Ribo-Proteome Pool Sizes
According to Michael T. Sykes, Ph.D., a post doctoral fellow at the Scripps Institute, protein pool sizes are determined by the balance of synthesis and utilization, reflecting the dynamics of protein turnover. Dr. Sykes and his coworkers have explored quantitation of pool size measurements using stable isotope pulse- labeling and liquid chromatography-coupled mass spectrometry. “Quantitative proteomic mass spectrometry involves comparison of the amplitudes of peaks resulting from different isotope labeling patterns, including fractional atomic and residue labeling,” he explained
Quantitative analysis of proteins from ribosomes recovered at different times allows delineation of the kinetics of incorporation of 15N into each individual ribosomal protein.
As Dr. Sykes discussed, estimated pool sizes range from 2% to 40% for proteins in the 30S subunit, though most weigh in with an approximately 10% pool size. As could be anticipated, proteins with unusually large pool sizes turn out to be those with multiple known functions.
Pool sizes generally correlate with the in vitro assembly order. Using this relationship along with an estimate of the total assembly time of a single ribosome, Dr. Sykes and his coworkers were able to partition out the relative contributions of free proteins and ribosomal intermediates to the total pool.
“Our research represents a generally applicable approach to monitoring pool sizes and protein pool dynamics of the ribo-proteome in vivo.”
He described a least-squares Fourier transform convolution approach that can be applied to many types of quantitative proteomic data, including data from stable isotope labeling by amino acids in cell culture and pulse-labeling experiments.
Eric Hwang, Ph.D., assistant professor in the department of biological science and technology at the National Chiao Tung University, Taiwan, presented a quantitative analytical study of the microtubule-associated proteome during neurite formation. The studies that Dr. Hwang discussed were carried out in collaboration with Shelley Halpain, Ph.D., professor of biological sciences, UC San Diego.
“Neuritogenesis is the process that underlies establishment and plasticity of neuronal networks,” Dr. Hwang explained. The initiation of neurite outgrowth and the elaboration of axonal and dendritic processes involves a profound morphological reorganization of the microtubule cytoskeleton. This transformation is, in turn, controlled by the microtubule-associated proteins (MAPs). Dr. Hwang’s goal was to perform a comprehensive MAP proteomic analysis in order to reveal new protein participants.
As a model system for their proteomics characterization, he and his collaborators took advantage of the P19 cell line, derived from an embryonal carcinoma induced in a C3H/He strain mouse. The line has the extremely useful property that it can be induced to differentiate into neuronal and glial cells in the presence of retinoic acid. Moreover, cellular aggregates can be forced to differentiate into cardiac and skeletal muscle by dimethyl sulfoxide.
By taking advantage of the technique of spectral counting in their investigations, they identified about 800 proteins from each sample. Spectral counting quantifies relative protein concentrations in pre-digested protein mixtures analyzed by liquid chromatography online with tandem MS.
The results derived from spectral counting were compared with in-gel stable isotope labeling studies with the auspicious outcome that the two data sets showed a high degree of correlation. When pre- and post-neurite induction proteomes were quantitatively compared, the overall functional profiles appeared quite similar; however, the specific composition of the proteome changed significantly.
The most abundant classes of proteins identified are known cytoskeleton-associated proteins, nucleic acid-binding proteins, and proteins involved in phosphorylation. Protein kinase A components and specific A-kinase anchoring proteins were strongly increased in the post-neurite induction proteome, emphasizing a central role for PKA signaling. Interestingly, collapsin-response mediator proteins, spectrins, and components of the dynactin complex were also significantly increased in the neuronal microtubules.
According to Dr. Hwang, these results provided a comprehensive picture of the changes in the microtubule-associated proteome during neuritogenesis. “The most significant finding is the identification of potential new players, such as tripartite motif protein 2, that regulate key aspect of this process,” he stated.
The conference participants stressed the dynamic nature of the proteome and the role of post-translational modifications in altering cellular expression. The use of MS and other advanced technologies including stable-isotope labeling, ICAT, iTRAQ and SILAC bring protein quantification to a new level of sophistication and allows an understanding of these changes and their role in disease states.
Currently there is an ongoing development of new technologies to profile and analyze the cellular proteomes at an even more refined level. Alterations in protein levels even in the absence of protein expression changes are often linked to cellular responses and disease states. Using these new approaches investigators will provide a system-wide understanding of the protease web and the web-sculpted proteome, with important implication for the understanding of pathological events and development of innovative pharmacological agents.