Hanging drop sample preparation improves sensitivity of spatial proteomics

Kwon, Yumi; Piehowski, Paul; Zhao, Rui; Sontag, Ryan; Moore, Ronald J.; Smith, Richard D.; Qian, Weijun; Zhu, Ying

doi:10.1039/d2lc00384h

Cited by 16 publications

(26 citation statements)

References 56 publications

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“…235 Recently, LCM-nanoPOTS was modified with a hanging drop format to find tissue-region specific or cell-type specific biomarkers of mouse uterine tissues. 236 Using this approach, the results showed proteins enriched in the luminal epithelium were related to ion channels and transporters, while extracellular matrix and collagen-related proteins were enriched in the stromal region. The applications of these approaches demonstrated the potential of proteome mapping with high spatial resolution across tissue regions to advance biomedical research.…”

Section: Applications Of Scp In Biological and Clinical Researchmentioning

confidence: 99%

Recent advances in microfluidics for single-cell functional proteomics

et al. 2023

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Section: Applications Of Scp In Biological and Clinical Researchmentioning

confidence: 99%

Recent advances in microfluidics for single-cell functional proteomics

et al. 2023

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“…The proteomic analysis of small cell populations must avoid sample-processing dilution and loss of precious minute analyte amounts. Kelly and co-workers developed a platform for small cell-population proteomics, which couples nanodroplets (<200 nL) with micro-dissected cells and LC–MS (LCM-nanoPOTS platform). − They successfully quantified >1000 proteins from rat-brain sections corresponding to 10–18 cells . They also reported on hanging-drop sample preparation for the LCM-nanoPOTS platform with protein recovery and sensitivity that allowed high-resolution proteome coverage of mouse uterine sections .…”

Section: Introductionmentioning

confidence: 99%

“…Kelly and co-workers developed a platform for small cell-population proteomics, which couples nanodroplets (<200 nL) with micro-dissected cells and LC–MS (LCM-nanoPOTS platform). − They successfully quantified >1000 proteins from rat-brain sections corresponding to 10–18 cells . They also reported on hanging-drop sample preparation for the LCM-nanoPOTS platform with protein recovery and sensitivity that allowed high-resolution proteome coverage of mouse uterine sections . However, by assessing the entire proteome, low-abundance proteins, as is the case of intracellular Aβ, can be masked by high-abundance ones.…”

Section: Introductionmentioning

confidence: 99%

“…31 They also reported on hanging-drop sample preparation for the LCM-nanoPOTS platform with protein recovery and sensitivity that allowed high-resolution proteome coverage of mouse uterine sections. 30 However, by assessing the entire proteome, lowabundance proteins, as is the case of intracellular Aβ, can be masked by high-abundance ones. A combination of nanoliter droplet arrays with MALDI-MS 35−37 was used for the analysis of proteins secreted by encapsulated cells.…”

Section: ■ Introductionmentioning

confidence: 99%

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Intracellular Amyloid-β Detection from Human Brain Sections Using a Microfluidic Immunoassay in Tandem with MALDI-MS

et al. 2023

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Alzheimer's disease (AD) currently affects more than 30 million people worldwide. The lack of understanding of AD's physiopathology limits the development of therapeutic and diagnostic tools. Soluble amyloid-β peptide (Aβ) oligomers that appear as intermediates along the Aβ aggregation into plaques are considered among the main AD neurotoxic species. Although a wealth of data are available about Aβ from in vitro and animal models, there is little known about intracellular Aβ in human brain cells, mainly due to the lack of technology to assess the intracellular protein content. The elucidation of the Aβ species in specific brain cell subpopulations can provide insight into the role of Aβ in AD and the neurotoxic mechanism involved. Here, we report a microfluidic immunoassay for in situ mass spectrometry analysis of intracellular Aβ species from archived human brain tissue. This approach comprises the selective laser dissection of individual pyramidal cell bodies from tissues, their transfer to the microfluidic platform for sample processing on-chip, and mass spectrometric characterization. As a proof-of-principle, we demonstrate the detection of intracellular Aβ species from as few as 20 human brain cells.

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“…These approaches are particularly attractive as they offer a comprehensive, quantitative protein profile without a priori knowledge of the proteins of interest. In our lab, we have successfully combined laser capture microdissection and the nanoPOTS ap-proach 34,35 , to enable in-depth proteome imaging. This initial effort resulted in a platform capable of quantifying >2000 proteins at 100 μm spatial resolution without the use of antibodies or labels 36 .…”

Section: Introductionmentioning

confidence: 99%

Proteome mapping of the human pancreatic islet microenvironment reveals endocrine-exocrine signaling sphere of influence

Gosline¹,

Veličković²,

Pino³

et al. 2022

Preprint

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The need for a clinically accessible method with the ability to match protein activity within heterogeneous tissues is currently unmet by existing technologies. Our proteomics sample preparation platform, named microPOTS (Microdroplet Processing in One pot for Trace Samples), can be used to measure relative protein abundance in micron-scale samples alongside the spatial location of each measurement, thereby tying biologically interesting proteins and pathways to distinct regions. However, given the smaller sample number and amount of tissue measu red, standard mass spectrometric analysis pipelines have proven inadequate. Here we describe how existing computational approaches can be adapted to focus on the specific biological questions asked in spatial proteomics experiments. We apply this approach to present an unbiased characterization of the human islet microenvironment comprising the entire complex array of tissues involved while maintaining spatial information and the degree of the islet's sphere of influence. We identify specific functional activity unique to the pancreatic islet cells and demonstrate how far their signature can be measured. Our results show that we can distinguish pancreatic islet cells from the neighboring exocrine tissue environment, recapitulate known biological functions of islet cells, and identify a spatial gradient in the expression of RNA processing proteins within the islet microenvironment.

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Hanging drop sample preparation improves sensitivity of spatial proteomics

Abstract: Spatial proteomics holds great promise for revealing tissue heterogeneity in both physiological and pathological conditions. However, one significant limitation of most spatial proteomics workflows is the requirement of large sample...

Cited by 16 publications

References 56 publications

Recent advances in microfluidics for single-cell functional proteomics

Recent advances in microfluidics for single-cell functional proteomics

Intracellular Amyloid-β Detection from Human Brain Sections Using a Microfluidic Immunoassay in Tandem with MALDI-MS

Proteome mapping of the human pancreatic islet microenvironment reveals endocrine-exocrine signaling sphere of influence

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