trypsin) is separated by MS. The ion corresponding to one peptide is selected in the
first analyser and collided with argon gas in a collision cell to generate fragment ions.
The fragment ions thus generated are then separated, according to mass, in a second
analyser, identified, and the sequence determined as described in Section 9.5.2.
A further method, ladder sequencing, has been developed, and combines the Edman
chemistry with MS. Edman sequencing is carried out using a mixture of PITC and
phenylisocyanate (PIC) (at about 5% of the concentration of PITC). N-terminal amino
groups that react with PIC are effectively blocked as they are not cleaved at the acid
cleavage step. Consequently, at each cycle, approximately 5% of the protein mol-
ecules are blocked. Thus, after 20 to 30 cycles of Edman degradation, a nested set of
peptides is produced, each differing by the loss of one amino acid. Analysis of the
mass of each of these polypeptides using ESI or MALDI allows the determination of
the molecular mass of each polypeptide and the difference in mass between each
molecule identifies the lost amino acid residue.Detection of disulphide linkages
For proteins that contain more than one cysteine residue it is important to determine
whether, and if so how many, cysteine residues are joined by disulphide bridges. The
most commonly used method involves the use of MS (Section 9.5.5). The native
protein (i.e. with disulphide bridges intact) is cleaved with a proteolytic enzyme (e.g.
trypsin) to produce a number of small peptides. The same experiment is also carried
out on proteins treated with dithiothreitol (DTT) which reduces (cleaves) the disulphide
bridges. MALDI spectra of the tryptic digest before and after reduction with DTT
allows identification of disulphide-linked peptides. Linked peptides from the native
protein will disappear from the spectrum of the reduced protein and reappear astwo
peptides of lower mass. Knowledge of the exact mass of each of the two peptides, and
knowledge of the cleavage site of the enzyme used, will allow easy identification of the
two peptides from the known protein sequence.Thus, if the mass of two disulphide-linked
peptides isM, and this is reduced to two separate chains of massesAandB, respectively,
thenAþB¼Mþ2. The extra two mass units derive from the fact that reduction of
the disulphide bond results in an increase of mass ofþ1 for both cysteine residues.-SS-!2 H
SHþHS-Hydrophobicity profile
Having determined the amino acid sequence of a protein, analysis of the distribution of
hydrophobic groups along the linear sequence can be used in a predictive manner. This
requires the products of a hydrophobicity profile for the protein, which graphs the
average hydrophobicity per residue against the sequence number. Averaging is achieved
by evaluating, using a predictive algorithm, the mean hydrophobicity within a moving
window that is stepped along the sequence from each residue to the next. In this way, a
graph comprising a series of curves is produced and reveals areas of minima and maxima
in hydrophobicity along the linear polypeptide chain. For membrane proteins, such
profiles allow the identification of potential membrane-spanning segments. For example,
an analysis of a thylakoid membrane protein revealed seven general regions of the protein333 8.4 Protein structure determination