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estimate of size and these methods are sufficient. The third method, mass spectrometry,

requires expensive specialist instruments and can give accuracy to0.001%. This kind of

accuracy is invaluable in detecting postsynthetic modification of proteins.

SDS-polyacrylamide gel electrophoresis (SDS-PAGE)

This form of electrophoresis, described in Section 10.3.1, separates proteins on the

basis of their shape (size), which in turn relates to their relative molecular masses.

A series of proteins of known molecular mass (molecular weight markers) are run on a

gel on a track adjacent to the protein of unknown molecular mass. The distance each

marker protein moves through the gel is measured and a calibration curve of logMr

versus distance moved is plotted. The distance migrated by the protein of unknownMr

is also measured, and from the graph its logMr and henceMris calculated.

The method is suitable for proteins covering a largeMrrange (10 000–300 000). The

method is easy to perform and requires very little material. If silver staining

(Section 10.3.7) is used, as little as 1 ng of protein is required. In practice SDS–PAGE

is the most commonly used method for determining proteinMrvalues.

Molecular exclusion (gel filtration) chromatography

The elution volume of a protein from a molecular exclusion chromatography column

having an appropriate fractionation range is determined largely by the size of the

protein such that there is a logarithmic relationship between protein relative molecu-

lar mass and elution volume (Section 11.7.1). By calibrating the column with a range

of proteins of knownMr, theMrof a test protein can be calculated. The method is

carried out on HPLC columns ( 1 30 cm) packed with porous silica beads. Flow

rates are about 1 cm^3 min^1 , giving a run time of about 12 min, producing sharp, well-

resolved peaks. A linear calibration line is obtained by plotting a graph of logMr

versusKdfor the calibrating proteins.Kdis calculated from the following equation:

Kd¼

ðVeVoÞ

ðVtVoÞ

whereVois the volume in which molecules that are wholly excluded from the column

material emerge (the excluded volume),Vtis the volume in which small molecules

that can enter all the pores emerge (the included volume) andVeis the volume in

which the marker protein elutes. This method gives values that are accurate to10%.

Mass spectrometry

Using either electrospray ionisation (ESI) (Section 9.2.4) or matrix-assisted laser

desorption ionisation (MALDI) (Section 9.3.8) intact molecular ions can be produced

for proteins and hence their masses accurately measured by mass spectrometry. ESI

produces molecular ions from molecules with molecular masses up to and in excess of

100kDa, whereas MALDI produces ions from intact proteins up to and in excess of

200kDa. In either case, only low picomole quantities of protein are needed. For

example,ab 2 crystallin gave a molecular mass value (20 2000.9), in excellent agree-

ment with the deduced mass of 20 201. However, in addition about 10% of the analysed

material produced an ion of mass 20072.2. This showed that some of the purified

protein molecules had lost their N-terminal amino acid (lysine). The deduced mass with

329 8.4 Protein structure determination