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Proteomics. Lecture 4 Proteases. Why to digest the proteins?. Modern MS instruments are capable of measuring the molecular weight of intact proteins with a fairly high degree of accuracy. Still it is if little use due to three reasons

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Proteomics

Proteomics

Lecture 4

Proteases


Why to digest the proteins
Why to digest the proteins?

  • Modern MS instruments are capable of measuring the molecular weight of intact proteins with a fairly high degree of accuracy.

  • Still it is if little use due to three reasons

    • Greater the mass of protein greater the magnitude of the error.

    • It is difficult to obtain mass measurements on very large and hydrophobic proteins.

    • The sensitivity of measurements of intact protein masses is not nearly as good as the sensitivity for peptide mass measurements and peptide tendom MS analysis.


What do we want digestion to accomplish
What do we want digestion to accomplish?

  • Peptide analysis is the choice rather than the protein.

  • The ideal protein digestion approach would cleave proteins at certain specific amino acid residues to yield fragments that are most compatible with MS analysis.

  • Peptide fragments between 6-20 amino acids are ideal for MS analysis and database comparison.


  • Peptides shorter than about 6 amino acids generally are too short to produce unique sequence matches in database searches.

  • It is difficult to obtain sequence information from peptides longer than 20 amino acids in tendom MS analysis.

  • The objective of protein digestion will be to produce the highest yield of peptides of optimal length for MS analysis.


Proteases
Proteases short to produce unique sequence matches in database searches.

  • Naturally occurring

  • Purified and characterized

  • Only in limited quantity

  • A number of proteases that meet these requirements have been used for proteomic analysis.


Proteases and their cleavage specificity
Proteases and their cleavage specificity short to produce unique sequence matches in database searches.

aCleavage after aspartate or glutamate in sodium phosphate buffer otherwise cleavage only after glutamate


Trypsin
Trypsin short to produce unique sequence matches in database searches.

  • Widely used in protein analysis

  • Serine protease

  • Obtained primarily from porcine or bovine pancreas and easily purified.

  • It can be modified with tosylphenylalanylchloromethane (TCPK) to inhibit residual chymotrypsin.




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