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introduction of fast atom bombardment (FAB) MS allowed the analysis of large,

charged molecules such as proteins and peptides to be achieved for the first time.

The further development of more sophisticated methods such as electrospray ionisa-

tion (ESI) and matrix-assisted laser desorption ionisation (MALDI) (see Chapter 9) has

led to the analysis of protein by mass spectrometry becoming routine. Although the

Edman degradation still has occasional applications in protein structure analysis,

mass spectrometry is now the method of choice for determining amino acid sequence

data. When peptides are fragmented by MS it is fortunate that cleavage occurs

predominantly at the peptide bond (although it must be noted that other fragmenta-

tions, such as internal cleavages, secondary fragmentations, etc. do occur, thus

complicating the mass spectrum). This means that the peptide fragments produced

each differ sequentially by the mass of one amino acid residue. The amino acid

sequence can thus be readily deduced. In particular, if side-chain modifications occur,

these can also be observed due to the corresponding increase in mass difference.

The use of mass spectrometry to obtain sequence data from proteins and peptides

is described more fully in Section 9.5. Tandem mass spectrometry (MS/MS or MS^2 )is

also increasingly being used to obtain sequence data. A digest of the protein (e.g. with

Table 8.5Specific cleavage of polypeptide

Reagent Specificity

Enzymic cleavage

Chymotrypsin C-terminal side of hydrophobic amino acid residues, e.g. Phe, Try,
Tyr, Leu

Endoproteinase Arg-C C-terminal side of arginine

Endoproteinase Asp-N Peptide bonds N-terminal to aspartate or cysteine residues

Endoproteinase Glu-C C-terminal side of glutamate residues and some aspartate residues

Endoproteinase Lys-C C-terminal side of lysine

Thermolysin N-terminal side of hydrophobic amino acid residues excluding Trp

Trypsin C-terminal side of arginine and lysine residues but Arg-Pro and
Lys-Pro poorly cleaved

Chemical cleavage

BNPS skatole

g

N-Bromosuccinimide C-terminal side of tryptophan residues

o-Iodosobenzoate

Cyanogen bromide C-terminal side of methionine residues

Hydroxylamine Asparagine–glycine bonds

2-Nitro-5-thiocyanobenzoate N-terminal side of cysteine residues

332 Protein structure, purification, characterisation and function analysis