Pores Formation on Cell Membranes by Hederacolchiside A1 Leads to a Rapid Release of Proteins for Cytosolic Subproteome Analysis

Abstract

Hederacolchiside A1 was used to progressively permeabilize the membrane of human melanoma MEL-5 cells. Holes formation was followed by Scanning Electron Microscopy and interaction of the saponin with cholesterol and phospholipids by TOF-SIMS. 2D-LC-MS/MS and 2D-SDS-PAGE show that the release of soluble proteins into serum-free culture media increases with time. This can lead to a new rapid and efficient strategy to analyze the cytosolic subproteome and it opens the door to get information from the cytosolic compartment for clinical proteomic studies.

Micelles Self-Assembled by 3-O-β-D-Glucopyranosyl Latycodigenin Enhance Cell Membrane Permeability, Promote Antibiotic Pulmonary Targeting and Improve Anti-infective Efficacy

“…Different from other saponins that induce membrane lysis and lead to the leakage of organelles [32,33]. For example, hederacolchiside A1 formed larger than 1 µm holes in MEL-5 cells and induced cell necrosis [34]. While GP-682 micelles can induce cell membrane perforation that consistent with a smaller size of GP-682 micelle itself (60-90 nm).…”

“…LDH, a stable cytoplasmic enzyme existing in all cells, is often used as a cytosolic marker in the cellular membrane damage test [34]. When the BEAS-2B cells were exposed to various GP-682 micelles concentrations, the LDH was released from the cells in a dose-dependent manner (Fig.…”

Micelles self-assembled by 3-O-β-D-glucopyranosyl latycodigenin enhances cell membrane permeability, promotes antibiotic pulmonary targeting and improves anti-infect efficacy

“…Different from other saponins that induce membrane lysis and lead to the leakage of organelles [31,32], GP-682 micelles maintained the cell integrity. For example, larger than 1 µM holes in MEL-5 cells were formed after treatment with 10 µM hederacolchiside A1 for 2 min, which induced cell necrosis [33]. The BEAS-2B cells incubated with 100 µg/mL GP-682 micelles for 30 min remained healthy.…”

“…LDH, which is often used as a cytosolic marker in the cellular membrane damage test, is a stable cytoplasmic enzyme existing in all cells [33]. BEAS-2B cells were exposed to various concentrations of GP-682 micelles for 30 min at 37 °C.…”

Analysis of the immune response of human dendritic cells to Mycobacterium tuberculosis by quantitative proteomics

“…Among these methods, the 60 % methanol-assisted trypsin digestion is relatively simple, and the use of a methanol-based buffer circumvents the need for reagents that interfere with chromatographic separation and ionization of the peptides (e.g., detergents, chaotropes, nonvolatile salts). For quantitative aspects, isotope-coded affinity tag [ 16 ], isotope coded protein labeling [ 17 ], 18 O labeling [ 18 ], stable isotope dimethyl labeling [ 19 ], stable isotope labeling by amino acids in cell culture [ 20 ] and isobaric tags for relative and absolute quantitation [ 15 , 21 ] have been introduced for use in comparative membrane proteomics as well as in identification of membrane proteins. Due to its simplicity, effectiveness, and—most importantly—organic solvent compatibility, dimethyl labeling can be efficiently used with 60 % methanol-assisted trypsin digestion of membrane proteins [ 18 ].…”