The quantitative proteome of a human cell line

Beck, Martin; Schmidt, Alexander; Malmstroem, Johan; Claassen, Manfred; Ori, Alessandro; Szymborska, Anna; Herzog, Franz; Rinner, Oliver; Ellenberg, Jan; Aebersold, Ruedi

doi:10.1038/msb.2011.82

Cited by 736 publications

(772 citation statements)

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“…We constructed yeast strains carrying endogenously TAP‐tagged versions of Elp1 and Elp6 and purified Elongator using previously established purification protocols 20. Consistent with sub‐stoichiometric cellular amounts of Elp456 in vivo 28, 36, 37, purifications of Elp1‐TAP resulted in an excess of Elp123 sub‐complex 20, whereas Elp6‐TAP purifications resulted in reduced quantities but yielded highly pure, complete, and stoichiometric Elongator complex. Large‐scale preparations yielded sufficient amounts of pure Elongator complex and Elp123 sub‐complex to analyze their overall architecture and shape by EM.…”

Section: Resultsmentioning

confidence: 96%

Architecture of the yeast Elongator complex

et al. 2016

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The highly conserved eukaryotic Elongator complex performs specific chemical modifications on wobble base uridines of tRNAs, which are essential for proteome stability and homeostasis. The complex is formed by six individual subunits (Elp1‐6) that are all equally important for its tRNA modification activity. However, its overall architecture and the detailed reaction mechanism remain elusive. Here, we report the structures of the fully assembled yeast Elongator and the Elp123 sub‐complex solved by an integrative structure determination approach showing that two copies of the Elp1, Elp2, and Elp3 subunits form a two‐lobed scaffold, which binds Elp456 asymmetrically. Our topological models are consistent with previous studies on individual subunits and further validated by complementary biochemical analyses. Our study provides a structural framework on how the tRNA modification activity is carried out by Elongator.

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Section: Resultsmentioning

confidence: 96%

Architecture of the yeast Elongator complex

et al. 2016

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“…Most of the centrosomal proteins studied here are expected to be expressed at low levels, and information about their abundance remains scarce. In comprehensive proteomics studies, key regulators of centriole duplication, including Plk4, Sas‐6, and STIL, were barely detectable, if they were detected at all (Beck et al , 2011; Nagaraj et al , 2011). For example, one extensive mass spectrometry‐based analysis of U2OS cells concluded that Sas‐6 is expressed at fewer than 500 copies per cell (Beck et al , 2011).…”

Section: Discussionmentioning

confidence: 99%

“…In comprehensive proteomics studies, key regulators of centriole duplication, including Plk4, Sas‐6, and STIL, were barely detectable, if they were detected at all (Beck et al , 2011; Nagaraj et al , 2011). For example, one extensive mass spectrometry‐based analysis of U2OS cells concluded that Sas‐6 is expressed at fewer than 500 copies per cell (Beck et al , 2011). In striking contrast, an antibody‐based study, correlating immunofluorescence signal intensities with semi‐quantitative Western blotting in the same cell line, estimated Sas‐6 to be present at 185,000 copies in early S phase and 885,000 copies in late G2 phase (Keller et al , 2014).…”

Section: Discussionmentioning

confidence: 99%

“…Comprehensive studies on the proteomes of cells, tissues, or organisms have been reported (e.g. Addona et al , 2009; Nilsson et al , 2010; Beck et al , 2011; Nagaraj et al , 2011; for review, see Bensimon et al , 2012; Kulak et al , 2014; Wilhelm et al , 2014; Hein et al , 2015; Richards et al , 2015), but accurate quantitative information on proteins expressed at low levels remains scarce. As a consequence, published estimates for the abundance of specific proteins sometimes vary over several orders of magnitude.…”

Section: Introductionmentioning

confidence: 99%

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Quantitative analysis of human centrosome architecture by targeted proteomics and fluorescence imaging

et al. 2016

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Centrioles are essential for the formation of centrosomes and cilia. While numerical and/or structural centrosomes aberrations are implicated in cancer, mutations in centriolar and centrosomal proteins are genetically linked to ciliopathies, microcephaly, and dwarfism. The evolutionarily conserved mechanisms underlying centrosome biogenesis are centered on a set of key proteins, including Plk4, Sas‐6, and STIL, whose exact levels are critical to ensure accurate reproduction of centrioles during cell cycle progression. However, neither the intracellular levels of centrosomal proteins nor their stoichiometry within centrosomes is presently known. Here, we have used two complementary approaches, targeted proteomics and EGFP‐tagging of centrosomal proteins at endogenous loci, to measure protein abundance in cultured human cells and purified centrosomes. Our results provide a first assessment of the absolute and relative amounts of major components of the human centrosome. Specifically, they predict that human centriolar cartwheels comprise up to 16 stacked hubs and 1 molecule of STIL for every dimer of Sas‐6. This type of quantitative information will help guide future studies of the molecular basis of centrosome assembly and function.

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“…Recent developments in methods and instruments for mass spectrometry enable quantitative proteomics analysis of complex samples with good coverage (1)(2)(3)(4). Several techniques for quantification by mass spectrometry exist, both using isotopic labeling and label free methods (5,6).…”

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confidence: 99%

Defining, Comparing, and Improving iTRAQ Quantification in Mass Spectrometry Proteomics Data

Hultin‐Rosenberg

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Branca

et al. 2013

Molecular & Cellular Proteomics

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The purpose of this study was to generate a basis for the decision of what protein quantities are reliable and find a way for accurate and precise protein quantification. To investigate this we have used thousands of peptide measurements to estimate variance and bias for quantification by iTRAQ (isobaric tags for relative and absolute quantification) mass spectrometry in complex human samples. A549 cell lysate was mixed in the proportions 2:2:1:1:2:2:1:1, fractionated by high resolution isoelectric focusing and liquid chromatography and analyzed by three mass spectrometry platforms; LTQ Orbitrap Velos, 4800 MALDI-TOF/TOF and 6530 Q-TOF. We have investigated how variance and bias in the iTRAQ reporter ions data are affected by common experimental variables such as sample amount, sample fractionation, fragmentation energy, and instrument platform. Based on this, we have suggested a concept for experimental design and a methodology for protein quantification. By using duplicate samples in each run, each experiment is validated based on its internal experimental variation. The duplicates are used for calculating peptide weights, unique to the experiment, which is used in the protein quantification. By weighting the peptides depending on reporter ion intensity, we can decrease the relative error in quantification at the protein level and assign a total weight to each protein that reflects the protein quantitation confidence. We also demonstrate the usability of this methodology in a cancer cell line experiment as well as in a clinical data set of lung cancer tissue samples. In conclusion, we have in this study developed a methodology for improved protein quantification in shotgun proteomics and introduced a way to assess quantification for proteins with few peptides. The experimental design and developed algorithms decreased the relative protein quantification error in the analysis of complex biological samples. Recent developments in methods and instruments for mass spectrometry enable quantitative proteomics analysis of complex samples with good coverage (1-4). Several techniques for quantification by mass spectrometry exist, both using isotopic labeling and label free methods (5, 6). Quantification by isotopic labeling can be done on precursor ion level or by quantifying isobaric label fragments in fragment spectra. Isotope-coded affinity tag (7), isobaric tags for relative and absolute quantification (iTRAQ) 1 (8), and stable isotope labeling by amino acids in cell culture (SILAC) (9) are among the most commonly used labeling methods based on stable isotopes. iTRAQ allows for simultaneous relative quantification of up to eight samples within a single run. Quantification by mass spectrometry is however a challenge, and several factors contribute to the uncertainty in the quantitative estimate; differences in labeling efficiency, protein digestion, precursor mixing, ion suppression, peak detection, data preprocessing, and data analysis (10). The quality of quantitation methods can be measured in terms of precision ...

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The quantitative proteome of a human cell line

Abstract: The majority of all proteins expressed in the human osteosarcoma cell line U2OS were absolutely quantified by mass spectrometry. The quantified proteins span a concentration range of seven orders of magnitude up to 20 000 000 copies per cell.

Cited by 736 publications

References 41 publications

Architecture of the yeast Elongator complex

Architecture of the yeast Elongator complex

Quantitative analysis of human centrosome architecture by targeted proteomics and fluorescence imaging

Defining, Comparing, and Improving iTRAQ Quantification in Mass Spectrometry Proteomics Data

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