An Overview of Advanced SILAC-Labeling Strategies for Quantitative Proteomics.

Comparative, quantitative mass spectrometry of proteins provides great insight to protein abundance and function, but some molecular characteristics related to protein dynamics are not so easily obtained. Because the metabolic incorporation of stable amino acid isotopes allows the extraction of distinct temporal and spatial aspects of protein dynamics, the SILAC methodology is uniquely suited to be adapted for advanced labeling strategies. New SILAC strategies have emerged that allow deeper foraging into the complexity of cellular proteomes. Here, we review a few advanced SILAC-labeling strategies that have been published during last the years. Among them, different subsaturating-labeling as well as dual-labeling schemes are most prominent for a range of analyses including those of neuronal proteomes, secretion, or cell-cell-induced stimulations. These recent developments suggest that much more information can be gained from proteomic analyses if the labeling strategies are specifically tailored toward the experimental design.

A caged Ab reveals an immediate/instructive effect of BDNF during hippocampal synaptic potentiation.

Neurotrophins have been shown to be involved in functional strengthening of central nervous system synapses. Although their general importance in this process is undisputed, it remains unresolved whether neurotrophins are truly mediators of synaptic strengthening or merely important cofactors. To address this question, we have devised a method to inactivate endogenous brain-derived neurotrophic factor (BDNF) with high time resolution by "caging" a function-blocking mAb against BDNF with a photosensitive protecting compound. Different assays were used to show that this inactivation of the Ab is reversible by UV light. Synaptic potentiation after theta-burst [corrected] stimulation in the CA1 region of acute hippocampal slices was significantly less when applying the unmodified Ab compared with the caged Ab. Importantly, photoactivation of the caged Ab during the time of induction of synaptic enhancement led to a marked decrease in potentiation. Our experiments therefore strengthen the view that endogenous BDNF has fast effects during induction of synaptic plasticity. The results additionally show that caged Abs can provide a tool for precise spatiotemporal control over endogenous protein levels.

Photoactivated gene expression for cell fate mapping and cell manipulation.

A long-standing goal of developmental biologists is to create developmental fate maps by tracking individual cells through development. Another objective is to perturb the behavior of selected cells and follow the ensuing effects. To this end, we have developed a technique that allows for spatial and temporal control of gene expression in single cells or patches of cells using light to induce gene expression. This technique relies on "caging" the activity of the potent transcriptional activator GAL4VP16 with a photolabile compound, which can be removed with a brief exposure to long-wavelength ultraviolet (UV) light. The caged GAL4VP16 is injected into early-stage embryos, which are aged to the desired point in development, and the cell(s) of interest are irradiated with a brief pulse of long-wavelength UV light. This method has been used extensively in Drosophila, Xenopus, and Zebrafish embryos. The methods for purifying, caging, injection, and photoactivation of the GAL4VP16 protein, and methods for the visualization of marked cells are described in detail.

Drosophila mitotic domain boundaries as cell fate boundaries.

Fate determination in Drosophila embryos is evidenced by the appearance of mitotic domains. To identify fate or fates of cells, individual cells in mitotic domains 2, 8, and 15 were marked and monitored through development. Comparison of the different fates indicated that domain boundaries are cell fate boundaries. Cells were marked by expression of GAL4-dependent transgenes after photoactivation of a caged GAL4VP16 analog that had its DNA binding activity inhibited with a photolabile blocking reagent. Caged GAL4VP16 was also used to induce gene expression in Xenopus embryos. Thus, photoactivated gene expression is a versatile tool for spatiotemporal control of gene expression.

Variation between centers in technique and guidelines for liver biopsy.

Hospitals have few published guidelines to follow when performing a liver biopsy. In 1992, we began revising our protocol in an effort to institute new guidelines for our teaching hospitals. To assess the current practice of liver biopsy, we sent 500 multilingual questionnaires to international academic centers, and 85 U.S. centers were surveyed by telephone. The survey assessed: 1) patient preparation, 2) technical aspects of the biopsy, and 3) post-procedural care. One hundred and eighty international centers and 85 U.S. centers responded (total = 265). We found a wide variation in the practice of this surgical procedure at both national and international centers. Many Asian centers (73%) performed a bleeding time prior to liver biopsy. This practice was seen in only 36% of the U.S. centers. Most centers preferred platelet counts of 50,000/mm3 and above. The aspiration needle was more widely used in the U.S. (74%) and in many international centers, but Asian centers (61%) preferred a cutting needle. Thirty percent of Japanese centers performed more than 50% of their liver biopsies laparoscopically. Few laparoscopies were done at other centers. While about a quarter of the reported U.S., European, Asian, and South American centers observed patients for 4-6 hours after a biopsy, the majority of centers observed patients 10 hours or more. In addition to the wide variation seen, this survey provided us with an academic view of the contemporary practice of liver biopsy and an insight into how to redefine our present guidelines.