Maxine Jonas

Mass Spectrometry in High Throughput Screening: A Case Study on Acetyl-Coenzyme A Carboxylase using RapidFire® – Mass Spectrometry (RF-MS)

In this review various technologies and approaches for the utilization of mass spectrometry in high-throughput analyses are discussed. The use of quadrupole-based mass spectrometry in the screening of chemical libraries against enzymatic targets for the identification of inhibitors and/or activators is highlighted. The RapidFire mass spectrometry system, an integrated on-line solid-phase extraction system interfaced to a triple-quadrupole mass spectrometer is described in detail, and the identification of a series of inhibitors of the acetyl-coenzyme A carboxylase (ACC) assay is described.

Cell Mechanics at Multiple Scales

The response of cells to mechanical stresses is a field of growing inquiry. It is well known that both the morphologic and molecular expression of cells depend, in part, on the local mechanical environment, especially for cells such as endothelial cells that experience shear stress, stretch, and pressures. To systematically study the large variety of responses of cells to physical forces (e.g., signaling, adhesion, or stiffness changes), a number of techniques have been developed and used. Here we present methods for three types of cell mechanical studies, from the multicellular to the subcellular scales, and describe the basic principle and main use of each technique along with some design and setup considerations.

Fluorescence laser tracking microrheology

Microrheology allows for the characterization of a material stress-strain relationship in sample volumes of less than a milliliter that are particularly adapted to biological studies. It enables one to elucidate the mechanical properties of the cytoskeleton of the cell, which are crucial for understanding biological responses such as cell migration and structural dynamics. To gain insight in this field, we choose to monitor the frequency-dependent complex shear modulus G*(/spl omega/) by a nondestructive method that does not incorporate any foreign body into the cell. This method of laser tracking extracts information from the Brownian motion of individual spherical particles embedded in the viscoelastic cytoskeletal mesh. We will use a differential detection system in a novel laser device to ascertain the trajectory of the particle with subnanometer and near-microsecond resolution, information that can be related to G*(/spl omega/). This technique provides a way to quantify the viscoelastic behavior of the cell with wide bandwidth (five decades of frequencies). Additionally, incorporating fluorescence in this assay will allow us to discriminate organelles or cellular locations of interest.