Analyses of in vitro nonenzymatic glycation of normal and variant hemoglobins by MALDI-TOF mass spectrometry.

MALDI-TOF mass spectrometry is used here to differentiate different glycoisoforms of normal and variant hemoglobins (Hbs) in nonenzymatic in vitro glycation. Single, double, and/or multiple glycation of the α-globin, ß-globin, and/or γ-globin is observed. Different glycation rates are observed for various Hbs, and the normal Hb A has the slowest rate. Although the Hb A is relatively stable upon condensation with glucose at 37°C, the variants Hb C, Hb E, Hb F, Hb Leiden, and Hb San Diego are less stable. In addition, data reveal that the number of glucose attached/Hb molecule (state of glycation) increases with longer incubation time, higher glucose concentration, and higher temperature. The pH dependence of the state of glycation is more complex and varies for different Hbs. Although pH has little effect on the state of glycation for Hb C, Hb E, and Hb Leiden, it increases for Hb A and Hb F upon changing the pH of the solution from phosphate buffer saline (pH 7.4) to carbonate buffer (pH 10). Results obtained in this study could lead to the inference that the linkage of Hbs with glucose occurs in diabetic conditions in vivo (37°C, ∼neutral pH, ∼0.007 M glucose), and the state of glycation is more severe in the individuals who carry abnormal Hbs.

Production of antipeptide antibodies.

Peptides (8-20 residues) are as effective as proteins in raising antibodies, both polyclonal and monoclonal with a titer above 20,000 easily achievable. A successful antipeptide antibody production depends on several factors such as peptide sequence selection, peptide synthesis, peptide-carrier protein conjugation, the choice of the host animal, and antibody purification. Peptide sequence selection is likely the most difficult and critical step in the development of antipeptide antibodies. Although the format for designing peptide antigens is not precise, several guidelines can help maximize the likelihood of producing high-quality antipeptide antibodies. Typically, 5-20 mg of peptide is enough for raising an antibody, for preparing a peptide affinity column, and for antibody titer determination using an enzyme-linked immunosorbent assay (ELISA). Usually, it takes 3 months to raise a polyclonal antipeptide antibody from a rabbit that yields ~90 mL of serum which translates into approximately 8-10 mg of the specific antibody after peptide affinity purification.

Modification of the immobilized metal affinity electrophoresis using sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Modification to the original immobilized metal affinity electrophoresis (IMAEP) technique is presented. SDS-PAGE is used instead of native PAGE for improved extraction of phosphoproteins from a mixture of proteins. Protein samples treated with 2% w/v SDS instead of native sample buffer ensure that proteins are negatively charged. These negative charges on the proteins assure that the proteins migrate electrophoretically towards the anode regardless of their pI values and hence pass through the region embedded with the metal ions. Another benefit of treating proteins with SDS is that it unfolds the phosphoproteins exposing the phosphate groups to facilitate the metal-phosphate interactions. Phosphorylated ovalbumin can only be extracted after SDS sample buffer treatment. Data show that there is no detrimental effect upon SDS treatment on the extraction of phosphoproteins from a mixture of proteins. Electrophoretic migration of phosphoproteins ceases upon encounter with metal ions like Al+3, Ti+3, Fe+3, Fe+2, and Mn+2 whereas non-phosphorylated proteins migrate freely.