Workflow from Scientific Research

Workflow of OcSILAC combined with subcellular fractionation in HT29-MD2 cells. Step 1: Metabolic labeling. Step 2: Cell stimulation; Light cells were treated with different conditions (cell culture medium time zero or T0, LPS, LPSMn1, LPS MnCl2,Mn1) in blue, and Heavy untreated cells (cell culture medium only) shown in red. Step 3: An equal number of Light and Heavy cells were mixed for each condition. Step 4: Subcellular fractionation and recovery of cytosolic and plasma membrane and organelles (PMO) fractions. Step 5: Protein extraction. Step 6: Cysteine derivatization (alkylation using iodoacetamide, reduction using 1,2-dithiothreitol (DTT), biotin-HPDP labeling). Step 7: Protein tryptic digestion. Step 8: Streptavidin affinity chromatography, biotinylated peptides are retained and eluted by DTT to constitute the enriched fraction containing oxidized peptides (S-Ox), and the flowthrough is the non-enriched fraction containing reduced peptides (S-Red), and peptides without cysteines (called no-S). Step 9: Liquid chromatography separation of peptides of the two fractions and MS/MS analysis. Step 10: Data Analysis. The intensities of the heavy and light peptides are used for quantification (L:H ratio). Abbreviations: CAM = carbamidomethyl, biotin-HPDP = N-[6-(Biotinamido)hexyl]-3'-(2'-pyridyldithio) propionamide.

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