Validation of Identifications

In PME, false identifications occur if a protein matches by chance some peptide masses detected in a spectrum. These peptide masses might stem from other 

SWISS-PROT Name Median Score Status Eliminatins Criterion 

Matches with matrix cluster and impurity peaks 

All these criteria are rules of thumb and the user has to check them using visualisation tools. The first one serves to eliminate identifications that are poorly localised and therefore do not look like a spot. The second tries to eliminate matches with matrix cluster [187] and impurity peaks. For the third criterion it is assumed that every peptide mass belongs to one matching protein. If a protein matches with a lower score and reuses two or more peptide masses of proteins that matched with a significantly higher score, these masses are discarded. If these masses are indispensable for the match of the former protein, that protein will be eliminated from the list. A detailed discussion of these criteria will be given in Miiller et. al. [188]. 

An important point in criteria 1) and 2) is that they take account of spatial correlation and distribution of the data. This allows an improvement in the results to a much greater extent than if only localised information was available. We beheve that this is one of the strongest features of the molecular scanner. 

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An investigation of specificity should be conducted during the validation of identification test, the determination of impurities, and the assay. The procedures used to demonstrate specificity will depend on the intended objective of the analytical procedure. It is not always possible to demonstrate that an analytical procedure is specific for a particular analyte (complete discrimina-... 

HPLC coupled with a mass detector has been shown to be suitable for determining minor differences in molecular masses, and thus can satisfactorily replace the UV-Visible detection method. In this destructive detection system, ideal separation (resolution below 1.5) is not required for completing the validation of identification and quantification procedures. However, the HPLC-mass-mass technique is not cost-effective and is more suitable for research centers. However, at present, the HPLC-UV technique is easily affordable in both developed and developing countries. 

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