Selected Recent PublicationsOct 2020Ly, T., Ahmad Y., Shlien, A., Soroka, D., Mills, A., Emanuele, M.J., Stratton, M.R., Lamond A.I. A proteomic chronology of gene expression through the cell cycle in human myeloid leukemia cells. View PublicationOct 2020*Ly, T., Whigham, A., Clarke, R., Brenes-Murillo, A.J., Estes, B., Madhessian, D., Lundberg, E., Wadsworth, P., *Lamond, A.I. *Co-corresponding authors Proteomic analysis of cell cycle progression in asynchronous cultures, including mitotic subphases, using PRIMMUS View PublicationOct 2020Ly, T., Endo, A., Lamond, A.I. Proteomic analysis of the response to cell cycle arrests in human myeloid leukemia cells View Publication We are interested in developing and applying new mass-spectrometry (MS)-based approaches to identify the proteomic changes that accompany and control cell state transitions during cell growth and division in human cells. The mitotic phase of the cell cycle is characterized by large-scale and rapid changes in the spatial organization, post-translational modifications (PTMs), and abundance of proteins. These changes are important in carrying out the essential process of precisely segregating chromosomes, organelles, and other cellular compartments into two daughter cells. In human cell lines, these changes occur in a relatively short window of time relative to the total doubling time. We aim to obtain a time-lapse view of proteome changes during an unperturbed mitosis using MS-based proteomics. Recent advances in this area enable quantitative analysis of protein parameters, including abundance, localization and PTMs in a proteome-wide manner. Using FACS, we can fractionate asynchronous cells into pure cell cycle populations (Ly et al. eLife 2017). A major challenge with the analysis of mitotic subphases is that they constitute a vanishingly small proportion of asynchronous cells, with the rarest subphases (late anaphase) present at ~0.02%. To overcome this challenge, we developed new methods, in-cell digest and AMPL, to greatly increase the sensitivity of MS-based proteomics for the analysis of rare cell subpopulations, enabling proteome-characterisation with 2,000 human lymphoblastoid cells (Fig. 1A). We first focus on the analysis of protein abundance changes during an unperturbed mitotic cell cycle using FACS to separate asynchronous cells into 16 cell cycle populations, 8 interphase and 8 mitotic (Fig. 1B). The set of 16 populations represents a pseudotimecourse, representing 16 cell cycle states ordered by progression through the cell cycle. We have used this approach to identify 119 periodic proteins, including known and novel cell cycle regulated proteins (Fig. 1C). We further showed that these proteins can be used as markers for cell cycle states, including the characterization of unknown cellular states (Fig. 1D). In future, these approaches will be used to characterise the biochemical state changes that occur during key transition from quiescence (G0) to proliferation (G1) in immune cell types. The significant gains in sensitivity afforded by the in-cell digest and AMPL have surprisingly enabled analysis of proteins from very low cell numbers, including single cells. We are currently working towards improving the sensitivity further with an aim of making advances in single cell protein analysis by MS.
