Hemoglobin peptide, analysis

Ijeucine aminopeptidase has been applied in many ways to particular problems in structural analysis of peptides and proteins. Sequences in the amino-terminal portion of a peptide can often be established by measurement of the order of appearance of amino acids that are released during hydrolysis. The procedure has been used with a variety of proteins and peptides, including ribonuclease (Hirs et al., 1960), hemoglobin (Konigsberg and Hill, 1962, 1963 Schroeder et al., 1963), cytochrome c (Margoliash, 1962 Matsubara and Smith, 1963), and lysozyme (Canfield, 1963). 

Figure 4 Mass spectrometry analysis of difference peptides from the dehydroascorbate modified p-globin subunit. Dehydroascorbate modified deoxy recombinant hemoglobin was prepared as described in Fig. 2. Trypsin digestion was performed as described in materials and methods and the p-globin N-terminal peptide (see upper panel Fig. 3) and the difference peptide (see lower panel Fig. 3) analyzed hy LC-MS. The spectrum in the upper panel represents the P-globin N-terminal peptide and the spectrum in the lower panel represents the difference peptide. A summary of the data is presented in Table 1.

Capillary isoelectric focusing (CIEF) is a high-resolution technique for protein and peptide separation performed at academic sites and in the biotechnology and pharmaceutical industries for the analysis and characterization of, for example, recombinant antibodies and other recombinant proteins, isoforms of glycoproteins, point mutations in hemoglobin, and peptide mapping. Also, hyphenation to mass spectrometry and chip-based CIEF (microfabrication) have shown promise. CIEF kits and specific recipes/application notes are available from vendors of capillary electrophoresis (CE) equipment, as are a vast amount of publications and handbooks of CE published over recent years. 

Capillary isoelectric focusing coupled to mass spectrometry has gained popularity in recent years (by analogy to two-dimensional gel electrophoresis). Additional information obtained from mass spectrometry includes not only precise molecular-weight determination but also the possiblity for peptide sequencing. Analysis of hemoglobin variants, recombinant proteins, and monoclonal antibodies have been demonstrated [1,7,8]. 

Reactive xenobiotic Intermediates may be covalently bound to amino acids, free or In proteins. For example the reactivity of epoxides have been studied In relation to formation of covalent bound products with the hemoglobin amino acids histidine, valine and to some extent also cysteine In dose monitor experiments (249,250). The hemoglobin must be hydrolyzed prior to analysis of histidine adducts while that Is not neccessary In order to characterize adducts to the N-termlnal valine (251). A few reference cysteine, histidine and valine xenobiotic adducts have been synthesized (250,252-254). The primary amino group of histidine Is occupied In the peptide bond, therefore this group must be protected In order, to favor tjeactlon at the other nucleo-... 

I 10. Blood is drawn from a child with severe anemia and the hemoglobin protein is degraded for peptide and amino acid analysis. Of the results below, which change in hemoglobin primary structure is most likely to correlate with the clinical phenotype of anemia ... 

Analysis of Blood Samples. Urinary metabolites undergo relatively rapid elimination from the body, whereas blood components offer biomarkers that have the potential to be used for verification of sulfur mustard exposure long after the exposure incident. Three different approaches have been used for blood biomarker analysis. The intact macromolecule such as protein or DNA with the sulfur mustard adducts still attached can be analyzed. To date, this approach has only been demonstrated for hemoglobin using in vitro experiments. For proteins, an alternate approach is to enzymatically digest them to produce a smaller peptide with the sulfur mustard adduct still attached. Methods of this type have been developed for both hemoglobin and albumin. A third approach has been to cleave the sulfur mustard adduct from the macromolecule and analyze in a fashion similar to that used for free metabolites found in the urine. The later two approaches have both been successfully used to verify human exposure of sulfur mustard. 

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