Hemoglobin, peptide mapping

This work uncovered the fact that a substitution of the amino acid alanine for valine at position 126 in the /3-chain of hemoglobin occurred in a hemato-logically normal adult of Lebanese extraction. The variation /3-globin was initially observed and subsequently purified by reverse-phase liquid chromatography (see Fig. 2-5). Also LC was used to isolate the variant tryptic peptide of /3-T13 that had alanine replacing the valine at amino-acid residue position 126. This is shown in Figure 2-6. (Peptide mapping is discussed later in this chapter.)... 

Figure 5. Peptide maps of tryptic digests of normal and sickle cell hemoglobins. Adapted from Ref. 73. The single peptide difference between the two hemoglobins is indicated by shying and...

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. 

Kobold U, Jeppsson JO, Duhfer T, Finke A, Hoelzel W, Miedema K. Candidate reference methods for hemoglobin Ale based on peptide mapping. Clin Chem 1997 43 1944-51. 

Lippincott J, Apostol I. Carbamylation of cysteine a potential artifact in peptide mapping of hemoglobins in the presence of urea. Anal Bioehem. 1999 267 57-64. doi 10.1006/abio. 1998.2970. 

Open tubular capillary microreactor Trypsin Covalent linking Trypsin immobilization by photo-initiated polymerization on a portion of silica capillary digestion performance tested using carbonic anhydrase, Cyt-C, hemoglobin substrates, then subjected to peptide mapping [117]... 

Peptide mapping has also been used to characterize abnormal variants in human hemoglobin.Table 4.3 lists the amino acid sequences, calculated masses, and masses observed using PDMS for the tryptic peptides from the a-chain of normal human hemoglobin. In this case, peptides were purified by reversed-phase HPLC prior to mass-spectral analysis, and a human a-chain variant (Hb Bobbi) had been identified in which the HPLC peak corresponding to aT6 eluted earlier than expected. A comparison of the PDMS spectra shown in Figure 4.14 for the normal and variant aT6 revealed a mass difference of 22 Da, which corresponds to an Asp - His substitution, or a single substitution (G -> C) in the codon. 

Fig. 2. Five fragment masses determined from the mass spectrum of the mock unknown protein (human hemoglobin a chain) tryptic digest form the basis of peptide mapping. The mass-to-charge ratio is labeled nih on the horizontal axis.

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