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Profiling membrane proteins would facilitate revealing disease mechanism and discovering new drug targets as they play essential roles in cellular signaling, substrate transport, and cell adhesion. However, the analysis of membrane proteins still remains a challenge due to their high hydrophobicity, as well as the suppression effect of high abundant soluble proteins. In this work, to achieve a membrane proteome profiling, a sample preparation strategy based on sequential extraction at the protein level assisted by a range of extraction reagents with different dissolving capabilities, followed by nano-RPLC-ESI-MS/MS analysis was developed and applied for HeLa cell line analysis. It was found that with progressively harsher extraction reagents (i.e., 2 M NaCl, 4 M urea, 0.1 M Na2CO3, and 10% 1-dodecyl-3- methyl-imidazolium chloride (C12ImCl) performed, much more high hydrophobic proteins and low abundant proteins were identified. With our developed strategy, 5553 of the identified proteins (4419 gene products) were annotated to be membrane proteins and 2573 proteins (2183 gene products) have at least one transmembrane domain, to our best knowledge, which is the most comprehensive membrane proteome dataset for HeLa cell line. Notably, 110 of the identified membrane proteins were discovered in the “missing proteins” list referred to those in the neXtProt database. All above results indicated that our strategy has great potential to tackle the difficult but relevant task of identifying and profiling membrane proteins.