High-throughput screening of organic reactions in microdroplets using desorption electrospray ionization mass spectrometry (DESI-MS): hardware and software implementation.

This study describes an automated system used for high throughput screening of reaction conditions based on accelerated reactions occurring in small volumes of reagents. Reaction mixtures are prepared in array format using a fluid handling robot and spotted on a flat polytetrafluoroethylene plate at densities up to 6144 per plate. The reaction and analysis steps are performed simultaneously using desorption electrospray ionization (DESI) to release microdroplets containing the reaction mixture from the plate for reaction prior to arrival at a mass spectrometer. Analysis rates are up to 1 reaction mixture per second and data are recorded in real time using an ion trap mass spectrometer. Beacon compounds are used to triangulate position on the plate and this allows tandem mass spectrometry (MS/MS) to be performed on confirm products of interest. Custom software allows the user to control the system. It is also used to receive data from the DESI mass spectrometer to screen the spectra for compounds of interest, to perform MS/MS and to save data. This custom software also communicates with the software controlling the fluid handling robot (Biomek i7) as well as the Beckman software used to prepare reaction mixtures and also the software that controls the solvent used as the DESI spray. Data were recorded for N-alkylation, N-acylation and N-sulfonylation reactions in three 8 hour experiments on successive days to establish the ruggedness and repeatability of the system. Repeatability is high (94-97%) over this period with false negative 6% (depending on noise threshold chosen). Plates containing 384 reaction mixtures are analyzed in 7 min by moving the DESI sprayer in steps under the sprayer instead of continuously.

Multiple reaction monitoring (MRM)-profiling for biomarker discovery applied to human polycystic ovarian syndrome.

RATIONALE: We describe multiple reaction monitoring (MRM)-profiling, which provides accelerated discovery of discriminating molecular features, and its application to human polycystic ovary syndrome (PCOS) diagnosis. The discovery phase of the MRM-profiling seeks molecular features based on some prior knowledge of the chemical functional groups likely to be present in the sample. It does this through use of a limited number of pre-chosen and chemically specific neutral loss and/or precursor ion MS/MS scans. The output of the discovery phase is a set of precursor/product transitions. In the screening phase these MRM transitions are used to interrogate multiple samples (hence the name MRM-profiling). METHODS: MRM-profiling was applied to follicular fluid samples of 22 controls and 29 clinically diagnosed PCOS patients. Representative samples were delivered by flow injection to a triple quadrupole mass spectrometer set to perform a number of pre-chosen and chemically specific neutral loss and/or precursor ion MS/MS scans. The output of this discovery phase was a set of 1012 precursor/product transitions. In the screening phase each individual sample was interrogated for these MRM transitions. Principal component analysis (PCA) and receiver operating characteristic (ROC) curves were used for statistical analysis. RESULTS: To evaluate the method's performance, half the samples were used to build a classification model (testing set) and half were blinded (validation set). Twenty transitions were used for the classification of the blind samples, most of them (N = 19) showed lower abundances in the PCOS group and corresponded to phosphatidylethanolamine (PE) and phosphatidylserine (PS) lipids. Agreement of 73% with clinical diagnosis was found when classifying the 26 blind samples. CONCLUSIONS: MRM-profiling is a supervised method characterized by its simplicity, speed and the absence of chromatographic separation. It can be used to rapidly isolate discriminating molecules in healthy/disease conditions by tailored screening of signals associated with hundreds of molecules in complex samples.

TRAP: automated classification, quantification and annotation of tandemly repeated sequences.

TRAP, the Tandem Repeats Analysis Program, is a Perl program that provides a unified set of analyses for the selection, classification, quantification and automated annotation of tandemly repeated sequences. TRAP uses the results of the Tandem Repeats Finder program to perform a global analysis of the satellite content of DNA sequences, permitting researchers to easily assess the tandem repeat content for both individual sequences and whole genomes. The results can be generated in convenient formats such as HTML and comma-separated values. TRAP can also be used to automatically generate annotation data in the format of feature table and GFF files.