Pitfalls of proteomics.

Lindholt et al1 used purified serum antibodies to C pneumoniae outer membrane protein (OMP) to probe protein extracts of abdominal aortic aneurysms separated by 1D and 2D electrophoresis. Although no specific signal for OMP was detected, strong staining was obtained for a 50-kDa protein, which subsequently was identified as immunoglobulin heavy chain through the use of mass spectrometry. The authors conclude that OMP antibodies cross-react with human immunoglobulins and that C pneumoniae might trigger an autoimmune reaction. We agree that autoimmune reactions are a possible link between infections and atherosclerosis.2,3 However, additional controls are essential for supporting their conclusion, ie, a negative control probing the blots with horseradish peroxidase– conjugated secondary antibody only. It is well established that immunoglobulin deposits accumulate during progression of atherosclerosis.4 We frequently have observed strong positive staining for immunoglobulin chains in protein extracts of human vessels when the blot was probed with anti-human secondary antibody only. Hence, it is possible that their secondary antibody has recognized immunoglobulins in protein extracts of abdominal aortic aneurysms irrespective of the primary antibody, and according to their mass spectrometry data, the conjugate might have been specific for gamma (heavy) chains. Furthermore, with regard to their experimental design: Silver staining only visualizes the most abundant proteins (detection limit 10 9 g) and more than one protein can be present in a single spot, which complicates data interpretation. Nevertheless, if mass spectrometry data suggest that one protein predominates in a particular spot on 2D gels, then it is likely that this protein also dominates the silver-staining pattern. In contrast, immunoblotting combined with enhanced chemiluminescence (ECL) detection amplifies the signal (detection limit 10 12 g). Consequently, ECL will visualize proteins at concentrations well below the limit of detection for silver staining and mass spectrometry. Because of this difference in sensitivity, one has to be extremely cautious to assume that spots detected with the ECL method correspond to a protein stained by silver and identified by mass spectrometry. The results might be accurate only if the protein of interest happens to be present in high abundance, as demonstrated for immunoglobulin heavy chains. For less abundant proteins, eg, C pneumoniae OMP, this approach is very likely to give wrong identifications. Thus, any potential cross-reactivity must be, at least, confirmed by independent methods. We appreciate that the use of proteomic techniques is a timely one,5 but we hope that our comments can increase the awareness for this important methodological limitation.