Relative and Absolute Quantitation in Mass Spectrometry–Based Proteomics

Ankney, John A.; Muneer, Adil; Chen, Xian

doi:10.1146/annurev-anchem-061516-045357

Cited by 157 publications

(111 citation statements)

References 160 publications

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“…To improve protein identification rates, alternative approaches to singleshot mass spectrometry can be performed, including fractionation (e.g., size exclusion chromatography, high reversed-phase pH) to reduce sample complexity in a single run. We also describe the use of label-free quantification for measurement of relative protein abundance in the samples; however, other labeling methods (e.g., metabolic and chemical) can also be used to promote multiplexing of sample measurements on the mass spectrometer (Ankney, Muneer, & Chen, 2018;Cox et al, 2014). Table 1 presents common problems encountered with preparing proteomic samples, along with possible causes and recommended approaches to avoid or overcome these problems.…”

Section: Critical Parametersmentioning

confidence: 99%

Quantitative Proteomic Profiling of Murine Ocular Tissue and the Extracellular Environment

et al. 2020

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Mass spectrometry‐based proteomics provides a robust and reliable method for detecting and quantifying changes in protein abundance among samples, including cells, tissues, organs, and supernatants. Physical damage or inflammation can compromise the ocular surface permitting colonization by bacterial pathogens, commonly Pseudomonas aeruginosa, and the formation of biofilms. The interplay between P. aeruginosa and the immune system at the site of infection defines the host's ability to defend against bacterial invasion and promote clearance of infection. Profiling of the ocular tissue following infection describes the nature of the host innate immune response and specifically the presence and abundance of neutrophil‐associated proteins to neutralize the bacterial biofilm. Moreover, detection of unique proteins produced by P. aeruginosa enable identification of the bacterial species and may serve as a diagnostic approach in a clinical setting. Given the emergence and prevalence of antimicrobial resistant bacterial strains, the ability to rapidly diagnose a bacterial infection promoting quick and accurate treatment will reduce selective pressure towards resistance. Furthermore, the ability to define differences in the host immune response towards bacterial invasion enhances our understanding of innate immune system regulation at the ocular surface. Here, we describe murine ocular infection and sample collection, as well as outline protocols for protein extraction and mass spectrometry profiling from corneal tissue and extracellular environment (eye wash) samples. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Murine model of ocular infection Basic Protocol 2: Murine model sample collection Basic Protocol 3: Protein extraction from eye wash Basic Protocol 4: Protein extraction from corneal tissue Basic Protocol 5: Mass spectrometry‐based proteomics and bioinformatics from eye wash and corneal tissue samples

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Section: Critical Parametersmentioning

confidence: 99%

Quantitative Proteomic Profiling of Murine Ocular Tissue and the Extracellular Environment

et al. 2020

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“…For the choice of the most suitable quantitative approach, see ref. [] for recent comprehensive comparison.…”

Section: What Is the Best Approach (For You)?mentioning

confidence: 99%

Uncovering the In Vivo Proxisome Using Proximity‐Tagging Methods

Santin

2019

BioEssays

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The development of new approaches is critical to gain further insights into biological processes that cannot be obtained by existing methods or technologies. The detection of protein-protein interaction is often challenging, especially for weak and transient interactions or for membrane proteins. Over the last decade, several proximity-tagging methodologies have been developed to explore protein interactions in living cells. Among those, the most efficient are based on protein partner modification, such as biotinylation or pupylation. Such technologies are based on engineered variants of enzymes like peroxidases or ligases that release reactive molecules, in the presence of specific substrates, that bind surrounding proteins. Fusing a protein of interest (POI) to these enzymes allows the definition of an unbiased "proxisome," that is, all of the proteins in interaction or in close vicinity of the POI. Here, the different proximity-labeling tools available are described and comprehensive comparison to discuss advantages and limitations is provided.

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“…To identify high-confidence chaperone binding partners of human HSC70 and HSP70, we used human osteosarcoma (U2OS) cell lines containing stably-integrated, doxycyclineinducible HSC70-UBAIT (wild-type and ΔGG) and HSP70-UBAIT (wild-type and ΔGG) chaperones and grew these cells for 3 days after chaperone induction. We performed 12 wildtype UBAIT isolations and an equal number of control ΔGG isolations, each starting with 3 mg of total cellular protein, which were all performed under denaturing conditions followed by labelfree quantification of the isolated material as well as the total cell lysates by mass spectrometry (LC-MS/MS)( Figure 1C) [26]. We compared each of the identified proteins in wild-type and control ∆GG isolations to generate an enrichment ratio, and used a bootstrapping method to calculate p-values for likelihood of interaction (see Materials and Methods).…”

Section: Identification Of Ubait Clientsmentioning

confidence: 99%

Comprehensive identification of HSP70/HSC70 Chaperone Clients in Human Cells

Ryu

Stewart

Pectol

et al. 2019

Preprint

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The HSP70 family of chaperones are the front-line of protection from stress-induced misfolding and aggregation of polypeptides in most organisms and are responsible for promoting the stability, folding, and degradation of clients to maintain cellular protein homeostasis. Here we demonstrate quantitative identification of HSP70 and HSC70 clients using an ubiquitin-mediated proximity tagging strategy and show that, despite their high degree of similarity, these enzymes have largely non-overlapping specificities. Both proteins show a preference for association with newly synthesized polypeptides but each responds differently to changes in the stoichiometry of proteins in obligate multi-subunit complexes. In addition, expression of an ALS-associated SOD1 mutant protein induces changes in HSP70 and HSC70 client association and aggregation toward polypeptides with predicted disorder, indicating that there are global effects from a single misfolded protein that extend to many clients within chaperone networks. Together these findings show that the ubiquitin-mediated UBAIT fusion system can efficiently isolate the complex interactome of HSP chaperone family proteins under normal and stress conditions.

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Relative and Absolute Quantitation in Mass Spectrometry–Based Proteomics

Cited by 157 publications

References 160 publications

Quantitative Proteomic Profiling of Murine Ocular Tissue and the Extracellular Environment

Quantitative Proteomic Profiling of Murine Ocular Tissue and the Extracellular Environment

Uncovering the In Vivo Proxisome Using Proximity‐Tagging Methods

Comprehensive identification of HSP70/HSC70 Chaperone Clients in Human Cells

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