Skyline: an open source document editor for creating and analyzing targeted proteomics experiments

MacLean, Brendan; Tomazela, Daniela M.; Shulman, Nicholas; Chambers, Matthew; Finney, Gregory L.; Frewen, Barbara; Kern, Randall; Tabb, David L.; Liebler, Daniel C.; MacCoss, Michael J.

doi:10.1093/bioinformatics/btq054

Cited by 4,112 publications

(3,795 citation statements)

References 9 publications

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“…The transition lists with optimized collision energies for all peptides were reported previously (Bauer et al , 2014). Ratios between heavy‐labeled AQUA peptides and light endogenous peptides were determined using the Skyline software (MacLean B. et al , Bioinformatics, 2010; version 1.3). Notably, the transition intensities of peptides found across multiple fractions were summed before ratio calculation.…”

Section: Methodsmentioning

confidence: 99%

“…After centrifugation at 20,000 g for 5 min at 4°C, peptides in the supernatant were desalted by solid‐phase extraction, purified using macro‐spin solid‐phase extraction C18 columns according to the manufacturer's instructions (The Nest Group, Southborough, MA, USA), dried at 45°C under vacuum, resuspended in an aqueous solution containing 5% ACN/0.15% formic acid, and subjected to SRM analysis in a TSQ Vantage mass spectrometer or shotgun analysis in a LTQ Velos mass spectrometer as described below (both Thermo Scientific, Waltham, MA, USA). Ratios between heavy‐labeled AQUA peptides and light endogenous peptides were determined using the Skyline software (MacLean et al , 2010). …”

Section: Methodsmentioning

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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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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

et al. 2016

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“…These meetings provide opportunities for users and developers to meet and exchange ideas while describing the newest algorithms and advances incorporated into the OpenMS project every year. The OpenMS project not only sees high activity from its direct contributors, but several other open-source tools such as MSStats [47] , aLFQ [48] and Skyline [49] have started to integrate their tools with OpenMS. While OpenMS provides expert users and developers with great flexibility, the complexity of OpenMS can present a challenge to novice users.…”

Section: Conclusion and Future Developmentsmentioning

confidence: 99%

OpenMS: a flexible open-source software platform for mass spectrometry data analysis

et al. 2016

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“…Signals were integrated using Skyline and exported for further analysis. 31 The concentration of each peptide was determined based on the signal ratio between the endogenous peptide and the spiked internal standard peptide. Subsequently, peptide concentrations were aggregated to the protein level by summing sequence variants and averaging peptides belonging to the same protein chain.…”

Section: ■ Experimental Sectionmentioning

confidence: 99%

Quantitative Measurement of Immunoglobulins and Free Light Chains Using Mass Spectrometry

et al. 2015

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Serum free light chain (sFLC) assays are well established in the diagnosis and monitoring of plasma cell disorders. However, current FLC immunoassays are subject to several analytical issues, which results in a lack of harmonized results. To facilitate sFLC standardization, we investigated the strengths and limitations of mass spectrometry as a novel technological platform for sFLC quantification. Stable isotope labeled reference peptides are added to serum samples for quantitation by selected reaction monitoring (SRM). The use of redundant peptide sets allows for quality control measures during data analysis. Measurements on serum provide information on intact immunoglobulins, but depletion of these intact molecules from the sera during sample processing permits the quantitation of sFLC. sFLC concentrations measured with SRM were comparable to those obtained by nephelometry and showed excellent linearity (r 2 > 0.99). In samples with high levels of sFLC, SRM data was more consistent with serum protein electrophoresis than nephelometric data and SRM is unaffected by antigen excess. The lower limits of quantitation were 3.8 and 2.7 mg/L for κ and λ sFLC. Errors due to polymorphic sequences were prevented by comparison of redundant peptide pairs. The application of stable isotope labeling combined with SRM can overcome many of the current potential analytical issues of sFLC analysis. We describe which hurdles still need to be taken to make SRM a robust and more accurate method for sFLC measurements.

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Skyline: an open source document editor for creating and analyzing targeted proteomics experiments

Abstract: Single-click, self-updating web installation is available at http://proteome.gs.washington.edu/software/skyline. This web site also provides access to instructional videos, a support board, an issues list and a link to the source code project.

Cited by 4,112 publications

References 9 publications

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

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

OpenMS: a flexible open-source software platform for mass spectrometry data analysis

Quantitative Measurement of Immunoglobulins and Free Light Chains Using Mass Spectrometry

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