Analysis of Insoluble Proteins

Trimpin, Sarah; Brizzard, Bill

doi:10.2144/000113168

Cited by 34 publications

(38 citation statements)

References 87 publications

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“…Thus, the ability to produce highly charged ions using a laser ablation process is readily available for advanced spectrometers with mass range limitations such as FT-ICR-MS and, as shown here, IMS-MS instruments. Solvent-free decongestion of protein mixtures using IMS-MS technology is especially promising for tissue imaging applications [1,3,14,15]. Additionally, we are evaluating methods that lower the thermal requirement for desolvation and charge production.…”

Section: Resultsmentioning

confidence: 99%

“…This is possible because ions are separated in the IMS dimension according to charge and cross-section (size and shape) [8][9][10][11]. One key benefit of this solvent-free gas-phase separation by IMS is that when combined with solvent-free sample preparation [12] achieves total solvent-free analysis by MS entirely decoupling ionization, separation, and mass analyses from the use of any solvent [13][14][15].We rationalized that to produce LSI multiply charged ions from the proposed highly charged matrix/analyte clusters [3], a device is required that efficiently desolvates the 2,5-dihydroxybenzoic acid (2,5-DHB) matrix to produce the highly charged molecular ions. One function of the Waters Company (Manchester, UK) ESI z-spray source design used with the IMS-MS SYNAPT G2 is to reduce the background arising from cluster ions.…”

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Laserspray ionization (LSI) ion mobility spectrometry (IMS) mass spectrometry

Inutan

Trimpin

2010

J. Am. Soc. Mass Spectrom.

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111

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A simple device is described for desolvation of highly charged matrix/analyte clusters produced by laser ablation leading to multiply charged ions that are analyzed by ion mobility spectrometry-mass spectrometry. Thus, for example, highly charged ions of ubiquitin and lysozyme are cleanly separated in the gas phase according to size and mass (shape and molecular weight) as well as charge using Tri-Wave ion mobility technology coupled to mass spectrometry. This contribution confirms the mechanistic argument that desolvation is necessary to produce multiply charged matrix-assisted laser desorption/ionization (MALDI) ions and points to how these ions can be routinely formed on any atmospheric pressure mass spectrometer. (J Am Soc Mass Spectrom 2010, 21, 1260 -1264 -3]. The principle of this ionization method is that the analyte/matrix sample is ablated by the use of a laser operating at atmospheric pressure (AP) and ions are subsequently formed from highly charged matrix/analyte clusters during a desolvation process. The free choice of charge state selection demonstrates the utility of LSI for the analysis of complex mixtures. Singly charged ions similar to those obtained with matrix-assisted laser desorption/ ionization (MALDI) or multiply charged ions similar to those produced by electrospray ionization (ESI) [2] can be selected using LSI. Multiply charged ions are especially beneficial for providing the ability to ionize by laser ablation larger molecules such as proteins and synthetic polymers on high-performance but mass range limited mass spectrometers such as the Orbitrap Exactive [2,3]. Full range mass spectra of bovine insulin were obtained on as little as 40 fmol when applying ion transfer capillary temperatures of ϳ350°C [3].The unique feature of LSI to produce highly charged ions using direct laser ablation of a solid surface enhances fragmentation as was demonstrated by obtaining nearly complete protein sequence coverage of ubiquitin using electron-transfer dissociation (ETD) technology on a LTQ mass spectrometer [3]. Initial laserspray applications demonstrated the ability to produce singly charged lipid ions from mouse brain tissue under ambient conditions [1]. Using transmission geometry the matrix treated tissue sections were passed free-hand through the focused laser beam in front of the mass spectrometer orifice (Orbitrap Exactive) obtaining a spatial resolution of Ͻ100 m [1]. The objective is to observe proteins from tissue.Ion mobility spectrometry (IMS) MS has many advantages compared with even high-resolution mass spectrometers because of its ability to extend the dynamic range and separate isomeric composition [4][5][6][7]. This is possible because ions are separated in the IMS dimension according to charge and cross-section (size and shape) [8][9][10][11]. One key benefit of this solvent-free gas-phase separation by IMS is that when combined with solvent-free sample preparation [12] achieves total solvent-free analysis by MS entirely decoupling ionization, separation, and mass analyses fro...

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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Laserspray ionization (LSI) ion mobility spectrometry (IMS) mass spectrometry

Inutan

Trimpin

2010

J. Am. Soc. Mass Spectrom.

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111

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“…The soluble fraction represents a measure for the de novo biosynthesis of soluble proteins, whereas the insoluble fraction corresponds to newly synthesized insoluble proteins (e.g. cell wall or plasma membrane proteins) (45). Relative to the wild type, the ⌬asc1 mutant showed elevated de novo biosynthesis of soluble proteins.…”

Section: De Novo Proteome Analysis: Hypoxic Energy Metabolism In ⌬Asc1mentioning

confidence: 99%

RACK1/Asc1p, a Ribosomal Node in Cellular Signaling

Rachfall

Schmitt

Bandau

et al. 2013

Molecular & Cellular Proteomics

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§ RACK1/Asc1p and its essential orthologues in higher eukaryotes, such as RACK1 in metazoa, are involved in several distinct cellular signaling processes. The implications of a total deletion have never been assessed in a comprehensive manner. This study reveals the major cellular processes affected in a Saccharomyces cerevisiae ⌬asc1 deletion background via de novo proteome and transcriptome analysis, as well as subsequent phenotypical characterizations. The deletion of ASC1 reduces iron uptake and causes nitrosative stress, both known indicators for hypoxia, which manifests in a shift of energy metabolism from respiration to fermentation in the ⌬asc1 strain. Asc1p further impacts cellular metabolism through its regulative role in the MAP kinase signal transduction pathways of invasive/filamentous growth and cell wall integrity. In the ⌬asc1 mutant strain, aberrations from the expected cellular response, mediated by these pathways, can be observed and are linked to changes in protein abundances of pathway-targeted transcription factors. Evidence of the translational regulation of such transcription factors suggests that ribosomal Asc1p is involved in signal transduction pathways and controls the biosynthesis of the respective final transcriptional regulators. Molecular & Cellular

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“…Mass spectrometry (MS) is increasingly being used for various types of IMP studies [1,[17][18][19][20][21][22][23][24][25][26][27][28]. Covalent labeling coupled with MS [29] can provide structural information on IMPs.…”

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Validation of Membrane Protein Topology Models by Oxidative Labeling and Mass Spectrometry

Pan

Ruan

Valvano

et al. 2012

J. Am. Soc. Mass Spectrom.

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Computer-assisted topology predictions are widely used to build low-resolution structural models of integral membrane proteins (IMPs). Experimental validation of these models by traditional methods is labor intensive and requires modifications that might alter the IMP native conformation. This work employs oxidative labeling coupled with mass spectrometry (MS) as a validation tool for computer-generated topology models. ⋅OH exposure introduces oxidative modifications in solvent-accessible regions, whereas buried segments (e.g., transmembrane helices) are non-oxidizable. The Escherichia coli protein WaaL (O-antigen ligase) is predicted to have 12 transmembrane helices and a large extramembrane domain (Pérez et al., Mol. Microbiol. 2008, 70, 1424. Tryptic digestion and LC-MS/MS were used to map the oxidative labeling behavior of WaaL. Met and Cys exhibit high intrinsic reactivities with ⋅OH, making them sensitive probes for solvent accessibility assays. Overall, the oxidation pattern of these residues is consistent with the originally proposed WaaL topology. One residue (M151), however, undergoes partial oxidation despite being predicted to reside within a transmembrane helix. Using an improved computer algorithm, a slightly modified topology model was generated that places M151 closer to the membrane interface. On the basis of the labeling data, it is concluded that the refined model more accurately reflects the actual topology of WaaL. We propose that the combination of oxidative labeling and MS represents a useful strategy for assessing the accuracy of IMP topology predictions, supplementing data obtained in traditional biochemical assays. In the future, it might be possible to incorporate oxidative labeling data directly as constraints in topology prediction algorithms.

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Analysis of Insoluble Proteins

Cited by 34 publications

References 87 publications

Laserspray ionization (LSI) ion mobility spectrometry (IMS) mass spectrometry

Laserspray ionization (LSI) ion mobility spectrometry (IMS) mass spectrometry

RACK1/Asc1p, a Ribosomal Node in Cellular Signaling

Validation of Membrane Protein Topology Models by Oxidative Labeling and Mass Spectrometry

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