Fast quantitative analysis of timsTOF PASEF data with MSFragger and IonQuant

Yu, Fengchao; Haynes, Sarah E.; Teo, Guo Ci; Avtonomov, Dmitry M.; Polasky, Daniel A.; Nesvizhskii, Alexey I.

doi:10.1101/2020.03.19.999334

Cited by 9 publications

(3 citation statements)

References 38 publications

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“…Data was searched using MSFragger with IonQuant (v3.2; Fragpipe v15.0) against the mouse extracellular matrix database (downloaded February 26, 2021), a subset database of 2976 entries with keywords used (collagen, elastin, aggrecan, gelatin, osteonectin, perlecan, plasminogen, and fibronectin). Parameters included unspecified proteolytic enzyme, precursor mass tolerance of ±20 ppm, and fragment mass tolerance ±0.8 Da.…”

Section: Methodsmentioning

confidence: 99%

Evaluation of Therapeutic Collagen-Based Biomaterials in the Infarcted Mouse Heart by Extracellular Matrix Targeted MALDI Imaging Mass Spectrometry

Clift

McLaughlin

Muñoz

et al. 2021

J. Am. Soc. Mass Spectrom.

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The goal of this study was to develop strategies to localize human collagen-based hydrogels within an infarcted mouse heart, as well as analyze its impact on endogenous extracellular matrix (ECM) remodeling. Collagen is a natural polymer that is abundantly used in bioengineered hydrogels because of its biocompatibility, cell permeability, and biodegradability. However, without the use of tagging techniques, collagen peptides derived from hydrogels can be difficult to differentiate from the endogenous ECM within tissues. Imaging mass spectrometry is a robust tool capable of visualizing synthetic and natural polymeric molecular structures yet is largely underutilized in the field of biomaterials outside of surface characterization. In this study, our group leveraged a recently developed matrixassisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) technique to enzymatically target collagen and other ECM peptides within the tissue microenvironment that are both endogenous and hydrogel-derived. Using a multimodal approach of fluorescence microscopy and ECM-IMS techniques, we were able to visualize and relatively quantify significantly abundant collagen peptides in an infarcted mouse heart that were localized to regions of therapeutic hydrogel injection sites. On-tissue MALDI MS/MS was used to putatively identify sites of collagen peptide hydroxyproline site occupancy, a posttranslational modification that is critical in collagen triple helical stability. Additionally, the technique could putatively identify over 35 endogenously expressed ECM peptides that were expressed in hydrogel-injected mouse hearts. Our findings show evidence for the use of MALDI-IMS in assessing the therapeutic application of collagen-based biomaterials.

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Section: Methodsmentioning

confidence: 99%

Evaluation of Therapeutic Collagen-Based Biomaterials in the Infarcted Mouse Heart by Extracellular Matrix Targeted MALDI Imaging Mass Spectrometry

Clift

McLaughlin

Muñoz

et al. 2021

J. Am. Soc. Mass Spectrom.

View full text Add to dashboard Cite

show abstract

“…Mass spectrometry raw files were processed with MsFragger [32]. For all searches, a protein sequence database of reviewed canine proteins (accessed 11/27/2019 from UniProt; 1886 entries including decoys and 115 common contaminant sequences) was used.…”

Section: Ms Data Analysismentioning

confidence: 99%

“…Decoy database hits, proteins identified as potential contaminants, and proteins identified exclusively by one site modification were excluded from further analysis. Label-free protein quantification was performed with the IonQuant algorithm [32].…”

Section: Ms Data Analysismentioning

confidence: 99%

Mapping transmembrane binding partners for E-cadherin ectodomains

Shafraz

Xie

Yamada

et al. 2020

Preprint

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We combine proximity labeling and single molecule binding assays, to discover novel transmembrane protein interactions in cells. We first screen for proximal membrane proteins by tagging the extracellular and cytoplasmic regions of the protein of interest with TurboID biotin ligase, and then test direct binding interactions using single molecule Atomic Force Microscopy.Using this approach, we identify previously unknown direct interactions between membrane proteins and the extracellular regions of cadherin adhesion proteins in epithelial cells.

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An ultra-potent synthetic nanobody neutralizes SARS-CoV-2 by locking Spike into an inactive conformation

Schoof

Faust

Saunders

et al. 2020

Preprint

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Without an effective prophylactic solution, infections from SARS-CoV-2 continue to rise worldwide with devastating health and economic costs. SARS-CoV-2 gains entry into host cells via an interaction between its Spike protein and the host cell receptor angiotensin converting enzyme 2 (ACE2). Disruption of this interaction confers potent neutralization of viral entry, providing an avenue for vaccine design and for therapeutic antibodies. Here, we develop single-domain antibodies (nanobodies) that potently disrupt the interaction between the SARS-CoV-2 Spike and ACE2. By screening a yeast surface-displayed library of synthetic nanobody sequences, we identified a panel of nanobodies that bind to multiple epitopes on Spike and block ACE2 interaction via two distinct mechanisms. Cryogenic electron microscopy (cryo-EM) revealed that one exceptionally stable nanobody, Nb6, binds Spike in a fully inactive conformation with its receptor binding domains (RBDs) locked into their inaccessible down-state, incapable of binding ACE2. Affinity maturation and structure-guided design of multivalency yielded a trivalent nanobody, mNb6-tri, with femtomolar affinity for SARS-CoV-2 Spike and picomolar neutralization of SARS-CoV-2 infection. mNb6-tri retains stability and function after aerosolization, lyophilization, and heat treatment. These properties may enable aerosol-mediated delivery of this potent neutralizer directly to the airway epithelia, promising to yield a widely deployable, patient-friendly prophylactic and/or early infection therapeutic agent to stem the worst pandemic in a century.

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Fast quantitative analysis of timsTOF PASEF data with MSFragger and IonQuant

Cited by 9 publications

References 38 publications

Evaluation of Therapeutic Collagen-Based Biomaterials in the Infarcted Mouse Heart by Extracellular Matrix Targeted MALDI Imaging Mass Spectrometry

Evaluation of Therapeutic Collagen-Based Biomaterials in the Infarcted Mouse Heart by Extracellular Matrix Targeted MALDI Imaging Mass Spectrometry

Mapping transmembrane binding partners for E-cadherin ectodomains

An ultra-potent synthetic nanobody neutralizes SARS-CoV-2 by locking Spike into an inactive conformation

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