related directly to an amino acid residue because the two flanking Y ions result from
cleavage of two adjacent amide bonds. Therefore, with a knowledge of the relative
molecular masses of each of the 20 naturally occurring amino acids, it is possible to
determine the presence of a particular residue at any point within the peptide. The
position of the assigned amino acid is deduced by virtue of them/zratio of the two
ions. By reading several amino acids it was possible to assemble a sequence of amino
acids, in this case (using the one-letter code) YWS. Database searching was then
possible using the peptide 1778 Da, the position of the lowerm/zY ion (1116.8), the
proposed amino acid sequence (YWS) and the higher Y ion atm/z1553.6. This
provides a sequence tag, which is written as (1116.8) YWS (1553.6).
A search of the SWISS-PROT database (Fig. 8.7), showed just two ‘hits’ from 40 000
entries, suggesting the protein is glyceraldehyde-3-phosphate dehydrogenase. The full
sequence of this peptide is LISWYDNEYGYSNR and the MS/MS fragmentation data
give a perfect match. Other peptides in the sample can also be analysed in the same
manner, confirming the identity of the protein.
A further development of 2-D PAGE has been the introduction of difference gel
electrophoresis (DIGE). This again allows the comparison of protein components of
similar mixtures, but has the advantage that only one 2-D gel has to be run rather than
two. In this method the two samples to be compared are each treated with one of two
different, yet structurally very similar, fluorescent dyes (cy3 and cy5). Each dye reacts
with amino groups, so that each protein is fluorescently labelled by the dye binding
to lysine residues and the N-terminal amino groups. The two protein solutions to be
compared are then mixed and run on asingle2-D gel. Thus every protein in oneFig. 8.7The PeptideSearchTMinput form and search result based on data obtained from nano-ESI MS2
of m/s 890 from RBL Spot 2. (Courtesy of Glaxo SmithKline, Stevenage, UK.)344 Protein structure, purification, characterisation and function analysis