass and function. Although significant progress has been made over the years to identify the molecular determinants of sarcopenia, the precise mechanisms underlying the age-related loss of contractile function remains unclear. Advances in omics technologies, including mass spectrometry-based proteomic and metabolomic analyses, offer great opportunities to better understand sarcopenia. Herein, we performed mass spectrometry-based analyses of the vastus lateralis from young, middle-aged, and older rhesus monkeys to identify molecular signatures of sarcopenia. In our proteomic analysis, we identified numerous proteins that change with age, including those involved in adenosine triphosphate and adenosine monophosphate metabolism as well as fatty acid beta oxidation. In our untargeted metabolomic analysis, we identified multiple metabolites that changed with age largely related to energy metabolism including fatty acid beta oxidation. Pathway analysis of age-responsive proteins and metabolites revealed changes in muscle structure and contraction as well as lipid, carbohydrate, and purine metabolism. Together, this study discovers new metabolic signatures and offer new insights into the molecular mechanism underlying sarcopenia for the evaluation and monitoring of therapeutic treatment of sarcopenia.
May 2022
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27 Reads
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14 Citations
Analytical Chemistry
Exosomes are small extracellular vesicles (EVs) secreted by all cells and found in biological fluids, which can serve as minimally invasive liquid biopsies with extremely high therapeutic and diagnostic potential. Mass spectrometry (MS)-based proteomics is a powerful technique to profile and quantify the protein content in exosomes, but the current methods require laborious and time-consuming multistep sample preparation that significantly limit throughput. Herein, we report a one-pot exosome proteomics method enabled by a photocleavable surfactant, Azo, to simplify exosomal lysis, effectively extract proteins, and expedite digestion. We have applied this method to exosomes derived from isolated mammary fibroblasts and confidently identified 3466 proteins and quantified 2288 proteins using a reversed-phase liquid chromatography coupled to trapped ion mobility spectrometry (TIMS) quadrupole time-of-flight mass spectrometer. Here, 3166 (91%) of the identified proteins are annotated in the exosome/EVs databases, ExoCarta and Vesiclepedia, including important exosomal markers, CD63, PDCD6IP, and SDCBP. This method is fast, simple, and highly effective at extracting exosomal proteins with high reproducibility for deep exosomal proteome coverage. We envision that this method could be generally applicable for exosome proteomics applications in biomedical research, therapeutic interventions, and clinical diagnostics.
March 2022
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20 Reads
Exosomes are small extracellular vesicles (EVs) secreted by all cells and found in biological fluids, which can serve as minimally invasive liquid biopsies with high therapeutic and diagnostic potential. Mass spectrometry (MS)-based proteomics is a powerful technique to profile and quantify the protein content of exosomes but the current methods require laborious and time-consuming multi-step sample preparation that significantly limit throughput. Herein, we report a one-pot exosome proteomics method enabled by a photocleavable surfactant, Azo, for rapid and effective exosomal lysis, protein extraction, and digestion. We have applied this method to exosomes derived from isolated mammary fibroblasts and confidently identified 3,466 proteins and quantified 2,288 proteins using reversed-phase liquid chromatography coupled to trapped ion mobility spectrometry (TIMS) quadrupole time-of-flight mass spectrometer. 3,166 (91%) of the identified proteins are annotated in the exosome/EVs databases, ExoCarta and Vesiclepedia, including important exosomal markers, CD63, PDCD6IP, and SDCBP. This method is fast, simple, and highly effective at extracting exosomal proteins with high reproducibility for deep exosomal proteome coverage. We envision this method could be generally applicable for exosome proteomics applications in biomedical research, therapeutic interventions, and clinical diagnostics.
July 2021
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169 Reads
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40 Citations
Analytical Chemistry
July 2021
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182 Reads
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44 Citations
Journal of Proteome Research
Global bottom-up mass spectrometry (MS)-based proteomics is widely used for protein identification and quantification to achieve a comprehensive understanding of the composition, structure, and function of the proteome. However, traditional sample preparation methods are time-consuming, typically including overnight tryptic digestion, extensive sample cleanup to remove MS-incompatible surfactants, and offline sample fractionation to reduce proteome complexity prior to online liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Thus, there is a need for a fast, robust, and reproducible method for protein identification and quantification from complex proteomes. Herein, we developed an ultrafast bottom-up proteomics method enabled by Azo, a photocleavable, MS-compatible surfactant that effectively solubilizes proteins and promotes rapid tryptic digestion, combined with the Bruker timsTOF Pro, which enables deeper proteome coverage through trapped ion mobility spectrometry (TIMS) and parallel accumulation-serial fragmentation (PASEF) of peptides. We applied this method to analyze the complex human cardiac proteome and identified nearly 4000 protein groups from as little as 1 mg of human heart tissue in a single one-dimensional LC-TIMS-MS/MS run with high reproducibility. Overall, we anticipate this ultrafast, robust, and reproducible bottom-up method empowered by both Azo and the timsTOF Pro will be generally applicable and greatly accelerate the throughput of large-scale quantitative proteomic studies. Raw data are available via the MassIVE repository with identifier MSV000087476.
May 2021
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110 Reads
Global bottom-up mass spectrometry (MS)-based proteomics is widely used for protein identification and quantification to achieve a comprehensive understanding of the composition, structure, and function of the proteome. However, traditional sample preparation methods are time-consuming, typically including overnight tryptic digestion, extensive sample clean-up to remove MS-incompatible surfactants, and offline sample fractionation to reduce proteome complexity prior to online liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Thus, there is a need for a fast, robust, and reproducible method for protein identification and quantification from complex proteomes. Herein, we developed an ultrafast bottom-up proteomics method enabled by Azo, a photocleavable, MS-compatible surfactant that effectively solubilizes proteins and promotes rapid tryptic digestion, combined with the Bruker timsTOF Pro, which enables deeper proteome coverage through trapped ion mobility spectrometry (TIMS) and parallel accumulation-serial fragmentation (PASEF) of peptides. We applied this method to analyze the complex human cardiac proteome and identified nearly 4,000 protein groups from as little as 1 mg of human heart tissue in a single one-dimensional LC-TIMS-MS/MS run with high reproducibility. Overall, we anticipate this ultrafast, robust, and reproducible bottom-up method empowered by both Azo and the timsTOF Pro will be generally applicable and greatly accelerate the throughput of large-scale quantitative proteomic studies. Raw data are available via the MassIVE repository with identifier MSV000087476.
... Proteins were extracted from cardiac left ventricular tissue as previously reported. 18,19 No reducing agents were added to the extraction buffers to preserve protein SSG. Following the extraction procedure, the protein-enriched cardiac tissue lysate was passed through a Titan3, 17mm PES membrane syringe filter (pre-soaked with LiCl extraction buffer) using a 5 mL Luer-lock syringe into an Eppendorf Protein Lo-Bind tube, snap-frozen in liquid nitrogen, and stored at -80 ºC until topdown proteomics analysis. ...
February 2024
Journal of the American Society for Mass Spectrometry
... [21][22][23] Various organisms have been used to establish muscle atrophy models, including rats, mice, Drosophila, Caenorhabditis elegans, zebrafish, African turquoise killifish, medaka, rhesus monkeys, rabbits, Yucatan minipigs, and so forth. [24][25][26][27][28] These models are induced using different methods and are instrumental in studying sarcopenia and cachexia ( Figure 1). In sarcopenia research, the natural aging model is the most commonly used, as it accurately reflects the physiological changes observed in age-related muscle atrophy in humans. ...
November 2023
Journal of Proteome Research
... First studies show promising results, for example, PASEF was first successfully employed to aid in the identification of more than 6000 protein groups in a 200 ng single-run for HeLa standard digest (Meier et al., 2018). Specifically, in case of small extracellular vesicles, one study used the PASEF method to identify 3466 unique protein groups in a triplicate run of cell-derived sEV (Buck et al., 2022). Serum-derived vesicles were characterized using this technique with 915 protein identifications in a study comparing samples from healthy donors and patients suffering from dermatomyositis or polymyositis (Meng et al., 2023). ...
May 2022
Analytical Chemistry
... 8 In conjunction with collision-induced unfolding (CIU), IM/MS has been applied to probe structures and stabilities of multi-domain proteins, including mAbs. [9][10][11][12] In combination with molecular dynamics (MD) simulations, IM/MS suggested that the broad peaks in IM/MS spectra of mAbs may be related to their hinge motions. 13 Nevertheless, conventional IM/MS methods conduct ensemble-type measurements that characterise the entirety of the mAb conformational space and hence characterise their structures or stabilities only by their ensemble averages. ...
July 2021
Analytical Chemistry
... The sensitivity of mass spectrometers has dramatically improved in recent years due to, for example, increased ionization efficiency, improved and enhanced ion transfer, and advances in DDA (data-dependent analysis) due to the introduction of new fragmentation approaches such as PASEF (parallel serial fragmentation) combined with ion mobility separation and fast scanning TOF (time-of-flight) detector [44,56,[66][67][68][69][70][71]. ...
July 2021
Journal of Proteome Research