Sequencing of Proteins from the C-Terminus
In 1992 we reported a new method of sequencing proteins from the carboxy-terminus (C-terminus) (Boyd et al., 1992). In the past 2 years, we have continued our investigations, including the mechanism of the initial activation of the C-terminal carboxyl group and the deliberate modifications of the reactive side-chains of the amino acids with the sequencing reagents. Through the selection of the reagents and reaction conditions used for our sequencing protocol, aspartic acid, glutamic acid, serine and threonine are derivatized. The amidation of aspartic and glutamic acid, and the acetylation of serine and threonine, have led to improved yields in sequencing these residues. Aspartic and glutamic acid are now categorized, as seen in Table 1, as amino acid residues that are readily sequenced. Our criteria for determining whether a residue is reliably called is the ability to sequence through and detect that residue when it is present in one nanomole of a protein sample. On average, it is possible to sequence 5 cycles on one nanomole of protein applied to polyvinylidene difluoride (PVDF) membrane if the amino acid sequence contains those residues listed in the “reliably called” column of Table 1. Our focus for the 1994 MPSA conference is to illustrate the current utility of this C-terminal sequencing method in the sequencing of proteins immobilized onto PVDF.
KeywordsGlutamic Acid Acetic Anhydride Aspartic Acid Residue Sequencing Protocol Glutamic Acid Residue
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