Higher-energy C-trap dissociation for peptide modification analysis

Olsen, Jesper V.; Maček, Boris; Lange, Oliver; Makarov, Alexander; Horning, Stevan; Mann, Matthias

doi:10.1038/nmeth1060

Cited by 890 publications

(803 citation statements)

References 15 publications

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“…Using a standard LTQ Orbitrap XL mass spectrometer [5,9] as a test-bed, higher power bake-out heaters were employed to raise bake-out temperature from the standard 110°C to 180°C. This allowed pressure in the Orbitrap compartment to be reduced below 10 Ϫ10 mbar level following just a 12-h bake-out after venting and ultimately down to 5 ϫ10 Ϫ11 mbar.…”

Section: Methodsmentioning

confidence: 99%

Dynamics of ions of intact proteins in the Orbitrap mass analyzer

Makarov

Denisov

2009

J. Am. Soc. Mass Spectrom.

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175

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While allowing analysis of intact proteins without a theoretical upper mass limit, the Orbitrap mass analyzer demonstrates reduced resolving power as ion mass increases even at a constant mass-to-charge ratio. It is shown that this effect comes from the effects of ion scattering on background gas molecules. The main mechanisms causing decay of acquired transient appear to be fragmentation as well as accelerated dephasing of ion packets. Isotopic resolution of proteins including bovine serum albumin (MW 66.4 kDa) and transferrin (MW 78 kDa) has also been demonstrated. As a part of this study, detection of individual multiply-charged ions of myoglobin (MW 16.9 kDa) has been demonstrated. Quantized distribution of signal intensities for ϩ20 myoglobin ions well above the noise threshold was observed, with high mass accuracy and resolution of recorded individual ions used as an independent confirmation of correct assignment of signal to ions rather than to noise. The latter also allowed us to benchmark the sensitivity of image-current detection and explore in detail factors responsible for signal decay. (J Am Soc

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Section: Methodsmentioning

confidence: 99%

Dynamics of ions of intact proteins in the Orbitrap mass analyzer

Makarov

Denisov

2009

J. Am. Soc. Mass Spectrom.

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175

231

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“…Furthermore, TiO 2 is extremely tolerant toward most buffers and salts used in biochemistry and cell biology laboratories [73]. This has resulted in a robust method for enrichment of phosphopeptides, which has already become a highly popular method for in large-scale phosphoproteomic studies [10,[77][78][79][80][81][82][83][84][85][86][87][88][89][90].…”

Section: Titanium Dioxide Chromatographymentioning

confidence: 99%

Analytical strategies for phosphoproteomics

2009

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Protein phosphorylation is a key regulator of cellular signaling pathways. It is involved in most cellular events in which the complex interplay between protein kinases and protein phosphatases strictly controls biological processes such as proliferation, differentiation, and apoptosis. Defective or altered signaling pathways often result in abnormalities leading to various diseases, emphasizing the importance of understanding protein phosphorylation. Phosphorylation is a transient modification, and phosphoproteins are often very low abundant. Consequently, phosphoproteome analysis requires highly sensitive and specific strategies. Today, most phosphoproteomic studies are conducted by mass spectrometric strategies in combination with phosphospecific enrichment methods. This review presents an overview of different analytical strategies for the characterization of phosphoproteins. Emphasis will be on the affinity methods utilized specifically for phosphoprotein and phosphopeptide enrichment prior to MS analysis, and on recent applications of these methods in cell biological applications.

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“…It is clear that the ion trap is faster for MS 2 data acquisition but can lead to many recorded spectra without confident peptide identifications. In a technique called ''higher energy dissociation,'' ions are fragmented in the intermediate ''C-trap'' between linear ion trap and orbitrap (44). This dissociation mode does not have a low mass cut-off and fragments are analyzed at high resolution in the orbitrap.…”

Section: Winds Of Change: High-resolution Tandem Mass Spectrometrymentioning

confidence: 99%

Precision proteomics: The case for high resolution and high mass accuracy

Mann

Kelleher

2008

Proc. Natl. Acad. Sci. U.S.A.

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Proteomics has progressed radically in the last 5 years and is now on par with most genomic technologies in throughput and comprehensiveness. Analyzing peptide mixtures by liquid chromatography coupled to high-resolution mass spectrometry (LC-MS) has emerged as the main technology for in-depth proteome analysis whereas two-dimensional gel electrophoresis, low-resolution MALDI, and protein arrays are playing niche roles. MS-based proteomics is rapidly becoming quantitative through both label-free and stable isotope labeling technologies. The latest generation of mass spectrometers combines extremely high resolving power, mass accuracy, and very high sequencing speed in routine proteomic applications. Peptide fragmentation is mostly performed in lowresolution but very sensitive and fast linear ion traps. However, alternative fragmentation methods and high-resolution fragment analysis are becoming much more practical. Recent advances in computational proteomics are removing the data analysis bottleneck. Thus, in a few specialized laboratories, ''precision proteomics'' can now identify and quantify almost all fragmented peptide peaks. Huge challenges and opportunities remain in technology development for proteomics; thus, this is not ''the beginning of the end'' but surely ''the end of the beginning.''

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Higher-energy C-trap dissociation for peptide modification analysis

Cited by 890 publications

References 15 publications

Dynamics of ions of intact proteins in the Orbitrap mass analyzer

Dynamics of ions of intact proteins in the Orbitrap mass analyzer

Analytical strategies for phosphoproteomics

Precision proteomics: The case for high resolution and high mass accuracy

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