[ccp4bb]: initial methionine lost

[Yadong Yu]

Hi,

This is a ccp4 irrelevant problem but it may be interesting to this group.

Recently I sent 2 freshly purified protein samples for MALDI mass spectra to determine their molecular weights. The results of these 2 protein both were ~135 Da lower than their theoretical molecular weights. It is certainly out of the range of normal error as my proteins have MW less than 20 kDa. The accuracy of these MALDI experiments is reliable, as evidenced by the exact 2-fold ratios between the single-charge value and double-charge value. The expression vector of these 2 proteins were sequenced. They were expressed in Rosetta(DE3), an E.coli strain, and purified in native conditions. Taken account of the size of this difference, it would not be cleavage of some small group, e.g. amino group of Gln/Asn, from certain residues, the more possible reason would be deprivation of amino acid at either end of the protein. In this secenario, the initial methionine may be lost because its peptide fragment (-H2O), of 131 Da in size, matches the difference very well.

Anyone experienced such problem? Is the head of protein already cut during expression, or just lost in MALDI experiments?

Yadong Yu
LCMN/NICHD/NIH
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