A peptide analysis

Acetylation is one of the most frequently used derivatization techniques in the mass spectral analysis of peptides. Reaction with freshly distilled acetic anhydride in methanol [27] results in acetylation of a-amino functionalities within one minute. Acetylation at other less reactive sites may be accelerated by the addition of a small amount of base, such as NaHC03 or triethylamine [28], Peptide hydroxyl and amino groups can be acety-lated by exposure to a 1 1 mixture of acetic anhydride/ pyridine for about 40 minutes. The net result of the reaction is a positive shift of 42 daltons for each acetyl group addition. The failure of an acetylation reaction in a peptide analysis may be diagnostic of a blocked N-terminus. 

Steiner, W., and Niederwieser, A. Peptide Analysis as Amino Alcohols... 

For gradient separations with a need to maximize the resolving power, the 10 cm X 2.1 mm ACQUITY UPLC column is used. A prime example of a separation that requires a high power is a peptide analysis, for example, a complex tryptic map. Such separations are typically executed over a time frame of 1-2 h. 

Fig. 2 Peptide analysis of inside-out vesicles in the SDS-polyacr lamide gel electrophoresis and result of Western-Blot analyses, a) Peptide analysis in the SDS-gel b-h) Nitrocellulose membranes with peptides of inside-out vesicles after reaction of the antisera to bi) 33 kDa peptide (OEEi) b2) 33 kDa peptide obtained by CaCh-washing of the reaction with the homologous antiserum c) LllCP-complex d) Dpcore peptide (the antiserum labels the 32 kDa Dppeptide and a 66 kDa aggregate formed with a 17 kDa fragment of this peptide) e) D2-core peptide f) Monogalactolipid g) Sulfolipid h) Violaxanthin...

Modern methods of amino-acid and peptide analysis, have enabled the complete amino-acid sequence of a number of proteins to be worked out. The grosser structure can be determined by X-ray diffraction procedures. Proteins have molecular weights ranging from about 6 000 000 to 5 000 (although the dividing line between a protein and a peptide is ill defined). Edible proteins can be produced from petroleum and nutrients under fermentation. 

Several chemical methods have been devised for identifying the N terminal ammo acid They all take advantage of the fact that the N terminal ammo group is free and can act as a nucleophile The a ammo groups of all the other ammo acids are part of amide linkages are not free and are much less nucleophilic Sanger s method for N terminal residue analysis involves treating a peptide with 1 fluoro 2 4 dimtrobenzene which is very reactive toward nucleophilic aromatic substitution (Chapter 23)... 

A major advance was devised by Pehr Edman (University of Lund Sweden) that has become the standard method for N terminal residue analysis The Edman degrada tion IS based on the chemistry shown m Figure 27 12 A peptide reacts with phenyl iso thiocyanate to give a phenylthwcarbamoyl (PTC) denvative as shown m the first step This PTC derivative is then treated with an acid m an anhydrous medium (Edman used mtromethane saturated with hydrogen chloride) to cleave the amide bond between the N terminal ammo acid and the remainder of the peptide No other peptide bonds are cleaved m this step as amide bond hydrolysis requires water When the PTC derivative IS treated with acid m an anhydrous medium the sulfur atom of the C=S unit acts as... 

This reaction forms the basis of one method of terminal residue analysis A peptide is treated with excess hydrazine in order to cleave all the peptide linkages One of the terminal amino acids is cleaved as the free amino acid and identified all the other ammo acid residues are converted to acyl hydrazides Which amino acid is identified by hydrazmolysis the N terminus or the C terminus ... 

The techniques described thus far cope well with samples up to 10 kDa. Molecular mass determinations on peptides can be used to identify modifications occurring after the protein has been assembled according to its DNA code (post-translation), to map a protein structure, or simply to confirm the composition of a peptide. For samples with molecular masses in excess of 10 kDa, the sensitivity of FAB is quite low, and such analyses are far from routine. Two new developments have extended the scope of mass spectrometry even further to the analysis of peptides and proteins of high mass. 

The specification development process is a data-driven activity that requires a validated analytical method. The levels of data needed include assay precision, replicate process results (process precision), and real-time stability profiles. A statistical analysis of these data is critical in setting a realistic specification. Most often, aggregation and fragmentation degradation mechanisms are common to protein and peptide therapeutics. Therefore, the SE-HPLC method provides a critical quality parameter that would need to be controlled by a specification limit. 

FIGURE 5.19 N-Tertninal analysis using Edman s reagent, phenylisothiocyanate. Phenylisothiocyanate combines with the N-terminus of a peptide under mildly alkaline conditions to form a phenylthiocarbamoyl substitution. Upon treatment with TFA (trifluo-roacetic acid), this cyclizes to release the N-terminal amino acid residue as a thiazolinone derivative, but the other peptide bonds are not hydrolyzed. Organic extraction and treatment with aqueous acid yield the N-terminal amino acid as a phenylthiohydantoin (PTH) derivative. 

Because the amount of time required for a given amino acid to elute from a standard column is reproducible, the identities of the amino acids in a peptide can be determined. The amount of each amino acid in the sample is determined by measuring the intensity of the purple color resulting from its reaction with ninhydrin. Figure 26.3 shows the results of amino acid analysis of a standard equimolar mixture of 17 a-amino acids. Typically, amino acid analysis requires about 100 picomoles (2-3 /xg) of sample for a protein containing about 200 residues. 

To determine the structure of a peptide or protein, the identity and amount of each amino acid present is first found by amino acid analysis. The peptide is... 

Figure 4.27. Arrhenius analysis The right-hand panel shows the assay-vs.-time data for an aqueous solution of a peptide. The regression lines are for storage temperatures of 80°, 73°, 60°, 50°, 40°, and 30°C. The left-hand panel gives the ln(-slope)-vs.-l/T Arrhenius plot.

DEAE 25W were operated in tandem to map the peptides generated on tryptic hydrolysis of very large proteins, such as albumin genetic variants.197 A useful review that covers many aspects of peptide analysis is available.198... 

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