Sonja Hess

Publications (83) View all

• Article: Evaluation and Optimization of Mass Spectrometric Settings during Data-Dependent Acquisition Mode: Focus on LTQ-Orbitrap Mass Analyzers.

  Anastasia Kalli, Geoffrey T Smith, Michael J Sweredoski, Sonja Hess
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  ABSTRACT: Mass-spectrometry-based proteomics has evolved as the preferred method for the analysis of complex proteomes. Undoubtedly recent advances in mass spectrometry instrumentation have greatly enhanced proteomic analysis. A popular instrument platform in proteomics research is the LTQ-Orbitrap mass analyzer. In this tutorial we discuss the significance of evaluating and optimizing mass spectrometric settings on the LTQ-Orbitrap during CID data-dependent acquisition (DDA) mode to improve protein and peptide identification rates. We focus on those MS and MS/MS parameters, which have been systematically examined and evaluated by several researchers and are commonly used during DDA. More specifically we discuss the effect of mass resolving power, preview mode for FTMS scan, monoisotopic precursor selection, signal threshold for triggering MS/MS events, number of microscans per MS/MS scan, number of MS/MS events, automatic gain control target value (ion population) for MS and MS/MS, maximum ion injection time for MS/MS, rapid and normal scan rate and prediction of ion injection time. We, furthermore, present data from the latest generation LTQ-Orbitrap system, the Orbitrap Elite, along with recommended MS and MS/MS parameters. The Orbitrap Elite outperforms the Orbitrap Classic in terms of scan speed, sensitivity, dynamic range, resolving power and resulting in higher identification rates. Several of the optimized MS parameters determined on the LTQ-Orbitrap Classic and XL were easily transferable to the Orbitrap Elite, whereas others needed to be reevaluated. Finally, the Q Exactive and HCD are briefly discussed, as well as, sample preparation, LC-optimization and bioinformatics analysis. We hope this tutorial will serve as guidance for researchers new to the field of proteomics and assist in achieving optimal results.
  Journal of Proteome Research 05/2013; · 5.11 Impact Factor
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  Available from: Sonja Hess

  Article: Mechanism of an ATP-independent protein disaggregase. I. Structure of a membrane protein aggregate reveals a mechanism of recognition by its chaperone.

  Thang X Nguyen, Peera Jaru-Ampornpan, Vinh Q Lam, Peigen Cao, Samantha Piszkiewicz, Sonja Hess, Shu-Ou Shan
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  ABSTRACT: Protein aggregation is detrimental to the maintenance of proper protein homeostasis in all cells. To overcome this problem, cells have evolved a network of molecular chaperones to prevent protein aggregation and even reverse existing protein aggregates. The most extensively studied disaggregase systems are ATP-driven macromolecular machines. Recently, we reported an alternative disaggregase system, in which the 38-kDa subunit of chloroplast Signal Recognition Particle (cpSRP43) efficiently reverses the aggregation of its substrates, the light-harvesting chlorophyll a/b-binding (LHC) proteins, in the absence of external energy input. To understand the molecular mechanism of this novel activity, here we used biophysical and biochemical methods to characterize the structure and nature of LHC protein aggregates. We show that LHC proteins form micellar, disc-shaped aggregates that are kinetically stable and detergent-resistant. Despite the non-amyloidal nature, the LHC aggregates have a defined global organization, displaying the chaperone recognition motif on its solvent-accessible surface. These findings suggest an attractive mechanism for recognition of the LHC aggregate by cpSRP43 and provide important constraints to define the capability of this chaperone.
  Journal of Biological Chemistry 03/2013; · 4.77 Impact Factor
• Article: Data Dependent Middle-Down nano-Liquid Chromatography-Electron Capture Dissociation Tandem Mass Spectrometry: An Application for the Analysis of Unfractionated Histones.

  Anastasia Kalli, Michael J Sweredoski, Sonja Hess
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  ABSTRACT: Middle-down mass spectrometry combined with electron capture dissociation (ECD) represents an attractive method for characterization of proteins and their posttranslational modifications (PTMs). Coupling on-line chromatographic separation with tandem mass spectrometry enables for a high throughput analysis, while improving sensitivity of the electrosprayed peptides and reducing sample amount requirements. However, middle-down ECD has not been so far coupled with on-line chromatographic separation. In this work we examine the feasibility of coupling middle-down ECD with on-line nanoflow-liqiud chromatography (nano-LC) for the analysis of large, > 3kDa, and highly modified polypeptides in a data-dependent acquisition mode. We evaluate the effectiveness of the method by analyzing peptides derived from Asp-N and Glu-C digestions of unfractionated histones from calf thymus and acid-extracted histones from HeLa, MCF-7 and Jurkat cells. Our results demonstrate that middle-down ECD is compatible with online chromatographic separation providing high peptide and protein sequence coverage while allowing precise mapping of PTM sites. The high mass accuracy, obtained by the ICR mass analyzer, for both the precursor and product ions greatly increases confidence in peptide identification, particularly for modified peptides. Overall, for all samples examined, several histone variants were identified and modification sites were successfully localized, including single, multiple and positional isomeric PTM sites. The vast majority of the identified peptides were in the mass range of 3 to 9 kDa. The data presented here highlight the feasibility and utility of nano-LC-ECD MS/MS for a high-throughput middle-down analysis.
  Analytical Chemistry 02/2013; · 5.86 Impact Factor
• Article: Cand1 Promotes Assembly of New SCF Complexes through Dynamic Exchange of F Box Proteins.

  Nathan W Pierce, J Eugene Lee, Xing Liu, Michael J Sweredoski, Robert L J Graham, Elizabeth A Larimore, Michael Rome, Ning Zheng, Bruce E Clurman, Sonja Hess, Shu-Ou Shan, Raymond J Deshaies
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  ABSTRACT: The modular SCF (Skp1, cullin, and F box) ubiquitin ligases feature a large family of F box protein substrate receptors that enable recognition of diverse targets. However, how the repertoire of SCF complexes is sustained remains unclear. Real-time measurements of formation and disassembly indicate that SCFFbxw7 is extraordinarily stable, but, in the Nedd8-deconjugated state, the cullin-binding protein Cand1 augments its dissociation by one-million-fold. Binding and ubiquitylation assays show that Cand1 is a protein exchange factor that accelerates the rate at which Cul1-Rbx1 equilibrates with multiple F box protein-Skp1 modules. Depletion of Cand1 from cells impedes recruitment of new F box proteins to pre-existing Cul1 and profoundly alters the cellular landscape of SCF complexes. We suggest that catalyzed protein exchange may be a general feature of dynamic macromolecular machines and propose a hypothesis for how substrates, Nedd8, and Cand1 collaborate to regulate the cellular repertoire of SCF complexes.
  Cell 02/2013; · 32.40 Impact Factor
• Article: Electron capture dissociation of hydrogen-deficient Peptide radical cations.

  Anastasia Kalli, Sonja Hess
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  ABSTRACT: Hydrogen-deficient peptide radical cations exhibit fascinating gas phase chemistry, which is governed by radical driven dissociation and, in many cases, by a combination of radical and charge driven fragmentation. Here we examine electron capture dissociation (ECD) of doubly, [M + H](2+•), and triply, [M + 2H](3+•), charged hydrogen-deficient species, aiming to investigate the effect of a hydrogen-deficient radical site on the ECD outcome and characterize the dissociation pathways of hydrogen-deficient species in ECD. ECD of [M + H](2+•) and [M + 2H](3+•) precursor ions resulted in efficient electron capture by the hydrogen-deficient species. However, the intensities of c- and z-type product ions were reduced, compared with those observed for the even electron species, indicating suppression of N-C(α) backbone bond cleavages. We postulate that radical recombination occurs after the initial electron capture event leading to a stable even electron intermediate, which does not trigger N-C(α) bond dissociations. Although the intensities of c- and z-type product ions were reduced, the number of backbone bond cleavages remained largely unaffected between the ECD spectra of the even electron and hydrogen-deficient species. We hypothesize that a small ion population exist as a biradical, which can trigger N-C(α) bond cleavages. Alternatively, radical recombination and N-C(α) bond cleavages can be in competition, with radical recombination being the dominant pathway and N-C(α) cleavages occurring to a lesser degree. Formation of b- and y-type ions observed for two of the hydrogen-deficient peptides examined is also discussed.
  Journal of the American Society for Mass Spectrometry 08/2012; 23(10):1729-40. · 4.00 Impact Factor