Analysis of peptides

Once the composition of the peptide is known, then the sequence is generally approached by starting at the ends. However, we should note that cyclic peptides are known, and these will show none of the characteristic reactions of either the N- or C-termini. If the peptide is treated with lithium borohydride, then the free carboxylic acid group is reduced to an alcohol. If the peptide is then hydrolyzed, the usual amino acids are obtained, except that the residue at the C-terminus now appears as an amino alcohol. There is also an enzyme, carboxypeptidase, that specifically cleaves the C-terminal amino acid, which can then be identified. However, this leaves a new C-terminus, so cleavage does continue down the chain. 

FIGURE 22.35 Sanger method for N-terminus determination in peptides. 

FIGURE 22.36 Reaction of a peptide with the Edman reagent. 

TABLE 22.2 Reagents for Selective Cleavage of Peptide Chains Reag nt Position for Cieavage 

cyanogen bromide Carboxyl side of methionine 

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Automated ammo acid analysis of peptides containing asparagine (Asn) and glutamine (Gin) residues gives a peak corresponding to ammonia Why" ... 

A typical TIC chromatogram from an analysis of peptides resulting from enzymatic digest of myoglobin. The peaks represent individual peptides eluting from an LC column and being mass measured by a spectrometer coupled to it through a dynamic-FAB inlet/ion source. 

Until 1981, mass spectrometry was limited, generally, to the analysis of volatile, relatively low-molecular-mass samples and was difficult to apply to nonvolatile peptides and proteins without first cutting them chemically into smaller volatile segments. During the past decade, the situation has changed radically with the advent of new ionization techniques and the development of tandem mass spectrometry. Now, the mass spectrometer has a well-deserved place in any laboratory interested in the analysis of peptides and proteins. 

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. 

Chapter 40 Analysis of Peptides and Proteins by Mass Spectrometry... 

Fast-atom bombardment (FAB) is an ionization technique that produces a protonated or deprotonated molecular ion, hence a molecular mass for the sample. It can be used for analysis of peptides up to m/z about 5000. 

The use of mass spectrometry for the analysis of peptides, proteins, and enzymes has been summarized. This chapter should be read in conjunction with others, including Chapter 45, An Introduction to Biotechnology, and Chapters 1 through 5, which describe specific ionization techniques in detail. 

FIGURE 10.9 Analysis of peptides on SynChopak GPC peptide. Mobile phase (a) 0.1 M potassium phosphate, pH 7 and (b) 0.1 M potassium phosphate, pH 7, 35% methanol. (From MICRA Scientific, Inc., with permission.)... 

A. J. Tomlinson and S. Naylor, Enhanced performance membrane preconcentration-capillary electiophoiesis-mass spectiometry (mPC-CE-MS) in conjunction with transient isotachophoresis for analysis of peptide mixtures, J. High Resolut. Chromatogr. 18 384-386(1995). 

In 1990, Bushey and Jorgenson developed the first automated system that eoupled HPLC with CZE (19). This orthogonal separation teehnique used differenees in hydrophobieity in the first dimension and moleeular eharge in the seeond dimension for the analysis of peptide mixtures. The LC separation employed a gradient at 20 p.L/min volumetrie flow rate, with a eolumn of 1.0 mm ID. The effluent from the ehromatographie eolumn filled a 10 p.L loop on a eomputer-eontrolled, six-port miero valve. At fixed intervals, the loop material was flushed over the anode end of the CZE eapillary, allowing eleetrokinetie injeetions to be made into the seeond dimension from the first. 

Figure 11.18 Schematic diagram of an in-line SPE unit for CE using (a) polyester wool frits to hold the sorbent, or (b) a paiticle-loaded membrane. Reprinted from Journal of Capillary Electrophoresis, 2, A. J. Tomlinson and S. Naylor, Enhanced performance membrane preconcenti ation-capillary electrophoresis-mass spectiometi y (mPC-CE-MS) in conjunction with ti ansient isotachophoresis for analysis of peptide mixtures, pp 225-233, 1995, with permission from ISC Teclmical Publications Inc.

Figure 26.4 MECHANISM Mechanism of the Edman degradation for N-terminal analysis of peptides.

Peterson, J. A., Lorenz, L. J., Risley, D. S., and Sandmann, B. J., Amino acid analysis of peptides using HPLC with evaporative light scattering detection, /. Liq. Chromatogr. Related Technol., 22, 1009, 1999. 

Opiteck, G. J. Jorgenson, J. W. Two-dimensional SEC/RPLC coupled to mass spectrometry for the analysis of peptides. Anal. Chem. 1997, 69, 2283-2291. 

Figure 12.3 M ALDI analysis of peptides from Bacillus subtilis sp. 168 vegetative cells prepared in situ.87 (a) Survey spectrum of peptide products. Protein assignments are listed in the figure, (b) Spectrum of product ions of unimolecular decomposition of the peptide with m z 2606.

Krimm, S. (2000). In Infrared Analysis of Peptides and Proteins Principles and Applications (B. R. Singh, ed.), pp. 38-53. American Chemical Society, Washington, D.C. 

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