Peptide Prophet

FIGURE 14.17 Ultrahigh resolution nano LC-MS separation of base peak chromatogram of 2351 peptides identified in trypsin digest of mouse brain lysate P2 fraction using Micro-Tech XtremeSimple nano-LC and Thermo Electron LTQ. Column 100 cm x 75 fim C18 column, 3 /mi, 8000 psi column head pressure. Solvent composition time 350 min gradient, 5 to 35% B. Solvent A 2% acetonitrile, 0.1% formic acid. Solvent B 95% acetonitrile, 0.1% formic acid. Data analysis Sequest, PeptideProphet, and Protein Prophet. 

The use of immobilised cells for industrial and analytical enzymic processes is prophetically advantageous, the problems of isolation of the enzyme(s) and separation of the enzyme(s) from the product being avoided. However, the majority of the reaction currently used for direct enzyme immobilisation would cause cell death if applied to cells. Our approach has been based on the ability of water-insoluble metal hydroxides to chelate and retain peptides, proteins, etc. including enzymes. From various studies it was concluded that gelatinous titanium and zirconium hydroxide matrices are effective matrices for enzyme etc. immobilisation. Their advantages include low cost, convenient preparation (which may be conducted in any location without specialised facilities), the absence of any need for pre-preparation, ability to couple enzyme at neutral pH, the high retentions of specific activity of the enzyme on immobilisation, and the ability of modification to exert microenvironmental effects on and thereby alter the characteristics of the immobilised enzyme. 

Band" Identifier Protein Description MW (kDa) AA Coverage (%) Protein Prophet Probability Total Peptides Unique Peptides- ... 

In a description of the utility of solid phase peptide synthesis Merrifield made the prophetic observation "A gold mine awaits discovery by organic chemists". Scarely ten years later Leznoff rather ruefully noted "Many gold nuggets have now been mined... and some iron pyrites". We are currently on the crest of what appears to be an ever-increasing wave and we would have to say that the task described by Fettes is certainly impossible. 

The raw peptide data is searched against known trypsin-digested prophetic peptides with specialized protein databases and the corresponding proteins can be identified within a confidence level and controlled false discovery rate. 

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