The MALDI-MES provides a rapid, sensitive and reproducible alternative approach to existing analytical techniques for the detection of enzymatic activities that does not require a chromophore or radiolabeling. An improved method is presented, by which enzymes with defined substrate specificities can be detected with a MALDI mass spectrometer in complex protein fractions. In order to demonstrate the utility of the new method, in this study we describe the use of MALDI-MES to detect proteolytic activities in a protein extract from porcine renal tissue, which contained several thousand proteins as visualized by 2D electrophoresis. The analytical procedure is based on covalent immobilization of proteins to beads. By immobilizing proteins, autolytic and proteolytic degradation is prevented and the removal of those molecules from the protein fraction is achieved, which otherwise would interfere with the mass spectrometric detection of the enzymatic reaction products. The enzymatic activity is determined by incubating the immobilized proteins with a reaction-specific probe, followed by the analysis of the reaction mixture with the MALDI-MS after defined incubation times. The presence of the target enzyme is validated by locating a signal, which fits the molecular mass of the expected reaction product in the mass spectrum. To demonstrate how to detect proteolytic activities in this system, the reactions catalyzed by endopeptidase, angiotensin-converting enzyme, kallikrein, renin, and urotensin-converting enzyme were monitored. The experiments showed that the MALDI-MES method is sufficient according the quantification to investigate the effects of inhibitors. This is demonstrated using a specific renin inhibitor to inhibit an angiotensin-I generating enzyme activity in a renal protein extract.
In this study we describe the transfer of a new and fully automated workflow for the cost effective drug screening of large populations based on the dried blood spot (DBS) technology. The method was installed at a routine poison control center and applied for DBS and dried urine spot (DUS) samples. A fast method focusing on the high interest drugs and an extended screening method were developed on the automated platform. The dried cards were integrated into the automated workflow, in which the cards were checked in a camera recognition system, spiked with deuterated standards via an in-built spraying module and directly extracted. The extract was transferred online to an analytical LC column and then to the electrospray ionization tandem mass spectrometry system. The target compounds were analyzed in positive multiple reaction monitoring mode. Before each sample batch or analysis day, calibration samples were measured to balance inter day variations and to avoid false negative samples. An internal standard was integrated prior the sample extraction to allow in process control. 28 target compounds were analyzed and directly extracted within 5 minutes per sample. This fast screening method was then extended to 20 minutes, enabling the usage of a Forensic Toxicology Database to screen over 1200 drugs. The method gives confident positive/negative results for all tested drugs at their individual cutoff concentration. Good precision (+/-15 %, respectively +/-20 % at LOQ) and correlation within the calibration range from 5 to 1000 ng/ml was obtained. The method was finally applied to real cases from the lab and cross checked with the existing methodologies.
Abstract:A new and fully automated newborn screening method for mass spectrometry was introduced in this paper. Pathological relevant amino acids, acylcarnitines, and certain steroids are detected within 4 min per sample. Each sample is treated in an automated and standardized workflow, where a mixture of deuterated internal standards is sprayed onto the sample before extraction. All compounds showed good linearity, and intra-and inter-day variation lies within the acceptance criteria (except for aspartic acid). The described workflow decreases analysis cost and labor while improving the sample traceability towards good laboratory practice.
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