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Phosphoproteome enrichment strategy using complementary chemistries of Fe-NTA and TiO2 followed by LC-MS/MS

ABSTRACT: The global phosphoproteome analysis by liquid chromatography-mass spectrometry (LC-MS) has emerged as an important tool to study cell signalling. However, the coverage of phosphoproteome by LC-MS is limited despite various approaches for phosphoprotein enrichment. Studies in literature have exploited the complementary chemistries of affinity materials like metal (immobilized metal ion affinity chromatography- IMAC) and metal oxides (metal oxide affinity chromatography- MOAC) independently and in a sequential elution approach to improve coverage of phosphoproteins. We demonstrate that further improvement in the enrichment of phosphoproteins can be achieved by using these metals and metal oxides independently and pooling the data.

INSTRUMENT(S):

ORGANISM(S): Bombyx Mori (silk Moth) Caenorhabditis Elegans Danio Rerio (zebrafish) (brachydanio Rerio) Brassica Oleracea Var. Botrytis (cauliflower) Candida Albicans (yeast) Pinus Radiata (monterey Pine) Drosophila Melanogaster (fruit Fly) Cercopithecus Aethiops (green Monkey) (grivet) Homo Sapiens (human) Bos Taurus (bovine) Cicer Arietinum (chickpea) (garbanzo) Brassica Napus (rape) Dictyostelium Discoideum (slime Mold)

TISSUE(S): Cell Suspension Culture

SUBMITTER: Neha Agrawal

LAB HEAD: Dr Rukmini Govekar

PROVIDER: PXD057032 | Pride | 2025-10-30

REPOSITORIES: Pride

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			Action	DRS
	FeNTA_ITGB1_KD_set1_FKD1.1_IDA-FKD1.1IDA.mzML	Mzml
	FeNTA_ITGB1_KD_set1_FKD1.1_IDA.group	Other
	FeNTA_ITGB1_KD_set1_FKD1.1_IDA.mgf	Mgf
	FeNTA_ITGB1_KD_set1_FKD1.1_IDA.mzML	Mzml
	FeNTA_ITGB1_KD_set1_FKD1.1_IDA.wiff	Wiff

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Phosphoproteome enrichment strategy using complementary chemistries of Fe-NTA and TiO2 followed by LC-MS/MS

Project description:The global phosphoproteome analysis by liquid chromatography-mass spectrometry (LC-MS) has emerged as an important tool to study cell signalling. However, the coverage of phosphoproteome by LC-MS is limited despite various approaches for phosphoprotein enrichment. Studies in literature have exploited the complementary chemistries of affinity materials like metal (immobilized metal ion affinity chromatography- IMAC) and metal oxides (metal oxide affinity chromatography- MOAC) independently and in a sequential elution approach to improve coverage of phosphoproteins. We demonstrate that further improvement in the enrichment of phosphoproteins can be achieved by using these metals and metal oxides independently and pooling the data.

2025-05-06 | PXD050933 | Pride

An Internal Standard for Assessing Phosphopeptide Recovery from Metal Ion/Oxide Enrichment Strategies

Project description:Phosphorylation-mediated signaling pathways have major implications in cellular regulation and disease. Mass spectrometry-based proteomics is a well-established approach for quantifying thousands of phosphorylation events in a single experiment. However, proteins with roles in signaling pathways are frequently less abundant and phosphorylation is often sub-stoichiometric. As such, the efficient enrichment and subsequent recovery of phosphorylated peptides is vital. To understand better phosphopeptide recovery from enrichment strategies, we designed a peptide internal standard-based assay directed toward sample preparation strategies for mass spectrometry analysis. We coupled mass-differential tandem mass tag (mTMT) reagents (specifically, TMTzero and TMTsuper-heavy), nine mass spectrometry-amenable phosphopeptides (phos9), and peak area measurements from extracted ion chromatograms to determine phosphopeptide recovery. We showcase this mTMT/phos9 recovery assay by evaluating three commercial phosphopeptide enrichment workflows. Our assay provides data on the recovery of phosphopeptides, which complement other metrics, namely the number of identified phosphopeptides and enrichment specificity. Our mTMT/phos9 assay is applicable to any enrichment protocol in a typical experimental workflow irrespective of sample origin or labeling strategy.

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Project description:Apolipoprotein E4 (APOE4), the main susceptibly gene for Alzheimer’s disease1–5, exerts cerebrovascular toxicity6 leading to blood-brain barrier (BBB) breakdown in humans7–9 and APOE4 transgenic mice10–13. Moreover, an early BBB dysfunction predicts cognitive decline in humans7. However, the comprehensive large-scale analysis of cell-specific mechanisms underlying APOE4 cerebrovascular disorder and how it relates to neuronal disorder is lacking. Using single-nucleus RNA-sequencing, phosphoproteome and proteome analysis14–17 here we show that APOE4 compared to APOE3 leads to early disruption of the BBB transcriptome in 2-3-month old APOE4 knock-in mice18 followed by dysregulation in protein signaling networks. This includes proteins regulating cell junctions, cytoskeleton, clathrin-mediated transport, and translation in brain endothelium, and transcription and RNA-splicing suggestive of DNA damage in pericytes. Changes in the BBB signaling mechanisms paralleled an early, progressive BBB breakdown and loss of pericytes starting at 2-3 months of age. The BBB breakdown preceded loss of neurites, post-synaptic interactome dysregulation, and behavioral deficits that developed 2-5 months later, and was associated with astrocyte and microglia response protecting BBB integrity. Thus, disruption of the BBB cell-specific signaling mechanisms could be an initial central contributor to APOE4-mediated neuronal disorder, and possibly a major target to correct APOE4-related cognitive deficits.

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