Peptide mapping fingerprints

Filamin 370 Filaria worms 24 Fimbriae 6. See also Pili Fingerprinting. See also Peptide mapping of DNA 259 of proteins 118, 360 First Law of Thermodynamics 282 First order reactions 457 Fischer, Edmond H. 84 Fischer, Emil H. 42, 83 Fischer projection formula 42 of monosaccharides 163 FK506 488 Flagella... 

In peptide mapping, a protein is enzimatically or chemically cleaved into small peptide fragments, and the resultant mixture is separated by HPLC to generate a peptide map, or fingerprint,... 

The capillary LC/MS-based approach for peptide mapping performed by Arnott and colleagues features miniaturized sampleloading procedures, which are routinely amenable to small quantities of peptides. The reliable characterization of protein/peptide mixtures in conjunction with the widely used 2-DGE methods offers a powerful fingerprinting approach in the pharmaceutical industry. Low femtomole detection limits (typically <50 femtomole) with a mass accuracy of +0.5Da provide unique advantages for protein identification. Liberal parameters for mass range and unmatched masses are used for the initial protein search, whereas more conservative parameters are used to reduce the number of matches and to improve the confidence in the search. 

The approaches described in the previous section enable the molecular-mass determination of intact proteins, generally with an accuracy better than 0.01%. Further stractural characterization of the protein requires determination of possible post-translational modifications (PTM) as well as the amino acid sequence. In addition, issues related to tertiary and quaternary stracture of the protein, the presence of cofactors, etc., may be relevant. LC-MS plays an important role in the primaiy and secondaiy stractural characterization of proteins, i.e., in terms of amino-acid sequencing and PTM. The procedure generally involves chemical or enzymatic treatment of the intact protein, acquisition of a peptide map or peptide mass fingerprint by either direct infusion (nano-)ESI-MS or RPLC-MS, and the amino-acid sequencing of individual peptides by means of product-ion MS-MS. Further experiments may be needed in relation to PTM, as outlined in more detail in Ch. 19. 

An important development in high-throughput protein identifieation is the introduction of protein database searching [111]. After separation on ID- or 2D-GE, the proteins were blotted onto a membrane and enzymatically digested after reduction and alkylation. The tryptic peptide mixture is analysed by MALDl-MS to achieve a peptide map or peptide mass fingerprint (PMF). The m/z information of the peptides is used to search the protein database, e.g., the Protein Identification Resource (PIR) database [112-114]. If the mass of just 4-6 tryptic peptides is accurately measured (between 0.1 and 0.01%), a useful database search can be performed. 

Peptide mass fingerprinting (PMF) of tryptic digests of both the modified and the tmmodified protein (complementary peptide mapping). By careful comparison of the two spectra, m/z shifts can be found, from which the identity of the modification may be elucidated, as well as the tryptic fragment(s) that are actrrally modified. When the amino-acid sequence of the protein is known (and vahdated), the position of the modification may be known. For example. 

Figure 7.1 The complexity of the proteins of the grape berry mesocarp of Vitis vinifera L. cv. Gamay noir. 2-DE map of proteins identified by MALDI-TOF Peptide-Mass-Fingerprint (PMF) (Reproduced from Proteomics, 2004, 4, 201-215, Sarry et al., with permission of Wiley-VCH)...

Sagliocco, F., Guillemot, J.-C., Monti hot, C., Capdevielle, J., Perrot, M., Ferran, E., Ferrara, P., and Boucherie, H. 1996. Identification of proteins of the yeast protein map using genetically manipulated strains and peptide-mass fingerprinting. Yeast 12,1519-1533. 

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