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The starch availability and nutritional value of corn (Zea mays L.) are affected by zein proteins. The aim of the study was to see whether the proposed reduction of γ-zeins during the fermentation of silages is a result of either the enzymatic proteolytic activity or of the acidic environment, and how this reduction affects starch availability and degradability in high-moisture corn. A mass spectrometry (MS) technique was used to quantify the 16- and 27-kDa γ-zeins. Briefly, two-dimensional gel electrophoresis (2-DE) was used for γ-zein separation, followed by densitometry for protein quantification and matrix-assisted laser desorption ionization time-of-flight MS (MALDI-TOF/TOF) for protein identification. The results show that the reduction in γ-zeins induced by the ensiling led to a more pronounced starch availability and in vitro degradation, and this reduction was dependent on the type of proteolysis. More specifically, the results indicate that the reduction of γ-zeins in the ensiled corn was primarily driven by the enzymatic proteolysis. Furthermore, we demonstrated that 2-DE followed by densitometric quantification and the mass spectrometry analysis for protein identification can be used as a state-of-the-art method for γ-zein evaluation both in fresh and fermented/ensiled corn samples.