Bioinformatic Tools in Crop Improvement - Crop Improvement: New Approaches and Modern Techniques - page 76

For example, the Swiss Institute of Bioinformatics SWISS-2DPAGE database, Ex-
pasy and the Kazusa DNA Research Institute Cyano2Dbase are important. Differ-
ent chromatography separation methods, such as gel filtration chromatography, ion
exchange chromatography and affinity chromatography can also be used, but are
used less often in plants (Wu et al. 2005 ; Frolich and Arnold 2006 ).
Identification Methods
To identify each protein or polypeptide found in a sample, peptide mass fingerprint-
ing has been widely employed. Currently the most efficient method available con-
sists of two steps; (i) enzymatic digestion of well separated proteins excised from
gels into smaller peptides, and (ii) accurate mass measurements (fingerprints) of the
peptide fractions using mass spectrometry (MS). Various 'in-gel' digestion methods,
and modifications of these have been used to separate protein samples using 2-DE,
and further developments of these methods will continue to play an important role
in proteomics. The MS equipment generally consists of a source to ionise samples
and a mass spectrometer(s) to detect the ionized samples. The matrix-assisted laser
desorption ionization (MALDI) method is used in combination with time of flight
(TOF) MS (as MALDI-TOF-MS), or the electrospray ionization (ESI) method is
used in combination with quadrupole (Q) or ion trap (IT) MS. More recent develop-
ments in MS procedures, such as Q-TOF MS, IT-TOF MS or MALDI Q-TOF MS,
have become popular. Furthermore, ion fragmentation by collision-induced dissoci-
ation (CID) using tandem MS such as Q-TOF MS or post-source decay (PSD) using
MALDI-TOF MS have been used to determine more correctly peptide amino acid
sequences. Eventually though, to identify the target proteins obtained, peptide mass
fingerprint data are searched against a database of theoretically predicted masses of
known amino acid sequences (Hirano et al. 2004 ; Newton et al. 2004 ). To aid in the
correct identification of proteins and polypeptides, pI and molecular mass informa-
tion from gels are used to check the accuracy of MS identification fingerprint data
(De Filippis and Magel 2012 ).
ShotgunProteomics
Conventional gel electrophoresis-based separation is by far the most common meth-
od used, however a gel-free separation method has been used from time to time;
sometimes being referred to as a 'shotgun proteomics' approach. In this gel-free
method, the protein mixture is directly digested into peptides and separated by one
of the separation and identification methods just described.