Use of mass spectrometry to ensure purity of recombinant proteins: a cautionary note

Electrospray mass spectrometry was used to confirm the identity and purity of the unlabeled and isotopically labeled vnd/NK2-A35T homeodomain protein prepared for NMR experiments. A series of events involving adduction by urea, carbamoylation of amino acid groups, and the presence of nickel ion resulted in protein masses shifted from their expected values, thus emphasizing that observed discrepancies in mass may have sources other than an error in amino acid sequence. Procedures used to detect and remedy these problems are described.