In this paper, we demonstrate a rapid and reproducible 1D LC-MS/MS workflow for fast quantitative proteomic research. We have optimized the LC-MS/MS conditions, including digestion and gradient conditions, sample loading amount, and MS parameter settings. As a result, we were able to obtain twice as many protein identifications compared with the LC-MS/MS conditions before optimization. More than 4500 protein groups and 50 000 peptides were identified in less than 8 h without any fractionation. This 1D workflow was then applied to the analysis of the MLN4924 treated/untreated human umbilical vein endothelial cell (HUVEC) samples with label-free quantification. In these experiments, a total of 179 proteins showed a statistically significant expression change after the MLN4924 treatment. Functional analysis showed that these proteins are associated with cell death and survival; gene expression; cell cycle; and DNA replication, recombination, and repair.