Both qualitative and quantitative proteome analyses are based on the results of peptides in all bottom-up proteomic strategies.A key assumption in peptide-based quantification is that the proteolytic digestion is sufficient and reproducible.However,few studies concern the incomplete protein digestion and the subsequent influence on large-scale proteome quantification.In this study,shortened digestion time,overdose chaotropic agent or detergent were applied to the digestion process,and their impacts on the accuracy of proteome quantification using stable isotope dimethyl labeling were investigated.We demonstrated that incomplete or hampered protein digestion compromised the accuracy of large-scale proteome quantification.Furthermore,the proteome composition might be changed in sample preparation procedures due to sample loss if minute amount of sample was analyzed,which would also decrease the accuracy of proteome quantification.