Qproteome albumin igg depletion kit

Sample pooling is a strategy commonly used to minimize individual variation in proteomic studies.12 (link) The proteins isolated from the lungs within the same group (n=6) were pooled: 24-hour control versus 24-hour ZnONP and 28-day control versus 28-day ZnONP. Albumin and IgG were depleted using a Qproteome Albumin/IgG Depletion Kit (Qiagen, Germantown, MD, USA). One hundred micrograms of each sample was precipitated with acetone at −20°C for 2 hours and then redissolved in 0.5 M triethylammonium bicarbonate (TEAB) as a cleanup process. Subsequent reduction, alkylation, digestion, and iTRAQ labeling (Applied Biosystems, Grand Island, NY, USA) were performed according to the manufacturer’s protocol. Briefly, the protein samples were reduced in 5 mM tris-(2-carboxyethyl)phosphine (TCEP) at 60°C for 1 hour, and the cysteine groups were blocked by incubation with a 10 mM methyl methanethiosulfonate (MMTS) solution at room temperature for 10 minutes. The samples were digested with 10 µg trypsin at 37°C for 16 hours and then labeled with the iTRAQ tag (114 for control and 115 for ZnONP exposure). The samples were combined, dried using a speedvac, redissolved in 50 µL of 5% acetone in 0.1% formic acid, and subjected to further analysis.