Shotgun strategy based proteomics has greatly developed in recent years and nowadays it is a routine to identify and quantify thousands of proteins in just one experiment.Stable isotope labeling by using chemical reagents is one of most used methods for accurate proteome quantification.However, unexpected variations and sample loss will introduce during the tedious sample processing and isotope labeling procedures inevitably, which will seriously influence the quantification accuracy, especially for low abundant proteins 1-3.Therefore, we developed solid-phase isotope labeling strategy combing with multidimensional separation for automated or semi-automated proteome quantification, which simplified the sample processing and labeling procedures and improved the quantification accuracy significantly.