2nd slide Peptide Mass Fingerprinting (PMF) is a technique used to identify proteins by matching their constituent fragment masses

(peptide masses) to the theoretical peptide masses generated from a protein or DNA database. The first step in PMF is that an intact, unknown protein is cleaved with a proteolytic enzyme to generate peptides. With PMF, heterogeneity is most commonly imparted to the unknown protein with a trypsin digestion. A PMF database search is usually employed following MALDI TOF mass analysis. The premise of peptide mass fingerprinting is that every unique protein will have a unique set of peptides and hence unique peptide masses. 4th slide Identification is accomplished by matching the observed peptide masses to the theoretical masses derived from a sequence database. PMF identification relies on observing a large number of peptides, 5 or more, from the same protein at high mass accuracy. This technique does well with 2D gel spots where the protein purity is high. PMF protein identification can run into difficulties with complex mixtures of proteins. Low level ID also becomes difficult due to commonplace contamination by keratin. 5th slide A mass spectrum of the peptide mixture resulting from the digestion of a protein by an enzyme provides a fingerprint of great specificity. So specific, that it is often possible to identify the protein from this information alone. This method of identification is much more reliable than using fingerprints based on PAGE migration patterns or HPLC retention times. However, peptide mass fingerprinting is limited to the identification of proteins for which sequences are already known, it is not a method of structural elucidation. 6th slide SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) is commonly used in the lab for the separation of proteins based on their molecular weight. Its one of those techniques that is commonly used but not frequently fully understood. So lets try and fix that. SDS-PAGE separates proteins according to their molecular weight, based on their differential rates of migration through a sieving matrix (a gel) under the influence of an applied electrical field. 8th slide Computer program used to convert graph data (from mass spectrum) to table data (list of peptide masses) Software programs cut all these proteins into peptides with the same enzyme used in the chemical cleavage 9th slide Figure 1. This spectrum was collected on a Voyager DE STR MALDI TOF mass spectrometer and was an average of 240 scans. Peptide peaks appear as [M+H]1+ ions. The peaks appearing at +22u are sodium adducts. 11th slide Peak list : the mass spectrometrical analysis produces a list of molecular weights