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Primary structure peptide mapping

Application of the analytical techniques discussed thus far focuses upon detection of proteinaceous impurities. A variety of additional tests are undertaken that focus upon the active substance itself. These tests aim to confirm that the presumed active substance observed by electrophoresis, HPLC, etc. is indeed the active substance, and that its primary sequence (and, to a lesser extent, higher orders of structure) conform to licensed product specification. Tests performed to verify the product identity include amino acid analysis, peptide mapping, N-terminal sequencing and spectrophotometric analyses. [Pg.185]

Figure 10.6 Peptide mapping of PIXY321. (A) Cleavage of the primary sequence with Lys-C endoprotease results in 14 theoretical fragments. (B) As detailed in the text, LC-MS analysis of the Lys-C digest shows that a few peptides, notably LI, L7, and L14, elute at several retention times due to heterogeneity of both their glycan structures and amino acid sequence. The insets illustrate the complexity of the mass spectra of glycopeptide LI eluting under peaks 17 (inset C) and 18 (inset D). Figure 10.6 Peptide mapping of PIXY321. (A) Cleavage of the primary sequence with Lys-C endoprotease results in 14 theoretical fragments. (B) As detailed in the text, LC-MS analysis of the Lys-C digest shows that a few peptides, notably LI, L7, and L14, elute at several retention times due to heterogeneity of both their glycan structures and amino acid sequence. The insets illustrate the complexity of the mass spectra of glycopeptide LI eluting under peaks 17 (inset C) and 18 (inset D).
Peptides extracted from casein with N, N-dimethyl formamide have complex electrophoretic patterns identical to those of the fraction first prepared by Long and co-workers and called X-casein (El-Negoumy 1973). These peptides are identical electrophoretically to those released by the action of plasmin, which is present in fresh raw milk, upon asr casein (Aimutis and Eigel 1982). Two of these peptides have tryptic peptide maps and molecular weights identical to those of a pair of the peptides produced by plasmin degradation of asl-casein. These peptides appear to be fragments of a8l-casein which are present in milk as the result of plasmin proteolysis. More definitive information on their primary structure is needed before nomenclature for these fragments can be established. [Pg.85]

A unique synthetic molecule that can be used as a carrier is the so-called multiple antigenic peptide (MAP) (Tam, 1988 Bosnetteta/., 1988). The MAP core structure is composed of a scaffolding of sequential levels of poly-L-lysine. The matrix is constructed from a divalent lysine compound to which two additional levels of lysine are attached. The final MAP compound consists of a symmetrical, octavalent primary... [Pg.448]

Once the structural features of a reference standard of the desired protein have been well characterized, lot-to-lot confirmation of identity can be conducted using a carefully selected group of tests, wherein the lot undergoing analysis is compared to the reference standard. Tests commonly employed for this purpose are listed in Table III. Peptide mapping is perhaps the most powerful and universally used technique since it provides relatively specific confirmation of correct primary sequence and, when non-reducing conditions are employed, can be used to confirm correct disulfide bond formation. Tertiary structure is difficult to address directly on a routine (lot-to-lot) basis, and the presence of correct biological activity is often used as evidence that the correct tertiary structure is maintained. [Pg.115]

Matrix-assisted laser desorption-ionization ionizes molecules with molecular masses of 100-1,000,000 Da for analysis by MS and provides high sensitivity, high throughput, and simplicity of operation. MALDI combined with enzymatic reactions and protein chemistry can provide very useful information on molecular masses, peptide maps, and primary structure.15 MALDI-TOF MS can quickly and accurately determine unfractionated mixtures at concentrations below 100 fmol per liter. The obtained data are then calibrated with internal standards, monoisotopic masses are assigned for all prominent peaks, and the peptide list thus generated is used to identify the protein by using a suitable database.16... [Pg.698]

Primary structure Amino acid sequencing, N-terminal Edman sequencing, peptide mapping... [Pg.49]

Mapping of the primary structure of CHO IL-4 was carried out by tryptic hydrolysis followed by measurement of the resulting peptide fragments by on-line HPLC/ESI-MS. Since the primary sequence of IL-4 was known, this MS mapping approach could confirm the cDNA-derived protein sequence,... [Pg.863]

Proteins are amino acid chains that fold into unique three-dimensional structures. The primary structure of proteins is the peptide sequence of the whole molecule, which can be characterized by the amino acid composition, N-terminal amino acid sequence, C-terminal amino acid sequence, and peptide mapping. [Pg.396]

Peptide Mapping. Peptide mapping is an important tool for protein identif-ication, primary structure determination, the detection of posttranslational modifications, the identification of genetic variants, and the determination of glycosylation and/or disulfide sites. For these reasons, peptide mapping is widely used for quality control and for the characterization of recombinant DNA-derived products. Moreover, the high resolution of CE makes it a powerful peptide mapping technique. [Pg.484]

The primary structure was assessed by peptide mapping and N- and C-terminal sequencing. N-terminal sequence analysis showed that a single sequence was detected, MKAIFVLNAA, which corresponds exactly to the first 10 amino acids at the N terminus of P40 as predicted from the DNA sequence. Reverse phase HPLC analysis of a digestion of P40 with a lysine-specific endopeptidase, i.e. endoproteinase Lys-C, was used for identification and primary structure confirmation (Fig. 9). Endoproteinase Lys-C hydolyzes peptide bonds at the carboxylic side of lysine residues. The seventeen peaks resolved were characterized by mass spectrometry, allowing the confirmation of 99 % of the primary sequence. [Pg.263]


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Peptide mapping

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