Update publication information. An increasing demand for the development of immunoglobin Y (IgY) illustrates the necessity of the component analysis in the process of conduction and quality control. This study investigated the proteome changes during IgY extraction (polyethylene glycol precipitation) and purification (a human mycoplasma protein-based affinity chromatography) from hen egg yolk. After confirming the extraction efficiency and purity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, bottom-up label-free liquid chromatography tandem-mass spectrometry (LC-MS/MS) was performed with samples including fresh yolk, IgY extracted product and purified product (all samples in triplicates). A total of 348 proteins were identified, with 36 proteins deleted and 209 newly detected proteins in the final IgY preparation compared to the intact egg yolk. The significantly reduced proteins mainly included phosvitin, albumin and apolipoprotein B whereas the significantly increased proteins were mainly IgY-related proteins and peptidase S1 domain-containing protein. GO functional analysis showed that compared to the PEG precipitated product, the differentially expressed protein in the IgY preparation obtained after protein M purification mainly had ATPase activity and purine ribonucleoside triphosphate binding activity and was mainly involved in purine and nucleic acid metabolism. This study will inevitably fasten the commercial application of IgY antibodies and is of greater significance for promotion, development and approval for new antibody derived drug products.