Analysis of prolyl 4-hydroxylation of P4ha1 KO MEFs and P4ha2 KO mouse kidneys

We established a mouse embryonic fibroblast (MEF) cell line derived from mice that have a genetic ablation of of P4ha1. Type I collagen produced by these cells in presence of ascorbate was collected and partially purified by precipitation. Site specific hydroxylation analysis was performed using mass spectrometry. In another setup, type IV collagen from kidneys of P4ha2-/- mice was partially purified using pepsin digestion in acetic acid. Gel bands corresponding type IV collagens were cut and analyzed by mass spectrometry.