Nano-electrospray Ionization Ion Research Articles

Taking Charge: Metal Ions Accelerate Amyloid Aggregation in Sequence Variants of α-Synuclein.

Αlpha-synuclein (αS) is an intrinsically disordered protein which exhibits a high degree of conformational heterogeneity. In vivo, αS experiences various environments which cause adaptation of its structural ensemble. Divalent metal ions are prominent in synaptic terminals where αS is located and are thought to bind to the αS C-terminal region. Herein, we used native nanoelectrospray ionization ion mobility-mass spectrometry to investigate changes in the charge state distribution and collision cross sections of wild-type N-terminally acetylated (NTA) αS, along with a deletion variant (ΔΔNTA) which inhibits amyloid formation and a C-terminal truncated variant (119NTA) which increases the rate of amyloid formation. We also examine the effect of the addition of divalent metal ions, Ca2+, Mn2+, and Zn2+, and correlate the conformational properties of the αS monomer with the ability to aggregate into amyloid, measured using Thioflavin T fluorescence and negative stain transmission electron microscopy. We find a correlation between the population of species with a low collision cross section and accelerated amyloid assembly kinetics, with the presence of metal ions resulting in protein compaction and causing ΔΔ to regain its ability to form an amyloid. The results portray how the αS conformational ensemble is governed by specific intramolecular interactions that influence its amyloidogenic behavior.

Effects of Detergent on α-Synuclein Structure. A Native MS-Ion Mobility Study.

The intrinsically disordered protein α-synuclein plays a major role in Parkinson’s disease. The protein can oligomerize resulting in the formation of various aggregated species in neuronal cells, leading to neurodegeneration. The interaction of α-synuclein with biological cell membranes plays an important role for specific functions of α-synuclein monomers, e.g., in neurotransmitter release. Using different types of detergents to mimic lipid molecules present in biological membranes, including the presence of Ca2+ ions as an important structural factor, we aimed to gain an understanding of how α-synuclein interacts with membrane models and how this affects the protein conformation and potential oligomerization. We investigated detergent binding stoichiometry, affinity and conformational changes of α-synuclein taking detergent concentration, different detergent structures and charges into account. With native nano-electrospray ionization ion mobility-mass spectrometry, we were able to detect unique conformational patterns resulting from binding of specific detergents to α-synuclein. Our data demonstrate that α-synuclein monomers can interact with detergent molecules irrespective of their charge, that protein-micelle interactions occur and that micelle properties are an important factor.

Collision-Induced Unfolding of Partially Metalated Metallothionein-2A: Tracking Unfolding Reactions of Gas-Phase Ions.

Metallothioneins (MTs) constitute a group of intrinsically disordered proteins that exhibit extreme diversity in structure, biological functionality, and metal ion specificity. Structures of coordinatively saturated metalated MTs have been extensively studied, but very limited structural information for the partially metalated MTs exists. Here, the conformational preferences from partial metalation of rabbit metallothionein-2A (MT) by Cd2+, Zn2+, and Ag+ are studied using nanoelectrospray ionization ion mobility mass spectrometry. We also employ collision-induced unfolding to probe differences in the gas-phase stabilities of these partially metalated MTs. Our results show that despite their similar ion mobility profiles, Cd4-MT, Zn4-MT, Ag4-MT, and Ag6-MT differ dramatically in their gas-phase stabilities. Furthermore, the sequential addition of each Cd2+ and Zn2+ ion results in the incremental stabilization of unique unfolding intermediates.

Ambient temperature nanoelectrospray ion mobility detector for high performance liquid chromatography in determining amines

A nanoelectrospray ionization ion mobility spectrometer (nanoESI–IMS) working at ambient pressure and ambient temperature was developed as a detector of high performance liquid chromatography (HPLC) to achieve sensitive detection of amines with no derivatization and meanwhile provide another dimension of separation. The easier desolvation property of the charged droplets formed in nanoESI source enabled complete desolvation of the product ions of sixteen amines and drugs using the nanoESI–IMS at ambient temperature. Working at ambient temperature was good for suppressing the dissociation of thermal volatile ions, such as only the proton adducted molecular ions were observed for morphine in the nanoESI–IMS. Besides, the resolving power of the nanoESI–IMS also showed an increasing tendency as lowering the working temperature, an increment of 19 percent and 10 percent was observed for diethylamine and triethylamine as the temperature dropped from 92°C to 32°C. The resolving power of the nanoESI–IMS at 32°C for the 16 tested compounds was amid 33–44. With the nanoESI–IMS coupled to HPLC, a six-compound mixture including isomers was successfully separated and detected without any derivatization. And linear response ranges of 1 to 20, 0.5 to 20, and 0.8 to 20μgml−1 and limits of detection of 0.25, 0.15, and 0.17μgml−1 for triethylamine, diethylamine, and butylamine, respectively, were obtained with the hyphenated system. These results showed the excellent performance of the two-dimensional separation and detection method in direct qualitative and quantitative analyses of amines.

2D LC/MS Analysis of Membrane Proteins from Breast Cancer Cell Lines MCF7 and BT474

Membrane proteins play a central role in the interaction of the cell with its environment and in the function of subcellular organelles. The current study focused on developing a better understanding of the membrane proteome of two well-characterized breast cancer cell lines. Membranes from osmotically lysed BT474 and MCF7 cells were treated with cyanogen bromide followed by a combination of trypsin and Staphylococcus V8 protease to obtain hydrophilic peptides from membrane proteins. The complex peptide mixtures obtained were separated by 2-dimensional liquid chromatography coupled online with a nano-electrospray ionization ion trap mass spectrometer (2D LC/nanoESI-MS). The strong cation exchange column used in the first dimension of the separation was eluted in an automated fashion using a series of salt steps of increasing concentration. Peptides eluted from each of the salt steps were separated using a capillary reversed-phase HPLC column, the output of which was directed through a nano-electrospray fused silica tip into the mass spectrometer. Peptides were fragmented by collision-induced dissociation (CID) and analyzed by data-dependent MS/MS followed by database searching using the Sequest algorithm. Analysis of the data revealed both similarities and expected differences between proteins identified from these cell lines. As demonstrated by others, mRNA and the HER2/neu protein tyrosine kinase-linked receptor in BT474 cells is up regulated compared to its level in MCF7, while the expression of the estrogen receptor alpha is known to be up regulated in MCF7 cells. As expected, our studies showed identification of peptides from HER2 in BT474 while estrogen receptor peptides were detected in the MCF7 line. A total of 604 proteins were identified from BT474 membranes while 313 proteins were found from MCF7. The results are discussed in terms of the known differences in both protein and mRNA expression between these two breast cancer cell lines and also in the context of other known phenotypic differences between these cells.

Comprehensive ultra-high pressure capillary liquid chromatography/ion mobility spectrometry

An ultra-high pressure liquid chromatograph was interfaced to a moderately high-resolution nanoelectrospray ionization ion mobility spectrometer operating at ambient temperature and pressure to achieve fast multidimensional separations. The potential of using ion mobility spectrometry as the second dimension in a comprehensive arrangement with liquid chromatography to offer improved qualitative information for compounds under specific operating conditions, is discussed. Separation and detection of selected benzodiazepines and triazine herbicides are demonstrated. Composite peak capacities of 39 and 33 for benzodiazepine and triazine herbicide mixtures, respectively, were achieved in less than 75 s using a 16.5 cm×50 μm internal diameter fused silica capillary liquid chromatographic column packed with 1.5 μm diameter ODS-bonded silica particles.

Structure characterization of flavonoid O-diglycosides by positive and negative nano-electrospray ionization ion trap mass spectrometry.

Isomeric flavonoid O-diglycosides were analyzed by positive and negative nano-electrospray ionization (ESI) ion trap mass spectrometry (ITMS) in order to evaluate whether the two most common interglycosidic linkage types, i.e. 1 --> 2 and 1 --> 6, found for glycosides containing a rhamnosylglucose glycan part can be differentiated. In the positive ion mode the degree of internal glucose residue loss was found to be strongly dependent on the aglycone type and was very pronounced for aglycones of the flavanone type. The relative abundance of the Y-type ions formed by fragmentation at glycosidic bonds only allows one to infer the interglycosidic linkage types in the case of flavone O-diglycosides. In contrast, the negative ion mode makes a clear differentiation between a rutinoside (1 --> 6) and a neohesperidoside (1 --> 2) glycan residue possible for all aglycone types. The neohesperidose-containing compounds could be characterized by additional product ions. When the compounds were dissolved in pure methanol a molecular radical ion was found to be the base peak in nano-ESI.

Capture and Analysis of Low Molecular Weight Ligands by Surface Plasmon Resonance Combined with Mass Spectrometry

The combination of biomolecular interaction analysis (BIA)by surface plasmon resonance (SPR)and nano-electrospray ionization ion trap mass spectrometry (nanoESI-Ion Trap MS)as well as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS)is demonstrated for the binding of low molecular weight inhibitors (∼ 600 Da) to HIV-1 protease. Inhibitors were captured on sensor chips of a manual or an automated SPR biosensor, to which HIV-1 protease was immobilized. Compounds and buffer components that bound unspecifically to the sensor surface were removed and the inhibitors were eluted in a minimal volume (3 μL), between air bubbles, in order to prevent dispersion of analyte into buffer eluent. Molecular weights were subsequently determined by mass spectrometry, structural information was obtained by MALDI-ToF post-source decay as well as by electrospray ionization tandem mass spectrometry (MS/MS)analysis. Furthermore, competition experiments, using a mixture of different ligands, demonstrated that the peak intensities in the MALDI-ToF spectrum could be used for relative quantification of the amount of the different ligands bound to the immobilized target. Methodology for automated capture and elution of analytes was developed and evaluated.

Sequencing of partially methyl-esterified oligogalacturonates by tandem mass spectrometry and its use to determine pectinase specificities.

Complex mixtures of acidic oligosaccharides were produced by enzymatic digestion of partially methyl-esterified pectin with Aspergillus niger pectin lyase, endopolygalacturonase II, and exopolygalacturonase. To determine the specificities of these pectolytic enzymes toward non-esterified and methyl-esterified galacturonic acid residues, we have studied the methyl esterification patterns of selected oligomers in unseparated pectin digests. Collision-induced dissociation in a nanoelectrospray ionization ion trap mass spectrometer was used to locate methyl-esterified galacturonic acid residues in oligomers up to a degree of polymerization of 10. Analysis of the methyl esterification patterns gave insight into the substrate specificities of these pectolytic enzymes. Isomeric fragment ions containing the reducing and nonreducing ends were differentiated by 18O-labeling of the reducing end.