In this study, differential protein expression was assessed during human atherosclerotic plaque progression. A multifaceted approach was used in which differential protein expression was studied by two-dimensional (2D) gel electrophoresis and validated in individual patients using western blotting and immunohistochemistry. 2D profiles of whole-mount advanced stable lesions were compared to those of plaques containing a thrombus. Mass spectrometry analysis identified vinexin-beta and alpha1-antitrypsin (AAT) in the same spot that was differentially expressed in plaques with a thrombus. Immunohistochemistry and western blotting showed limited expression of both vinexin-beta and AAT in early lesions, whereas high expression of both proteins was found in advanced lesions. Differential expression of vinexin-beta in lesions with a thrombus compared to stable plaques could not be confirmed, indicating the importance of validation of proteomic analysis. For AAT, western blotting of 2D gels revealed expression of six isoforms in advanced plaques, one of which was confirmed to be solely expressed in thrombus-containing plaques. In conclusion, vinexin-beta is expressed in advanced human atherosclerotic plaques, but differential expression of this protein in lesions with a thrombus versus stable plaques could not be confirmed. However, this analysis revealed expression of six isoforms of AAT in advanced plaques, one of which was uniquely expressed in thrombus-containing plaques.