Proton pump inhibitors (PPIs), drugs that are widely used for treatment of acid related diseases, are either substituted pyridylmethylsulfinyl benzimidazole or imidazopyridine derivatives. They are all prodrugs that inhibit the acid-secreting gastric (H(+), K(+))-ATPase by acid activation to reactive thiophiles that form disulfide bonds with one or more cysteines accessible from the exoplasmic surface of the enzyme. This unique acid-catalysis mechanism had been ascribed to the nucleophilicity of the pyridine ring. However, the data obtained here show that their conversion to the reactive cationic thiophilic sulfenic acid or sulfenamide depends mainly not on pyridine protonation but on a second protonation of the imidazole component that increases the electrophilicity of the C-2 position on the imidazole. This protonation results in reaction of the C-2 with the unprotonated fraction of the pyridine ring to form the reactive derivatives. The relevant PPI pK(a)'s were determined by UV spectroscopy of the benzimidazole or imidazopyridine sulfinylmethyl moieties at different medium pH. Synthesis of a relatively acid stable analogue, N(1)-methyl lansoprazole, (6b), allowed direct determination of both pK(a) values of this intact PPI allowing calculation of the two pK(a) values for all the PPIs. These values predict their relative acid stability and thus the rate of reaction with cysteines of the active proton pump at the pH of the secreting parietal cell. The PPI accumulates in the secretory canaliculus of the parietal cell due to pyridine protonation then binds to the pump and is activated by the second protonation on the surface of the protein to allow disulfide formation.
Mass spectrometric peptide mapping of proteins separated by two-dimensional gel electrophoresis can be routinely performed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) which has become a standard tool. Since MALDI-MS detection relies heavily on the quality of the MALDI target, development of an efficient sample preparation technique for removal of sample contaminants is necessary. To date, among the several sample preparation techniques for MALDI targets available, multistep perfusion chromatography (MSPC) using Poros R2 and Oligo R3 has been most commonly used. However, MSPC requires at least four working steps and is not efficient for high-throughput analysis and recovery of low abundance proteins. During the course of proteomic analysis of a large set of rat liver tissues and the immortalized human sebaceous gland cells (SZ95 cells), we were interested in developing an alternative to MSPC. Here, we describe a single-step perfusion chromatography (SSPC) method for MALDI target preparation, which uses a tiny column packed with a mixture of Poros R2 and Oligo R3 resins. The SSPC method significantly improves not only detection of peptides but also efficiency of sample handling, thus enabling high-throughput sample preparation for analyzing large set of samples with high resolution and reproducibility.
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