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The routine method adopted by
BCPS for protein sequencing is based on the Edman degradation. The sequencing
is done in a
Applied Biosystems 477A automated protein sequencer.
Such method allows sequencing at low protein amounts
(approximately 10 pmol), and identification of over 70 residues per sample.
When planning sample submission it is important to consider the desired
sequence length, because the longer the sequence, the larger sample amount
needed.
At these links (Campbell)
and (MIT)
you will find more detailed information about sequencing
methods and strategies.
Mass spectometry is also
very frequently used in protein and peptide sequencing.
The methods frequently used by BCPS are mainly two:
One of them involves manual hydrolysisof the protein in HCl vapour followed
by automatic separation and post column derivatization with ninhydrin
made in a Hitachi
L-8500 automatic analyser.
The other analysis method is fully automated
by the Applied
Biosystems 420A amino acid analyzer, which does automated hydrolysis in
HCl vapour followed by PITC derivatization, being the amino acid mixture then
submitted to RP-HPLC separation for quantification.
At these links (Campbell)
and (MIT)
you will find more detailed information about amino acid composition determination.
The routine method adopted
by BCPS for macromolecule analysis usnig mass spectrometry is the electrospray
ionization followed by m/z selection and fragmentation in a triple quadrupole.
The analyses are made in a Perkin-Elmer
Sciex API 300 instrument.
This method allows analyses at the subpicomol level of samples and the precise molecular mass identification. Many techniques are used in ESMS, providing a large amount of information about the sample, such as amino acid sequence, dissulfide bonds determination, phosphorialtion and glycosilation sites determination, blocked N-terminus, molecule interaction and others.
