Divergent role of Mitochondrial Amidoxime Reducing Component 1 (MARC1) in human and mouse.

Recent human genome-wide association studies have identified common missense variants in MARC1, p.Ala165Thr and p.Met187Lys, associated with lower hepatic fat, reduction in liver enzymes and protection from most causes of cirrhosis. Using an exome-wide association study we recapitulated earlier MARC1 p.Ala165Thr and p.Met187Lys findings in 540,000 individuals from five ancestry groups. We also discovered novel rare putative loss of function variants in MARC1 with a phenotype similar to MARC1 p.Ala165Thr/p.Met187Lys variants. In vitro studies of recombinant human MARC1 protein revealed Ala165Thr substitution causes protein instability and aberrant localization in hepatic cells, suggesting MARC1 inhibition or deletion may lead to hepatoprotection. Following this hypothesis, we generated Marc1 knockout mice and evaluated the effect of Marc1 deletion on liver phenotype. Unexpectedly, our study found that whole-body Marc1 deficiency in mouse is not protective against hepatic triglyceride accumulation, liver inflammation or fibrosis. In attempts to explain the lack of the observed phenotype, we discovered that Marc1 plays only a minor role in mouse liver while its paralogue Marc2 is the main Marc family enzyme in mice. Our findings highlight the major difference in MARC1 physiological function between human and mouse.

Maximizing hydrophobic peptide recovery in proteomics and antibody development using a mass spectrometry compatible surfactant.

Peptide loss due to surface absorption can happen at any step in a protein analysis workflow and is sometimes especially deleterious for hydrophobic peptides. In this study, we found the LC-MS compatible surfactant, n-Dodecyl-ß-D-maltoside (DDM), can maximize hydrophobic peptide recovery in various samples including single cell digests, mAb clinical PK samples, and mAb peptide mapping samples. In HeLa single cell proteomics analysis, more than half of all unique peptides identified were found only in DDM prepared samples, most of which had significantly higher hydrophobicities compared to peptides in control samples. In clinical PK studies, DDM enhanced hydrophobic complementarity-determining region (CDR) peptide signals significantly. The fold change of CDR peptides' intensity enhancement in DDM added samples compared to controls correlate with peptide retention time and hydrophobicity, providing guidance for surrogate peptide selection and peptide standard handling in PK studies. For peptide mapping analysis of mAbs, DDM can improve hydrophobic peptide signal and solution stability over 48 h in an autosampler at 4 °C, which can aid method qualification and transfer during drug development. Lastly, maximizing hydrophobic peptide recovery from samples dried in vacuo was achieved by DDM reconstitution, which provided higher signal for later eluting peaks and higher proteome coverage overall.

Challenges in urine bioanalytical assays: overcoming nonspecific binding.

Dr Allena Ji is the Director of Bioanalytical Services, XenoBiotic Laboratories, Inc., NJ, USA. She has worked in the bioanalytical field for many years and accumulated rich experience in LC-MS/MS method development, method validation and sample analysis under GLP compliance in large pharmaceutical company and contract laboratory settings. In the past 10 years, Allena worked at Pfizer (Legacy of Wyeth) and investigated many small-molecule drug candidates for their nonspecific binding in urine assays. Nonspecific binding of compounds results in a severe underestimation of the compounds' concentrations and poor precision and accuracy in urine bioanalytical assays. To overcome nonspecific binding in urine assays, Allena and her colleagues developed a series of practical approaches for urine method development. By adding an appropriate anti-adsorptive agent at its optimum concentration to the urine collection containers, the nonspecific binding can be blocked. Urine assays have much higher hurdles than plasma assays due to nonspecific binding and variability of urine pH, salt concentration, volume and solubility of drug(s) in urine. A simple and systematic approach for urine method development is emphasized in this paper. Nonspecific binding is a very serious issue in bioanalytical urine assays where a compound(s) adsorbs to the container wall. The adsorption happens frequently in urine assays because urine lacks proteins and lipids that can bind to the analytes or solubilize lipophilic analytes. Therefore, urine bioanalytical assays tend to suffer from analyte losses more often than plasma assays. In the past decade, there have been many methods described to overcome nonspecific adsorption in urine assays based on individual analyte characteristics. However, a common and simple method development approach for various analytes has not been discussed and summarized. In this article we demonstrate, discuss and summarize a common approach to urine method development with a focus on overcoming adsorption issues. The advantages and limitations of commonly used anti-adsorptive agents, such as bovine serum albumin, zwitterionic detergents such as CHAPS, sodium dodecyl benzene sulfonate, ß-cyclodextrin, Tween 80 and Tween 20 are discussed.