To increase grain quality, optimal nutrient management is desirable. Recently, atmospheric S emissions from industry have decreased leading to reduced grain quality. Fertilization of N primarily affects protein concentration and biomass production while S fertilization affects the fine tuning of storage protein composition. Additionally, high N supply increases the N/S ratio to the extent that the grains could be classified as S-deficient. Although total protein amounts were not changed by the fertilizer treatments, the amounts of individual gluten proteins were altered. High resolution proteomic analysis of S-containing gluten protein fractions demonstrates that under high N supply, low or high S fertilization significantly changed 41 and 66 of the gliadin and the glutenin proteins. In particular, this change may lead to different dough and baking quality. In conclusion, a high N fertilization induces S deficiency together with a change in gluten proteins and a loss of nutritional quality of grains.