Measuring Consequences of Protein Misfolding and Cellular Stress Using OMICS Techniques - Protein Misfolding and Cellular Stress in Disease and Aging - page 123

altered in intensity, in the comparison of different samples, is then
identified by MS, typically MALDI-TOF. Since modern tandem
mass spectrometry (MS/MS) instruments are able to identify and
quantitate more than hundred proteins in a mixture in one run,
the labor-intensive 2D-gel electrophoresis has largely been
replaced by liquid chromatography (LC) directly coupled (in-line)
to the MS instrument. For both gel electrophoresis and LC, there
exist several operation principles which can be applied at the pro-
tein or peptide level (Table 1 ) and often are two or more separation
methods combined. When different LC separations are com-
bined, it is referred to as multidimensional LC or Multidimensional
Protein Identification Technology (MudPit) ( 23, 24 ). In multidi-
mensional separations, the LC methods are chosen so that the
separation methods are based on totally different principles
(orthogonal principles) yielding efficient resolution of samples
with high complexity. The most common combination is cation
exchange followed by reverse phase chromatography, where the
latter is coupled directly to the MS instrument.
The MS measures the mass to charge ratio (m/z) of peptides,
and the most widespread method to prepare the peptide mixtures is
by enzymatic cleavage of the proteins by trypsin which cleaves after
lysine and arginine residues. Tandem mass spectrometry (MS/MS)
consists of two steps in the instrument. The first step is a determina-
tion of the mass of the intact peptide, and the second is an amino
acid sequencing of the peptide. Step two is done by fragmentation
of the peptide and determination of the fragment's masses
compared to a database of all proteins from the respective organism.
On basis of the full genome sequence of the respective organism,
the MS data confer identification of the peptides, and two peptides

Table 1
Examples of the most common separation methods applied
prior to MS detection

Method Separation principle References

In gel SDS-PAGE Size

Isoelectric focusing Isoelectric point

In liquid Anion or cation LC Charge ( 50 )

Reverse phase LC Hydrophobicity ( 51 )

Free flow electrophoresis Isoelectric point ( 52 )

Capillary electrophoresis Charge to mass ratio ( 53 )

Most of the methods can be adapted to separate either proteins or peptides, with the
exception of SDS-polyacrylamide gel electrophoresis (PAGE), which cannot separate
peptides. Within brackets is the biochemical property according to which the
proteins/peptides are separated