Quantitative, Time-Resolved Proteomic Analysis Using Bio-Orthogonal Non-Canonical Amino Acid Tagging

Bio-orthogonal non-canonical amino acid tagging (BONCAT) is an analytical method that allows the selective analysis of the subset of newly synthesized cellular proteins produced in response to a biological stimulus. In BONCAT, cells are treated with the non-canonical amino acid L-azidohomoalanine (Aha), which is utilized in protein synthesis in place of methionine by wild-type translational machinery. Nascent, Aha-labeled proteins are selectively ligated to affinity tags for enrichment and subsequently identified via mass spectrometry. The work presented in this thesis exhibits advancements in and applications of the BONCAT technology that establishes it as an effective tool for analyzing proteome dynamics with time-resolved precision.

Chapter 1 introduces the BONCAT method and serves as an outline for the thesis as a whole. I discuss motivations behind the methodological advancements in Chapter 2 and the biological applications in Chapters 2 and 3.

Chapter 2 presents methodological developments that make BONCAT a proteomic tool capable of, in addition to identifying newly synthesized proteins, accurately quantifying rates of protein synthesis. I demonstrate that this quantitative BONCAT approach can measure proteome-wide patterns of protein synthesis at time scales inaccessible to alternative techniques.

In Chapter 3, I use BONCAT to study the biological function of the small RNA regulator CyaR in Escherichia coli. I correctly identify previously known CyaR targets, and validate several new CyaR targets, expanding the functional roles of the sRNA regulator.

In Chapter 4, I use BONCAT to measure the proteomic profile of the quorum sensing bacterium Vibrio harveyi during the time-dependent transition from individual- to group-behaviors. My analysis reveals new quorum-sensing-regulated proteins with diverse functions, including transcription factors, chemotaxis proteins, transport proteins, and proteins involved in iron homeostasis.

Overall, this work describes how to use BONCAT to perform quantitative, time-resolved proteomic analysis and demonstrates that these measurements can be used to study a broad range of biological processes.

Retinal adaptation of visual processing time delays.

A significant proportion of the processing delays within the visual system are luminance dependent. Thus placing an attenuating filter over one eye causes a temporal delay between the eyes and thus an illusion of motion in depth for objects moving in the fronto-parallel plane, known as the Pulfrich effect. We have used this effect to study adaptation to such an interocular delay in two normal subjects wearing 75% attenuating neutral density filters over one eye. In two separate experimental periods both subjects showed about 60% adaptation over 9 days. Reciprocal effects were seen on removal of the filters. To isolate the site of adaptation we also measured the subjects' flicker fusion frequencies (FFFs) and contrast sensitivity functions (CSFs). Both subjects showed significant adaptation in their FFFs. An attempt to model the Pulfrich and FFF adaptation curves with a change in a single parameter in Kelly's [(1971) Journal of the Optical Society of America, 71, 537-546] retinal model was only partially successful. Although we have demonstrated adaptation in normal subjects to induced time delays in the visual system we postulate that this may at least partly represent retinal adaptation to the change in mean luminance.