Proteomics Lecture 4 Proteases.

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Presentation transcript:

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 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? 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 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 Enzymes Cleavage specificity Trypsin /K-, /R-,\P chymotrypsin /W-,/Y-,/F-,\P Glu C(V8 protease) /E-,/Da-, \P Lys C /K-, \P Asp N /D- aCleavage after aspartate or glutamate in sodium phosphate buffer otherwise cleavage only after glutamate

Trypsin 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.

It cleaves protein at lysine and arginine residues, unlesseither of these is followed by a proline residue in the C-terminal direstion. Resulting peptides are of a length well suited to MS analysis.

This dual specificity means that trypsin wil cut proteins more frequently than will a protease that cuts only at one amino acid residue. A 50kDa protein will yield 30 tryptic peptides. It displays good activity both in solution and in “in gel” digestion protocols.