Textbook of Personalized Medicine - Second Edition [2015]

75

2D PAGE and Mass Spectrometry

The essence of 2D PAGE (poly-acrylamide gel electrophoresis) approach is to sepa-
rate proteins from a specifi c cell or tissue type, record the pattern, and then produce a
Western Blot. Proteins in the blot are digested with a proteolytic enzyme, which has
well-defi ned cleavage specifi city. Peptide fragments can be analyzed by Matrix-
Assisted Laser Desorption Mass Spectrometry (MALDI-MS). The resulting peptide
masses are then compared with theoretical masses calculated from amino-acid
sequence databases. This technique has been used successfully to identify yeast pro-
teins. For completely sequenced genomes, 90 % of the proteins can be identifi ed
rapidly and automatically by searching databases with lists of peptide masses obtained
by 2D gel technique and matrix-assisted laser description ionization. This study
established that mass spectrometry provides the required throughput, the certainty of
identifi cation, and the general applicability to serve as the method of choice to con-
nect genome and proteome. Nanoelectrospray tandem MS is then used to character-
ize novel proteins either by searching EST databases with peptide sequence tags, or
by generating suffi cient sequence for cloning. This approach can be automated.

Comparison of Proteomic and Genomic Approaches

in Personalized Medicine

Although proteomic and genomic approaches can be complementary, there are
some similarities and differences that are shown in Table 2.4.

2D Electrophoresis

Biochip / Microfluidics

Mass Spectroscopy

High Performance Liquid

Chromatography

Bioinformatics

Protein Identification

© Jain PharmaBiotech

Fig. 2.7 The central role of spectrometry in proteomics

Application of Proteomics in Molecular Diagnosis