Dataset Information

A magnetic bead-based workflow for sensitive and streamlined cell surface proteomics

ABSTRACT: Cell-surface proteins represent an important class of molecules for therapeutic targeting and defining cellular phenotypes. However, their enrichment and detection via mass spectrometry-based proteomics remains challenging due to low abundance, posttranslational modifications, hydrophobic regions, and processing requirements. To improve the identification of cell-surface proteins via their corresponding N-linked glycopeptides (N-glycopeptides), we optimized a Cell-Surface Capture (CSC) workflow which incorporates magnetic bead-based processing. Using this approach, we evaluated labeling conditions (biotin tags and catalysts), enrichment specificity (streptavidin beads), missed cleavages (lysis buffers), non-enzymatic deamidation (digestion and de-glycosylation buffers), and data acquisition methods. Our findings support the use of alkoxyamine-PEG4-biotin plus 5-methoxy-anthranilic acid and streptavidin magnetic beads for maximal N-glycopeptide detection. Furthermore, single-pot solid-phased-enhanced sample-preparation (SP3) circumvented the need to isolate cell membranes by affording the use of strong detergents and chaotropes for protein extraction. Notably, with semi-automated processing, sample handling was simplified and between ~600-900 N-glycoproteins were identified from only 25-200µg of HeLa protein. Overall, the improved efficiency of the magnetic-based CSC workflow allowed us to identify both previously reported and novel N-glycosites with less material and high reproducibility, and should help advance the field of surfaceomics by providing insight in cellular phenotypes not previously documented.

INSTRUMENT(S): Orbitrap Eclipse, Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

TISSUE(S): B Cell, Uterus, Epithelial Cell, Cell Culture, Colon

DISEASE(S): Colon Cancer

SUBMITTER: Dylan Dieters-Castator

LAB HEAD: Matt Rardin

PROVIDER: PXD043678 | Pride | 2024-01-21

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	200721_ddc_2D_200ug_SP3-mCSC_TMT10_HeLa_biotin_comp_gene_5MA_04.raw	Raw
	200721_ddc_2D_200ug_SP3-mCSC_TMT10_HeLa_biotin_comp_gene_aniline_03.raw	Raw
	200721_ddc_2D_200ug_SP3-mCSC_TMT10_HeLa_biotin_comp_sera_5MA_02.raw	Raw
	200721_ddc_2D_200ug_SP3-mCSC_TMT10_HeLa_biotin_comp_sera_aniline_01.raw	Raw
	200725_ddc_2D_200ug_SP3-mCSC_TMT10_HeLa_biotin_comp_5MA_gene_02.raw	Raw

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Publications

Magnetic Bead-Based Workflow for Sensitive and Streamlined Cell Surface Proteomics.

Dieters-Castator Dylan Z DZ Manzanillo Paolo P Yang Han-Yin HY Modak Rucha V RV Rardin Matthew J MJ Gibson Bradford W BW

Journal of proteome research 20240116 2

Cell surface proteins represent an important class of molecules for therapeutic targeting and cellular phenotyping. However, their enrichment and detection via mass spectrometry-based proteomics remains challenging due to low abundance, post-translational modifications, hydrophobic regions, and processing requirements. To improve their identification, we optimized a Cell-Surface Capture (CSC) workflow that incorporates magnetic bead-based processing. Using this approach, we evaluated labeling co ...[more]

PMID: 38226771

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Project description:Exposure to Paraquat and RNA interference knockdown of mitochondrial superoxide dismutase (Sod2) are known to result in significant lifespan reduction, locomotor dysfunction, and mitochondrial degeneration in Drosophila melanogaster. Both perturbations increase the flux of superoxide, a progenitor reactive oxygen species, but the molecular underpinnings of the resulting phenotypes are poorly understood. Improved understanding of such processes could lead to advances in the treatment of numerous age-related disorders. Superoxide toxicity can act through protein carbonylation. Analysis of carbonylated proteins is attractive since reactive carbonyl groups are not present in the twenty canonical amino acids and are amenable to labeling and enrichment strategies. Here, carbonylated proteins were labeled with biotin hydrazide and enriched on streptavidin-coated beads. On-bead digestion was used to release carbonylated protein peptides, with relative abundance ratios versus controls obtained using the iTRAQ mass spectrometry-based proteomics approach. While Paraquat exposure and Sod2 knockdown have similar phenotypes, differences in protein carbonylation were anticipated because Paraquat exposure was expected to increase the concentration of superoxide throughout the cell while Sod2 knockdown was only expected to raise the concentration of superoxide in the mitochondrial matrix. Paraquat exposure resulted in widespread increases in carbonylated protein relative abundance: the median Paraquat-exposed to control carbonylated protein relative abundance ratio was 1.53. For Sod2 knockdown, in contrast, the median carbonylated protein relative abundance ratios were 1.13 versus the RNA interference driver control and 1.05 versus the RNA interference transgene control. However, some proteins did show large increases in carbonylated protein relative abundance on Sod2 knockdown, most notably cytochrome c oxidase subunit Vb, possibly providing some indication of the molecular basis of the Sod2-knockdown phenotype.