Eden P. Go

A mass spectrometry plate reader: monitoring enzyme activity and inhibition with a Desorption/Ionization on Silicon (DIOS) platform.

A surface‐based laser desorption/ionization mass spectrometry assay that makes use of Desorption/Ionization on Silicon Mass Spectrometry (DIOS‐MS) has been developed to monitor enzyme activity and enzyme inhibition. DIOS‐MS has been used to characterize inhibitors from a library and then to monitor their activity against selected enzyme targets, including proteases, glycotransferase, and acetylcholinesterase. An automated DIOS‐MS system was also used as a high‐throughput screen for the activity of novel enzymes and enzyme inhibitors. On two different commercially available instruments, a sampling rate of up to 38 inhibitors per minute was accomplished, with thousands of inhibitors being monitored. The ease of applying mass spectrometry toward developing enzyme assays and the speed of surface‐based assays such as DIOS for monitoring inhibitor effectiveness and enzyme activity makes it attractive for a broad range of screening applications.

Phosphonium labeling for increasing metabolomic coverage of neutral lipids using electrospray ionization mass spectrometry

Mass spectrometry has become an indispensable tool for the global study of metabolites (metabolomics), primarily using electrospray ionization mass spectrometry (ESI-MS). However, many important classes of molecules such as neutral lipids do not ionize well by ESI and go undetected. Chemical derivatization of metabolites can enhance ionization for increased sensitivity and metabolomic coverage. Here we describe the use of tris(2,4,6,-trimethoxyphenyl)phosphonium acetic acid (TMPP-AA) to improve liquid chromatography (LC)/ESI-MS detection of hydroxylated metabolites (i.e. lipids) from serum extracts. Cholesterol which is not normally detected from serum using ESI is observed with attomole sensitivity. This approach was applied to identify four endogenous lipids (hexadecanoyl-sn-glycerol, dihydrotachysterol, octadecanol, and alpha-tocopherol) from human serum. Overall, this approach extends the types of metabolites which can be detected using standard ESI-MS instrumentation and demonstrates the potential for targeted metabolomics analysis.

Metabolomics relative quantitation with mass spectrometry using chemical derivatization and isotope labeling

Comprehensive detection and quantitation of metabolites from a biological source constitute the major challenges of current metabolomics research. Two chemical derivatization methodologies, butylation and amination, were applied to human serum for ionization enhancement of a broad spectrum of metabolite classes, including steroids and amino acids. LC-ESI-MS analysis of the derivatized serum samples provided a significant signal elevation across the total ion chromatogram to over a 100-fold increase in ionization efficiency. It was also demonstrated that derivatization combined with isotopically labeled reagents facilitated the relative quantitation of derivatized metabolites from individual as well as pooled samples.

Monitoring EDTA and endogenous metabolite biomarkers from serum with mass spectrometry

We describe a quantitative method for the determination of ethylenediaminetetraacetic acid (EDTA) in human serum by gas chromatography mass spectrometry (GC-MS), liquid chromatography electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS), and desorption ionisation on silicon mass spectrometry (DIOS-MS). In the initial stages of the analysis, endogenous metabolites (1-palmitoyl-sn-glycero-3-phosphocholine and 1-stearoyl-sn-glycero-3-phosphocholine) were readily observed in LC-ESI-MS and DIOS-MS however, direct analysis of the EDTA free acid had limited sensitivity. In order to improve EDTA detection we employed a straightforward esterification derivatization. The most successful derivatization procedure converted EDTA to its methyl ester and, since 13C isotopes of these reagents are readily available, internal standards could be easily generated for quantitative analysis. This approach provided a limit of detection of 0.5 and 0.1 μM for GC-MS and LC-ESI-MS, and offers a viable method for the EDTA detection.