Quantifying proteomes and their post-translational modifications by stable isotope label-based mass spectrometry

Merrill, Anna E.; Coon, Joshua J.

doi:10.1016/j.cbpa.2013.06.011

Cited by 22 publications

(22 citation statements)

References 55 publications

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“…2A) with or without 3-MB-PP1 challenge. Isobaric labeling with 6-plex tandem mass tags (TMTs) allowed us to run three replicates each of AS cells with and without 3-MB-PP1 in a single MS experiment (43)(44)(45)(46). The G 1 /S boundary was selected for this analysis ( Fig.…”

Section: Cep131 Is a Substrate Of Plk4mentioning

confidence: 99%

Polo-like kinase 4 maintains centriolar satellite integrity by phosphorylation of centrosomal protein 131 (CEP131)

Denu

Sass

Johnson

et al. 2019

Journal of Biological Chemistry

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Edited by Xiao-Fan WangThe centrosome, consisting of two centrioles surrounded by a dense network of proteins, is the microtubule-organizing center of animal cells. Polo-like kinase 4 (PLK4) is a Ser/Thr protein kinase and the master regulator of centriole duplication, but it may play additional roles in centrosome function. To identify additional proteins regulated by PLK4, we generated an RPE-1 human cell line with a genetically engineered "analog-sensitive" PLK4 AS , which genetically encodes chemical sensitivity to competitive inhibition via a bulky ATP analog. We used this transgenic line in an unbiased multiplex phosphoproteomic screen. Several hits were identified and validated as direct PLK4 substrates by in vitro kinase assays. Among them, we confirmed Ser-78 in centrosomal protein 131 (CEP131, also known as AZI1) as a direct substrate of PLK4. Using immunofluorescence microscopy, we observed that although PLK4-mediated phosphorylation of Ser-78 is dispensable for CEP131 localization, ciliogenesis, and centriole duplication, it is essential for maintaining the integrity of centriolar satellites. We also found that PLK4 inhibition or use of a nonphosphorylatable CEP131 variant results in dispersed centriolar satellites. Moreover, replacement of endogenous WT CEP131 with an S78D phosphomimetic variant promoted aggregation of centriolar satellites. We conclude that PLK4 phosphorylates CEP131 at Ser-78 to maintain centriolar satellite integrity.

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Section: Cep131 Is a Substrate Of Plk4mentioning

confidence: 99%

Polo-like kinase 4 maintains centriolar satellite integrity by phosphorylation of centrosomal protein 131 (CEP131)

Denu

Sass

Johnson

et al. 2019

Journal of Biological Chemistry

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“…Large-scale proteomics studies on model organisms such as yeast report complete proteome coverage with ß4000 proteins [2,3], while studies on mammalian cells report >10 000 proteins [4]. The ability to detect large numbers of PTMs has also increased [5][6][7][8][9]. Proteomic studies routinely analyse extracts derived from hundreds of thousands to millions of cells.…”

Section: Introductionmentioning

confidence: 99%

Micro‐proteomics with iterative data analysis: Proteome analysis in C. elegans at the single worm level

et al. 2016

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Proteomics studies typically analyze proteins at a population level, using extracts prepared from tens of thousands to millions of cells. The resulting measurements correspond to average values across the cell population and can mask considerable variation in protein expression and function between individual cells or organisms. Here, we report the development of micro‐proteomics for the analysis of Caenorhabditis elegans, a eukaryote composed of 959 somatic cells and ∼1500 germ cells, measuring the worm proteome at a single organism level to a depth of ∼3000 proteins. This includes detection of proteins across a wide dynamic range of expression levels (>6 orders of magnitude), including many chromatin‐associated factors involved in chromosome structure and gene regulation. We apply the micro‐proteomics workflow to measure the global proteome response to heat‐shock in individual nematodes. This shows variation between individual animals in the magnitude of proteome response following heat‐shock, including variable induction of heat‐shock proteins. The micro‐proteomics pipeline thus facilitates the investigation of stochastic variation in protein expression between individuals within an isogenic population of C. elegans. All data described in this study are available online via the Encyclopedia of Proteome Dynamics (http://www.peptracker.com/epd), an open access, searchable database resource.

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“…Both the “top down” and “bottom up” proteomics are highly dependent on separation technologies [80, 81] to: (a) ensure extensive proteome coverage in a given time; (b) achieve higher analytical throughput; and (c) cover large dynamic concentration range, including even trace proteins. In some the most remarkable separations of complex peptide mixtures, multidimensional LC [81], small-particle technologies [82] and monolithic capillaries [84] were used. The high-power MS instrumentation, such as FTMS or Orbitrap, is additionally important in achieving the most comprehensive results.…”

Section: Problem-oriented Investigations: Integration Of Modern Anmentioning

confidence: 99%

Development of capillary liquid chromatography: A personal perspective

Novotný

2017

Journal of Chromatography A

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This is a historical account on the development of capillary LC from its beginning to the present day. The first investigations into the viability of capillary LC date back to the late 1970s, a decade after the pioneering efforts in HPLC. The drastically reduced column dimensions were required to counter the slow solute diffusion in liquids. There were numerous instrumental difficulties with sample introduction and detection in the picoliter or even femtoliter volumes. High-efficiency separations were needed in the analysis of complex biological mixtures. Miniaturization brought distinct advantages in spectroscopic and electrochemical detection. Since the 1980s, column technologies underwent significant changes: (a) from glass-drawn microcapillaries to slurry-packed, small-diameter fused silica columns; and (b) in microcapillaries packed alternatively with sub-2-μm particles or monoliths. The viability of LC-MS combination has dramatically promoted the use of small-diameter capillaries. Through “omics technologies”, capillary LC/tandem MS accounts for most applications in proteomics, glycomics and metabolomics.

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Quantifying proteomes and their post-translational modifications by stable isotope label-based mass spectrometry

Cited by 22 publications

References 55 publications

Polo-like kinase 4 maintains centriolar satellite integrity by phosphorylation of centrosomal protein 131 (CEP131)

Polo-like kinase 4 maintains centriolar satellite integrity by phosphorylation of centrosomal protein 131 (CEP131)

Micro‐proteomics with iterative data analysis: Proteome analysis in C. elegans at the single worm level

Development of capillary liquid chromatography: A personal perspective

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