Caracterització proteïnes, Apuntes de Biotecnología

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Characterization ofbiomolecules^ Molecular weightPeptide maps

Sequencing

Classical methods to measure

molecular weight

• Ultracentrifugation• Gel filtration• SDS Electrophoresis^ – When applied to known proteins, it is used toidentify quaternary structure (oligomers,complexes…)

CENTRIFUGAL FORCE

Rotation Axis

Size exclusion chromatography

Gel electrophoresis^ _

Electroforesi SDS-PAGE 1.4 g SDS/ g proteïna (aprox. 1 mol SDS/2 residus)

Electroforesi SDS-PAGE The difference of mobility of proteins with different molecular weightsdepends on the concentration of the gel and extrapolate to a commonvalue at zero concentration. This experiment shows that theseparation by molecular weight is exclusively due to the filteringeffect of the gel.

• Calibrations,

bovine thyroid thyroglobulin

horse spleen ferritin

bovine liver catalase

rabbit muscle aldolase

bovine serum albumin (BSA)

hen egg ovalbumin

bovine pancreas chymotrypsinogen A

bovine pancreas ribonuclease A

Stokes' Radius (A) MW (kDa) Source Protein

Mass spectrometryAccurate characterization of molecular weightIdentification of post-translational modificationsMonitoring of reactionsIdentification of proteins from fragmentsSequencingTechnical enabling techniquesMatrix Assisted Laser Desorption and Ionization (MALDI)Electrospray ionization (ES)Time of flight (TOF) analyzersQuadrupolar filtersTandem experiments….

TOF

Where:

•m= mass of analyte ioni^ •z= charge on analyte ioni^ •E = extraction field•t= time-of-flight of ioni^ •l= length of the sources^ •l= length of the field-free drift regiond^ • e^ = electronic charge (1.6022x10-19 C)

Electrospray ionization

Copyright, National High Magnetic Field Laboratory, 2010.