MS-DAP Platform for Downstream Data Analysis of Label-Free Proteomics Uncovers Optimal Workflows in Benchmark Data Sets and Increased Sensitivity in Analysis of Alzheimer’s Biomarker Data

Koopmans, Frank; Li, Ka Wan; Klaassen, Remco V.; Smit, August B.

doi:10.1021/acs.jproteome.2c00513

Cited by 29 publications

(21 citation statements)

References 52 publications

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“…Output was filtered at 0.01 FDR (Demichev et al, 2020). The Mass Spectrometry Downstream Analysis Pipeline (MS-DAP) (version beta 0.2.5.1) was used for quality control and candidate discovery (Koopmans et al, 2022). Differential abundance analysis between groups was performed on log transformed protein abundances.…”

Section: Lc-ms/ms Proteomic Analysis -Search and Data Analysismentioning

confidence: 99%

Luminescence-based screening for extracellular vesicle release modulators reveals a role for PI4KIIIβ in exosome biogenesis upon lysosome inhibition

Bebelman

Crudden

Snieder

et al. 2023

Preprint

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Dysregulated extracellular vesicle (EV) release has been implicated in various pathologies, including cancer, neurodegenerative disease and osteoarthritis. Despite clear therapeutic potential, drug screening for EV release modulators has yielded limited success due to the lack of a sensitive and scalable EV read-out system. Here, we employed CRISPR-Cas9 to engineer HEK293 cells expressing HA-NanoLuciferase-(NL)-tagged endogenous CD63. We found that under basal culture conditions, CD63-containing EVs are released via a mechanism that is independent of the exocytic SNARE protein SNAP23, presumably by direct budding from the plasma membrane. Endo-lysosome inhibition by chemical or genetic perturbation of vATPase strongly increased SNAP23 and nSmase2-dependent exosome secretion from intracellular compartments. Proteomic analysis revealed these exosomes are enriched for early- and late endosomal markers, but also for autophagosomal proteins. This suggests that a proportion of these exosomes originate from amphisomes, although chemical inhibition of canonical autophagy did not affect exosome secretion upon lysosome inhibition. Using a broad-spectrum kinase inhibitor screen, we identified and subsequently validated the lipid kinase PI4KIIIβ as a critical mediator of exosome secretion and amphisome-mediated secretory autophagy, upon lysosome inhibition. We conclude that tagging of endogenous CD63 with NanoLuciferase represents a sensitive, scalable reporter strategy that enables identification of (druggable) modulators of EV biogenesis and release under physiological and pathological conditions.

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Section: Lc-ms/ms Proteomic Analysis -Search and Data Analysismentioning

confidence: 99%

Luminescence-based screening for extracellular vesicle release modulators reveals a role for PI4KIIIβ in exosome biogenesis upon lysosome inhibition

Bebelman

Crudden

Snieder

et al. 2023

Preprint

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“…For the downstream analysis, we used MS-DAP (version beta 0.2.5.1 [19]) for quality control and candidate discovery. Differential abundance analysis between groups was performed on log-transformed protein abundances.…”

Section: Discussionmentioning

confidence: 99%

Suspension TRAPping Filter (sTRAP) Sample Preparation for Quantitative Proteomics in the Low µg Input Range Using a Plasmid DNA Micro-Spin Column: Analysis of the Hippocampus from the 5xFAD Alzheimer’s Disease Mouse Model

Thanou

Koopmans

Pita-Illobre

et al. 2023

Cells

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Suspension TRAPping filter (sTRAP) is an attractive sample preparation method for proteomics studies. The sTRAP protocol uses 5% SDS that maximizes protein solubilization. Proteins are trapped on a borosilicate glass membrane filter, where SDS is subsequently removed from the filter. After trypsin digestion, peptides are analyzed directly by LC-MS. Here, we demonstrated the use of a low-cost plasmid DNA micro-spin column for the sTRAP sample preparation of a dilution series of a synapse-enriched sample with a range of 10–0.3 µg. With 120 ng tryptic peptides loaded onto the Evosep LC system coupled to TimsTof Pro 2 mass spectrometer, we identified 5700 protein groups with 4% coefficient of variation (CoV). Comparing other sample preparation protocols, such as the in-gel digestion and the commercial Protifi S-TRAP with the plasmid DNA micro-spin column, the last is superior in both protein and peptide identification numbers and CoV. We applied sTRAP for the analysis of the hippocampal proteome from the 5xFAD mouse model of Alzheimer’s disease and their wildtype littermates, and revealed 121 up- and 54 down-regulated proteins. Protein changes in the mutant mice point to the alteration of processes related to the immune system and Amyloid aggregation, which correlates well with the known major Alzheimer’s-disease-related pathology. Data are available via ProteomeXchange with the identifier PXD038443.

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“…All other settings were left as default. MS-DAP 1.0.6 59 was used for downstream analyses of the DIA-NN results. Filtering and normalization were applied to respective samples per statistical contrast.…”

Section: Methodsmentioning

confidence: 99%

SFRP1 upregulation causes hippocampal synaptic dysfunction and memory impairment

Pereyra,

Mateo,

Martin-Bermejo

et al. 2024

Preprint

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Decreased dendritic complexity and impaired synaptic function are strongly linked to cognitive decline in Alzheimer's disease (AD), and precede the emergence of other neuropathological traits that establish a harmful cycle exacerbating synaptic dysfunction. SFRP1, a glial-derived protein regulating cell-cell communication, is abnormally elevated in the brain of AD patients and related mouse models already at early disease stages. Neutralization of SFRP1 activity in mice reduces the occurrence of protein aggregates, neuroinflammation and prevents the loss of synaptic long-term potentiation (LTP). In this study, we generated transgenic mice that overexpress Sfrp1 in astrocytes to investigate whether LTP loss is due to an early influence of SFRP1 on synaptic function or results from other alterations driving disease progression. We report that SFRP1-overexpressing mice show reduced dendritic complexity and spine density in dentate gyrus granule cells during early adulthood, prior to a significant deficit in LTP response and late onset cognitive impairment. Ultrastructural analysis revealed the loss of small-sized synapses and presynaptic alterations in transgenic mice. Analysis of proteomic changes points to a general decrease in protein synthesis and modifications in the synaptic proteome, particularly of proteins related to synaptic vesicle cycle and synaptic organizers, like neurexin and neuroligin. We propose a model wherein SFRP1 directly impacts on synaptic function, by increasing the availability of synaptic organizing molecules at the synapse. These observations, combined with documented SFRP1 effects on APP processing and microglial activation, imply that SFRP1 contributes to multiple pathological effects in AD, emerging as a promising therapeutic target for this devastating disease.

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MS-DAP Platform for Downstream Data Analysis of Label-Free Proteomics Uncovers Optimal Workflows in Benchmark Data Sets and Increased Sensitivity in Analysis of Alzheimer’s Biomarker Data

Cited by 29 publications

References 52 publications

Luminescence-based screening for extracellular vesicle release modulators reveals a role for PI4KIIIβ in exosome biogenesis upon lysosome inhibition

Luminescence-based screening for extracellular vesicle release modulators reveals a role for PI4KIIIβ in exosome biogenesis upon lysosome inhibition

Suspension TRAPping Filter (sTRAP) Sample Preparation for Quantitative Proteomics in the Low µg Input Range Using a Plasmid DNA Micro-Spin Column: Analysis of the Hippocampus from the 5xFAD Alzheimer’s Disease Mouse Model

SFRP1 upregulation causes hippocampal synaptic dysfunction and memory impairment

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