Universal Solid-Phase Protein Preparation (USP<sup>3</sup>) for Bottom-up and Top-down Proteomics

Dagley, Laura F.; Infusini, Giuseppe; Larsen, Rune H; Sandow, Jarrod J.; Webb, Andrew I.

doi:10.1021/acs.jproteome.9b00217

Cited by 44 publications

(75 citation statements)

References 43 publications

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“…Line Samples-To hydrolyze DNA and reduce viscosity, cell lysates were heated at 95°C for 5 min and TFA was added to a final concentration of 1% (9). Quenching was performed using 3 M Tris, pH 10 (final concentration of ;195 mM, pH 7.8).…”

Section: Sp3 Protein Digestion Tmt Labeling and Fractionation Of Cellmentioning

confidence: 99%

Data, Reagents, Assays and Merits of Proteomics for SARS-CoV-2 Research and Testing

Zecha

Lee

Bayer

et al. 2020

Molecular & Cellular Proteomics

142

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As the SARS-CoV-2 pandemic continues to spread, thousands of scientists around the globe have changed research direction to understand better how the virus works and to find out how it may be tackled. The number of manuscripts on preprint servers is soaring and peer-reviewed publications using mass spectrometry-based proteomics are beginning to emerge. To facilitate proteomic research on SARS-CoV-2, this report presents deep-scale proteomes (10,000 proteins; >130,000 peptides) of common cell line models, notably Vero E6, Calu-3, Caco-2, and ACE2-A549 that characterize their protein expression profiles including viral entry factors such as ACE2 or TMPRSS2. Using the 9 kDa protein SRP9 and the breast cancer oncogene BRCA1 as examples, we show how the proteome expression data can be used to refine the annotation of protein-coding regions of the African green monkey and the Vero cell line genomes. Monitoring changes of the proteome upon viral infection revealed widespread expression changes including transcriptional regulators, protease inhibitors, and proteins involved in innate immunity. Based on a library of 98 stable-isotope labeled synthetic peptides representing 11 SARS-CoV-2 proteins, we developed PRM (parallel reaction monitoring) assays for nano-flow and micro-flow LC-MS/MS. We assessed the merits of these PRM assays using supernatants of virus-infected Vero E6 cells and challenged the assays by analyzing two diagnostic cohorts of 24 (+30) SARS-CoV-2 positive and 28 (+9) negative cases. In light of the results obtained and including recent publications or manuscripts on preprint servers, we critically discuss the merits of mass spectrometry-based proteomics for SARS-CoV-2 research and testing.

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Section: Sp3 Protein Digestion Tmt Labeling and Fractionation Of Cellmentioning

confidence: 99%

Data, Reagents, Assays and Merits of Proteomics for SARS-CoV-2 Research and Testing

Zecha

Lee

Bayer

et al. 2020

Molecular & Cellular Proteomics

142

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“…Samples were stored at −80 • C for future use. Samples were prepared for mass spectrometry analysis using the modified SP3 method as previously described (34). Briefly, exosomes were subjected to reduction/alkylation with 2 M dithiothreitol (DTT, 50 mM final concentration) for 1 h at 37 • C followed by 1 M iodoacetamide (IAM, 100 mM final concentration) for 30 min in the dark at room temperature (RT).…”

Section: Proteomic Analysis Of Exosomesmentioning

confidence: 99%

Airway Exosomes Released During Influenza Virus Infection Serve as a Key Component of the Antiviral Innate Immune Response

et al. 2020

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Exosomes are extracellular vesicles secreted by cells that have an important biological function in intercellular communication by transferring biologically active proteins, lipids, and RNAs to neighboring or distant cells. While a role for exosomes in antimicrobial defense has recently emerged, currently very little is known regarding the nature and functional relevance of exosomes generated in vivo, particularly during an active viral infection. Here, we characterized exosomes released into the airways during influenza virus infection. We show that these vesicles dynamically change in protein composition over the course of infection, increasing expression of host proteins with known anti-influenza activity, and viral proteins with the potential to trigger host immune responses. We show that exosomes released into the airways during influenza virus infection trigger pulmonary inflammation and carry viral antigen that can be utilized by antigen presenting cells to drive the induction of a cellular immune response. Moreover, we show that attachment factors for influenza virus, namely α2,3 and α2,6-linked sialic acids, are present on the surface of airway exosomes and these vesicles have the ability to neutralize influenza virus, thereby preventing the virus from binding and entering target cells. These data reveal a novel role for airway exosomes in the antiviral innate immune defense against influenza virus infection.

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“…Furthermore, when our paper was under review, a recent study described the use of a KingFisher liquid handling system to perform SP3 in conjunction with subsequent enrichment of phosphopeptides, to facilitate automated sample processing for phosphoproteomic studies (Leutert et al , ). On the other side of the spectrum, MS analysis of intact proteins purified by SP3 was recently shown (Dagley et al , ), opening the perspective that the use of autoSP3 might be extended to fit in a workflow for top‐down proteomics.…”

Section: Discussionmentioning

confidence: 99%

Automated sample preparation with SP 3 for low‐input clinical proteomics

Müller

Kalxdorf

Longuespée

et al. 2020

Molecular Systems Biology

181

158

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High-throughput and streamlined workflows are essential in clinical proteomics for standardized processing of samples from a variety of sources, including fresh-frozen tissue, FFPE tissue, or blood. To reach this goal, we have implemented single-pot solid-phaseenhanced sample preparation (SP3) on a liquid handling robot for automated processing (autoSP3) of tissue lysates in a 96-well format. AutoSP3 performs unbiased protein purification and digestion, and delivers peptides that can be directly analyzed by LCMS, thereby significantly reducing hands-on time, reducing variability in protein quantification, and improving longitudinal reproducibility. We demonstrate the distinguishing ability of autoSP3 to process low-input samples, reproducibly quantifying 500-1,000 proteins from 100 to 1,000 cells. Furthermore, we applied this approach to a cohort of clinical FFPE pulmonary adenocarcinoma (ADC) samples and recapitulated their separation into known histological growth patterns. Finally, we integrated autoSP3 with AFA ultrasonication for the automated end-to-end sample preparation and LCMS analysis of 96 intact tissue samples. Collectively, this constitutes a generic, scalable, and cost-effective workflow with minimal manual intervention, enabling reproducible tissue proteomics in a broad range of clinical and non-clinical applications.

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Universal Solid-Phase Protein Preparation (USP³) for Bottom-up and Top-down Proteomics

Cited by 44 publications

References 43 publications

Data, Reagents, Assays and Merits of Proteomics for SARS-CoV-2 Research and Testing

Data, Reagents, Assays and Merits of Proteomics for SARS-CoV-2 Research and Testing

Airway Exosomes Released During Influenza Virus Infection Serve as a Key Component of the Antiviral Innate Immune Response

Automated sample preparation with SP 3 for low‐input clinical proteomics

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Universal Solid-Phase Protein Preparation (USP3) for Bottom-up and Top-down Proteomics

Cited by 44 publications

References 43 publications

Data, Reagents, Assays and Merits of Proteomics for SARS-CoV-2 Research and Testing

Data, Reagents, Assays and Merits of Proteomics for SARS-CoV-2 Research and Testing

Airway Exosomes Released During Influenza Virus Infection Serve as a Key Component of the Antiviral Innate Immune Response

Automated sample preparation with SP 3 for low‐input clinical proteomics

Contact Info

Product

Resources

About

Universal Solid-Phase Protein Preparation (USP³) for Bottom-up and Top-down Proteomics