When the 34 kDa kinase domain of human spleen tyrosine kinase (Syk-KD) was expressed as a C-terminally His-tagged protein in baculovirus-infected Sf-21 insect cells, the purified protein included two forms that migrated slightly differently in SDS-polyacrylamide gel electrophoresis. Intact mass analysis and LC-MS/MS peptide mapping showed that the major and faster-migrating product had the intended amino-acid sequence and 0-6 phosphorylations. This material accounted for about 95% of the purified protein. The minor product was Syk-KD with a 26 amino-acid N-terminal extension. The result suggested the existence of an upstream alternative site for the initiation of translation, and this proved to be an ACG codon derived from the pBacPAK9 vector used to express Syk-KD. The ACG codon was preceded and followed by Kozak-type sequence elements (a purine in the 3 position and a G in the +4 position) that would have enhanced the viability of initiation at ACG. The initiating amino-acid residue was Met for both minor and major products, and both forms of the protein were alpha-N-acetylated. For the minor product, protein intact mass analysis and peptide mapping both gave results in agreement with the sequence predicted from the DNA. A similar result with the same underlying cause was obtained with insect cell expression of full-length Syk. It appears that similar results are possible whenever this vector is used. (C) 2010 Elsevier Inc. All rights reserved.