Thesis final - after defense-7 - Jacobs University

Chapter 1
molecular weight impurities and buffer ions during electrophoresis for which the MALDI-
ToF has more sensitivity. Another advantage of the gel based approach is that polyacrylamide
materials act like safe containers to handle and excise very small amount of the proteins. The
protocols for the gel based approach are well established in comparison to gel free approach
(80, 81). The mass spectrometers consist of three essential parts such as ionizer, analyzer and
detector. The ionization source converts molecules into gas-phase ions. This ionization is
facilitated by the energy provided by laser. As ions exit the ion source, the individual mass to
charge (m / z) ratios are separated by a second device, a mass analyzer. The mass analyzer
separates ions on the basis of mass to charge ratios. In MALDI-TOF-MS, the molecules are
given mass values based on their movement or time of flight in the drift tube. Lighter ions fly
faster than heavier ions and when it reaches to the third part of the mass spectrometer called
detector. The signals are recorded on the detector and a mass spectrum is obtained. The
detector records the charge produced during the hitting of ions on the surface. The spectrum is
produced based on mass charge values. The experimental masses can be then compared to the
theoretical masses available in the proteome of specific organism (80). The peptide mass
fingerprinting approach can be used for searching protein databases. The principle behind
PMF is very simple; the identified protein is the protein has the best match between the
experimental and theoretical mass data. A theoretical digest of all the proteins sequences in
the database by a specific enzyme can be used for predicting the individual peptides. These
theoretical peptides can be searched to match peptides derived from the experimental mass
data and then ideally one protein has to match the protein of interest. The protein will be
considered as identified if the score is greater than the score fixed by Mascot with a p value
less than 0.05. In addition, the experimental and theoretical values of the molecular weight
and isoelectric point of a protein must be in the similar range. The databases such as NCBI
and Swiss Prot can be searched for the identifications of proteins (82).
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