Analysis of protein complexes is of increasing interest in the field of proteomics. A challenge is to develop methods for monitoring changes in the quantity and subunit composition of protein complexes on a proteome-wide scale. Here, we describe the combination of 1-D blue native polyacrylamide gel electrophoresis (BN-PAGE) with stable isotope labelling of amino acids in cell culture (SILAC) and tandem mass spectrometry (MS/MS). Cleared lysates from normal and perturbed samples, one incorporating heavy stable isotopes and the other light isotopes, are co-separated by blue native PAGE and then analysed and quantitated with MS/MS and appropriate software. This permits the analysis of cytoplasmic complexes. To demonstrate this technique, we explored how the 20S proteasome changes when the Pre9/alpha 3 subunit, the only nonessential subunit of this complex, was deleted. Our results showed that Delta Pre9/alpha 3 cells can form the 20S proteasome complex, although with reduced efficiency. This involves an increase in expression of the alpha 4 subunit. Our findings suggest this technique as an approach for the study of quantitative and qualitative differences in protein complexes, from cleared cell lysates.