Identification of acetylated proteins was performed via identifying BSA-ac by two kinds of mass spectrometers including ESI-MS and MALDI-MS. The lysine-acetylated proteins can be confirmed from the observation of a specific ion at m/z 126.1 or a mass difference of 170 Da between the adjacent b-ions or y-ions. The LTQ-Orbitrap hybrid spectrometer (ESI-MS) comprising an LC pre-concentration system and an ESI ionization source could acquire much more information than the others. However, the ion at m/z 126.1 could not be detected with a linear ion trap analyzer, which was well observed through a 4700 Proteomic analyzer (MALDI-TOF-TOF). This diagnostic ion can also reduce the false positive rate. It has been demonstrated that ESI and MALDI MS are complementary to each other in analyzing acetylated proteins. Accordingly, it can be concluded from the fact that different results would be obtained with different mass spectrometers, and matched protocols should be used for the identification of the acetylated proteins.