Contrary to antibodies, proteomics does not rely on recognition of one specific epitope and can be applied across species. Moreover, the use of MS allows for determination of changes that occur at the protein level (i.e. amino acid modifications) (Figure 1). For example, we demonstrated using MS that the C-terminus of decorin, a small leucine-rich proteoglycan, is often cleaved in the left atrium but not in the ventricle.52 MS data provided an explanation why the use of different antibodies for the same target protein yielded very different results (Figure 2). Figure 1Antibody limitations. Detection by antibodies relies on binding to specific regions (epitopes) of the target protein. PTMs such as glycosylation or fragmentation may hinder epitope accessibility. Antibody A recognises non-glycosylated regions and always yields detection independently of sugar removal (left panel). Antibody B recognises epitopes in the vicinity of glycosylated regions. Therefore, recognition is only achieved after deglycosylation. Similarly, if protein fragmentation occurs, only antibody C, which recognises an intact portion, reveals a degradation pattern. Antibody D targets a region affected by fragmentation and can only detect the intact epitope. Consequently, information about degradation is missed. Proteomics interrogates peptides across the whole sequence and allows for consideration of variable modifications at the amino acid level. Different protein forms can therefore be identified and quantified. GAG, glycosaminoglycan; Pan-DG, pan-deglycosylation. Figure 2MS to explain discrepancies between different antibodies. An antibody against a C-terminal epitope (green on left panels, red on right panels) results in less intense staining for decorin (DCN) in the atrium compared to the left ventricle. An antibody against a different epitope (green on right panel) shows no such difference in staining intensities. This may be explained by cleavage of decorin at the C-terminus, which was detected in the atrium using MS.