Comparison of two glutaraldehyde immobilization techniques for solid‐phase tryptic peptide mapping of human hemoglobin by capillary zone electrophoresis and mass spectrometry

Abstract

Stabilization of proteolytic enzymes, especially by immobilization, is of considerable interest because of their potential applications in medicine and the chemical and pharmaceutical industries. We report here a detailed comparison of two procedures for trypsin immobilization using the same homobifunctional agent, glutaraldehyde, for the purpose of peptide mapping. These methods include covalent coupling either to controlled pore glass (solid support) or via a cross-linking reaction (without any solid support). The immobilized trypsin preparations were characterized by the determination of immobilization efficiency, which ranged from 68 to > 95%, and measurement of apparent kinetic parameters toward a synthetic peptide-like substrate. Batch digestions of whole denaturated human normal adult hemoglobin (HbA) were performed to obtain peptide maps by capillary zone electrophoresis (CZE). Migration time reproducibility of the CZE maps was excellent, with a mean relative standard deviation of 1.5%. Moreover, the two immobilized enzyme preparations showed excellent reproducibility for repeated digestions. Matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry was also used for peptide mass mapping of denaturated HbA digested using the two immobilized trypsin preparations. Even though the two immobilized trypsin preparations do not behave identically, similar sequence coverages of 57% and 61% (for the two HbA chains merged) were achieved for the support-based and cross-linked trypsin preparations, respectively.

Advances in the analysis of proteins and peptides by capillary electrophoresis with matrix‐assisted laser desorption/ionization and electrospray‐mass spectrometry detection

“…Photopolymerized sol-gel monoliths (PSG) with pepsin encapsulated in the gel matrix enlarged the surface and facilitated the accessibility of enzymatic sites for large proteins [253]. Moreover, supportless immobilization by enzyme cross-linking through glutaraldehyde was used [251]. Enhanced enzymatic activity in combination with reaction chambers of minute volume and elevated substrate concentrations considerably reduces incubation time [252].…”

“…When executed in solution, long incubation times, single use, and rapid loss of catalytic activity are frequently encountered [251]. As an alternative, digestion can also take place in enzymatic microreactors, with the enzymes immobilized on a solid support, such as CPG or monolithic phases [251][252][253].…”

CE‐based analysis of hemoglobin and its applications in clinical analysis

“…Okafo et al [53] then separated tryptic peptides of globin chains by using phytic acid as an additive in the buffer. Migneault et al [54] separated the Hb digests by CZE using three given batches of immobilized method: (i) glutaraldehydetrypsin, (ii) glutaraldehyde-cross-linked trypsin, and (iii) free trypsin. Recently, Lin et al [55] developed a simple and rapid procedure for mapping of Hb D-Ouled Rabah, Hb Marseille, Hb G-Philadelphia, and Hb Ube-2 from globin chains of total a-and b-or the individual a-or b-chains, as shown in Fig.…”

Preparation of Chitosan Beads for Trypsin Immobilization

“…Glutaraldehyde has been used for many years for enzyme immobilization (27). It reacts with several functional protein groups, such as amine, thiol, phenol, and imidazole, since nucleophilic amino acid residues are the most reactive ones.…”