In proteomic study, top-down and bottom-up approaches have been widely adopted.However, each approach has its own weaknesses.For example, the challenge in protein separation/fragmentation largely restricts the widespread usage of top-down approach and bottom-up approach is limited in complex peptide mixture analysis which may result in false positive protein identification results by MS.Therefore, combining top-down and bottom-up approaches, i.e.combining protein separation, on-line digestion and peptide separation may be a good solution to reduce weaknesses each.