AbstractMultiple spotting due to protein speciation might increase a protein's chance of being captured in a random selection of 2‐DE spots. We tested this expectation in new (PXD015649) and previously published 2‐DE/MS data of porcine and human tissues. For comparison, we included bottom‐up proteomics studies (BU‐LC/MS) of corresponding biological materials. Analyses of altogether ten datasets proposed that amino acid modification fosters multispotting in 2‐DE. Thus, the number of 2‐DE spots containing a particular protein more tightly associated with a peptide diversity measure accounting for amino acid modification than with an alternative one disregarding it. Furthermore, every 11th amino acid was a post‐translational modification candidate site in 2‐DE/MS proteins, whereas in BU‐LC/MS proteins this was merely the case in every 21st amino acid. Alternative splicing might contribute to multispotting, since genes encoding 2‐DE/MS proteins were found to have on average about 0.3 more transcript variants than their counterparts from BU‐LC/MS studies. Correspondingly, resolution completeness as estimated from the representation of transcript variant‐rich genes was higher in 2‐DE/MS than BU‐LC/MS datasets. These findings suggest that the ability to resolve proteomes down to protein species can lead to enrichment of multispotting proteins in 2‐DE/MS. Low sensitivity of stains and MS instruments appears to enhance this effect.