Adsorption of unfolded Cu/Zn superoxide dismutase onto hydrophobic surfaces catalyzes its formation of amyloid fibrils

Intracellular aggregates of superoxide dismutase 1 (SOD1) are associated with amyotrophic lateral sclerosis. In vivo, aggregation occurs in a complex and dense molecular environment with chemically heterogeneous surfaces. To investigate how SOD1 fibril formation is affected by surfaces, we used an in vitro model system enabling us to vary the molecular features of both SOD1 and the surfaces, as well as the surface area. We compared fibril formation in hydrophilic and hydrophobic sample wells, as a function of denaturant concentration and extraneous hydrophobic surface area. In the presence of hydrophobic surfaces, SOD1 unfolding promotes fibril nucleation. By contrast, in the presence of hydrophilic surfaces, increasing denaturant concentration retards the onset of fibril formation. We conclude that the mechanism of fibril formation depends on the surrounding surfaces and that the nucleating species might correspond to different conformational states of SOD1 depending on the nature of these surfaces

Detection of Crosslinks within and between Proteins by LC-MALDI-TOFTOF and the Software FINDX to Reduce the MSMS-Data to Acquire for Validation

Lysine-specific chemical crosslinking in combination with mass spectrometry is emerging as a tool for the structural characterization of protein complexes and protein-protein interactions. After tryptic digestion of crosslinked proteins there are thousands of peptides amenable to MSMS, of which only very few are crosslinked peptides of interest. Here we describe how the advantage offered by off-line LC-MALDI-TOF/TOF mass spectrometry is exploited in a two-step workflow to focus the MSMS-acquisition on crosslinks mainly. In a first step, MS-data are acquired and all the peak list files from the LC-separated fractions are merged by the FINDX software and screened for presence of crosslinks which are recognized as isotope-labeled doublet peaks. Information on the isotope doublet peak mass and intensity can be used as search constraints to reduce the number of false positives that match randomly to the observed peak masses. Based on the MS-data a precursor ion inclusion list is generated and used in a second step, where a restricted number of MSMS-spectra are acquired for crosslink validation. The decoupling of MS and MSMS and the peptide sorting with FINDX based on MS-data has the advantage that MSMS can be restricted to and focused on crosslinks of Type 2, which are of highest biological interest but often lowest in abundance. The LC-MALDI TOF/TOF workflow here described is applicable to protein multisubunit complexes and using 14N/15N mixed isotope strategy for the detection of inter-protein crosslinks within protein oligomers

Proteomic Analysis of Primary Graft Dysfunction in Porcine Lung Transplantation Reveals Alveolar-Capillary Barrier Changes Underlying the High Particle Flow Rate in Exhaled Breath

Primary graft dysfunction (PGD) remains a challenge for lung transplantation (LTx) recipients as a leading cause of poor early outcomes. New methods are needed for more detailed monitoring and understanding of the pathophysiology of PGD. The measurement of particle flow rate (PFR) in exhaled breath is a novel tool to monitor and understand the disease at the proteomic level. In total, 22 recipient pigs underwent orthotopic left LTx and were evaluated for PGD on postoperative day 3. Exhaled breath particles (EBPs) were evaluated by mass spectrometry and the proteome was compared to tissue biopsies and bronchoalveolar lavage fluid (BALF). Findings were confirmed in EBPs from 11 human transplant recipients. Recipients with PGD had significantly higher PFR [686.4 (449.7–8,824.0) particles per minute (ppm)] compared to recipients without PGD [116.6 (79.7–307.4) ppm, p = 0.0005]. Porcine and human EBP proteins recapitulated proteins found in the BAL, demonstrating its utility instead of more invasive techniques. Furthermore, adherens and tight junction proteins were underexpressed in PGD tissue. Histological and proteomic analysis found significant changes to the alveolar-capillary barrier explaining the high PFR in PGD. Exhaled breath measurement is proposed as a rapid and non-invasive bedside measurement of PGD

Sampling and Selective Detection of Peptides and Proteins Emphazing Microdialysis,Bio-recognition Assays and MALDI-TOF MS

The use of microdialysis sampling and flow immunochemical detection as well as MALDI-TOF MS for peptide and protein analysis is described. Sampling of peptides and smaller proteins was investigated with different membranes. Cyclodextrines additives and an on-line push-pull microdialysis sampling method was used with the result of a higher sampling efficiency compared to conventional microdialysis sampling. Real-time flow immuno assays systems were developed for the quantification of Interleukin-8 and Interleukin10 in cell samples. Optimisation with regards to affinity support selection as well as carrier buffer composition were performed. Connection of capillary LC with MALDI-TOF MS was made utilising an m-dispenser technique enabling sample handling of picoliter volumes. A novel sample preparation technique for the uniform coating of MALDI targets was developed

Liquid chromatography-tandem mass spectrometry approach for quantification of mucins from sputum using C-13,N-15-labeled peptides as internal standards

Mucins are of great interest owing their involvement in physiological and pathological processes in the airways. A method which allows accurate quantification of such proteins in sputum samples may be helpful for research in this field. A liquid chromatographic single reaction monitoring (SRM) method was developed for the quantification of two mucins, MUC5AC and MUC5B, in induced sputum samples. Sample preparation for the assay included solubilization, reduction and alkylation prior to tryptic digestion. Solid phase extraction using C(18) sorbent was used for sample clean up prior to the LC-MS/MS analysis. A cysteine containing peptide was selected for quantification of MUC5AC protein, whereas a non-cysteine peptide was used for the quantification of MUC5B protein. Stable isotope-labeled synthetic peptides were used as internal standards and linear calibration curves were constructed in the range of 0.3 - 40 pmol/L. Both mucins could be determined with a precision of 6-19% and an accuracy of 98-114%. The method is transferable to robotics and is suitable to be run in a 96-well format

Cu/Zn Superoxide Dismutase Forms Amyloid Fibrils under Near-Physiological Quiescent Conditions : The Roles of Disulfide Bonds and Effects of Denaturant

Cu/Zn superoxide dismutase (SOD1) forms intracellular aggregates that are pathological indicators of amyotrophic lateral sclerosis. A large body of research indicates that the entry point to aggregate formation is a monomeric, metal-ion free (apo), and disulfide-reduced species. Fibril formation by SOD1 in vitro has typically been reported only for harsh solvent conditions or mechanical agitation. Here we show that monomeric apo-SOD1 in the disulfide-reduced state forms fibrillar aggregates under near-physiological quiescent conditions. Monomeric apo-SOD1 with an intact intramolecular disulfide bond is highly resistant to aggregation under the same conditions. A cysteine-free variant of SOD1 exhibits fibrillization behavior and fibril morphology identical to those of disulfide-reduced SOD1, firmly establishing that intermolecular disulfide bonds or intramolecular disulfide shuffling are not required for aggregation and fibril formation. The decreased lag time for fibril formation resulting from reduction of the intramolecular disulfide bond thus primarily reflects the decreased stability of the folded state relative to partially unfolded states, rather than an active role of free sulfhydryl groups in mediating aggregation. Addition of urea to increase the amount of fully unfolded SOD1 increases the lag time for fibril formation, indicating that the population of this species does not dominate over other factors in determining the onset of aggregation. Our results contrast with previous results obtained for agitated samples, in which case amyloid formation was accelerated by denaturant. We reconcile these observations by suggesting that denaturants destabilize monomeric and aggregated species to different extents and thus affect nucleation and growth

Osteopontin is increased in cystic fibrosis and can skew the functional balance between ELR-positive and ELR-negative CXC-chemokines.

The glycoprotein osteopontin plays important roles in several states of disease associated with inflammation, for example by recruiting neutrophils but its expression and possible roles in cystic fibrosis (CF) have not been investigated

Quantitative high-performance liquid chromatography-tandem mass spectrometry method for the analysis of free desmosines in plasma and urine

A rapid method for the determination of the sum of free desmosine and isodesmosine in human plasma and urine is described. Efficient sample clean-up prior to LC-MS/MS analysis is mandatory for detection of free desmosines in plasma samples. The combination of ultra-filtration and a two-step solid phase extraction minimizes the sample complexity and ion suppression effects. The flow through from the ultra filtration is passed through a C18 resin and then the target analytes are trapped and enriched on a mixed mode solid phase extraction material. The combination of these three orthogonal sample preparation steps allows detection of endogenous free desmosines in plasma from healthy individuals. An analytical column packed with porous graphitic carbon material enables the retention of the polar desmosine analytes, which are measured by electrospray ionization tandem mass spectrometry. Deuterium labeled isodesmosine is added as internal standard and a linear calibration curve was constructed in the range of 0.1-2.0 nmol/L for plasma samples and 5-200 nmol/L for urine samples. These results demonstrate that the described LC-MS/MS method provides sensitive, repeatable and accurate quantification of free desmosines in plasma and urine samples. (C) 2013 Elsevier B.V. All rights reserved

Probing the transient interaction between the small heat-shock protein Hsp21 and a model substrate protein using crosslinking mass spectrometry.

Small heat-shock protein chaperones are important players in the protein quality control system of the cell, because they can immediately respond to partially unfolded proteins, thereby protecting the cell from harmful aggregates. The small heat-shock proteins can form large polydisperse oligomers that are exceptionally dynamic, which is implicated in their function of protecting substrate proteins from aggregation. Yet the mechanism of substrate recognition remains poorly understood, and little is known about what parts of the small heat-shock proteins interact with substrates and what parts of a partially unfolded substrate protein interact with the small heat-shock proteins. The transient nature of the interactions that prevent substrate aggregation rationalize probing this interaction by crosslinking mass spectrometry. Here, we used a workflow with lysine-specific crosslinking and offline nano-liquid chromatography matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry to explore the interaction between the plant small heat-shock protein Hsp21 and a thermosensitive model substrate protein, malate dehydrogenase. The identified crosslinks point at an interaction between the disordered N-terminal region of Hsp21 and the C-terminal presumably unfolding part of the substrate protein

Electrophile-Induced Conformational Switch of the Human TRPA1 Ion Channel Detected by Mass Spectrometry

The human Transient Receptor Potential A1 (hTRPA1) ion channel, also known as the wasabi receptor, acts as a biosensor of various potentially harmful stimuli. It is activated by a wide range of chemicals, including the electrophilic compound N-methylmaleimide (NMM), but the mechanism of activation is not fully understood. Here, we used mass spectrometry to map and quantify the covalent labeling in hTRPA1 at three different concentrations of NMM. A functional truncated version of hTRPA1 (Δ1-688 hTRPA1), lacking the large N-terminal ankyrin repeat domain (ARD), was also assessed in the same way. In the full length hTRPA1, the labeling of different cysteines ranged from nil up to 95% already at the lowest concentration of NMM, suggesting large differences in reactivity of the thiols. Most important, the labeling of some cysteine residues increased while others decreased with the concentration of NMM, both in the full length and the truncated protein. These findings indicate a conformational switch of the proteins, possibly associated with activation or desensitization of the ion channel. In addition, several lysines in the transmembrane domain and the proximal N-terminal region were labeled by NMM, raising the possibility that lysines are also key targets for electrophilic activation of hTRPA1