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Your search keyword '"Alejandro J. Brenes"' showing total 2 results
Start Over Author "Alejandro J. Brenes" Topic mass spectrometry
2 results on '"Alejandro J. Brenes"'

1. DIA label-free proteomic analysis of murine bone-marrow-derived macrophages

Author

Christa P. Baker, Iain R. Phair, Alejandro J. Brenes, Abdelmadjid Atrih, Dylan G. Ryan, Roland Bruderer, Albena T. Dinkova-Kostova, Douglas J. Lamont, J. Simon C. Arthur, and Andrew J.M. Howden

Subjects
Bioinformatics, Immunology, Protein biochemistry, Proteomics, Mass spectrometry, Science (General), Q1-390
Abstract

Summary: Here, we describe an optimized protocol to analyze murine bone-marrow-derived macrophages using label-free data-independent acquisition (DIA) proteomics. We provide a complete step-by-step protocol describing sample preparation utilizing the S-Trap approach for on-column digestion and peptide purification. We then detail mass spectrometry data acquisition and approaches for data analysis. Single-shot DIA protocols achieve comparable proteomic depth with data-dependent MS approaches without the need for fractionation. This allows for better scaling for large sample numbers with high inter-experimental reproducibility.For complete details on the use and execution of this protocol, please refer to Ryan et al. (2022). : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Published
2022
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2. Proteomic analysis of cell cycle progression in asynchronous cultures, including mitotic subphases, using PRIMMUS

Author

Tony Ly, Arlene Whigham, Rosemary Clarke, Alejandro J Brenes-Murillo, Brett Estes, Diana Madhessian, Emma Lundberg, Patricia Wadsworth, and Angus I Lamond

Subjects
Proteomics, Proteome, QH301-705.5, Science, FACS, Mass Spectrometry, Cell Line, proteomics, Biochemistry and Chemical Biology, cell biology, biochemistry, Humans, human, Biology (General), mitosis, Cell Cycle, phosphoproteomics, Cell Biology, Flow Cytometry, Immunohistochemistry, Tools and Resources, Medicine, cell cycle, Human
Abstract

The temporal regulation of protein abundance and post-translational modifications is a key feature of cell division. Recently, we analysed gene expression and protein abundance changes during interphase under minimally perturbed conditions (Ly et al., 2014, 2015). Here, we show that by using specific intracellular immunolabelling protocols, FACS separation of interphase and mitotic cells, including mitotic subphases, can be combined with proteomic analysis by mass spectrometry. Using this PRIMMUS (PRoteomic analysis of Intracellular iMMUnolabelled cell Subsets) approach, we now compare protein abundance and phosphorylation changes in interphase and mitotic fractions from asynchronously growing human cells. We identify a set of 115 phosphorylation sites increased during G2, termed 'early risers'. This set includes phosphorylation of S738 on TPX2, which we show is important for TPX2 function and mitotic progression. Further, we use PRIMMUS to provide the first a proteome-wide analysis of protein abundance remodeling between prophase, prometaphase and anaphase.

Published
2017

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