Secondary Structures of Ubiquitin Ions Soft-Landed onto Self-Assembled Monolayer Surfaces

The secondary structures of multiply charged ubiquitin ions soft-landed onto self-assembled monolayer (SAM) surfaces were studied using in situ infrared reflection–absorption spectroscopy (IRRAS). Two charge states of ubiquitin, 5+ and 13+, were mass selected separately from a mixture of different charge states produced by electrospray ionization (ESI). The low 5+ charge state represents a nativelike folded state of ubiquitin, while the high 13+ charge state assumes an extended, almost linear conformation. Each of the two charge states was soft-landed onto a CH₃- and COOH-terminated SAM of alkanethiols on gold (HSAM and COOH-SAM). HSAM is a hydrophobic surface known to stabilize helical conformations of soft-landed protonated peptides, whereas COOH-SAM is a hydrophilic surface that preferentially stabilizes β-sheet conformations. IRRAS spectra of the soft-landed ubiquitin ions were acquired as a function of time during and after ion soft-landing. Similar to smaller peptide ions, helical conformations of ubiquitin are found to be more abundant on HSAM, while the relative abundance of β-sheet conformations increases on COOH-SAM. The initial charge state of ubiquitin also has a pronounced effect on its conformation on the surface. Specifically, on both surfaces, a higher relative abundance of helical conformations and a lower relative abundance of β-sheet conformations are observed for the 13+ charge state compared to the 5+ charge state. Time-resolved experiments indicate that the α-helical band in the spectrum of the 13+ charge state slowly increases with time on the HSAM surface and decreases in the spectrum of the 13+ charge state on COOH-SAM. These results further support the preference of the hydrophobic HSAM surface toward helical conformations and demonstrate that soft-landed protein ions may undergo slow conformational changes during and after deposition

Molecular Characterization of Nitrogen-Containing Organic Compounds in Biomass Burning Aerosols Using High-Resolution Mass Spectrometry

Although nitrogen-containing organic compounds (NOC) are important components of atmospheric aerosols, little is known about their chemical composition. Here we present detailed characterization of the NOC constituents of biomass burning aerosol (BBA) samples using high-resolution electrospray ionization mass spectrometry (ESI/MS). Accurate mass measurements combined with MS/MS fragmentation experiments of selected ions were used to assign molecular structures to individual NOC species. Our results indicate that N-heterocyclic alkaloid compounds (species naturally produced by plants and living organisms) comprise a substantial fraction of NOC in BBA samples collected from test burns of five biomass fuels. High abundance of alkaloids in test burns of ponderosa pine (a widespread tree in the western U.S. areas frequently affected by large scale fires) suggests that N-heterocyclic alkaloids in BBA may play a significant role in dry and wet deposition of fixed nitrogen in this region

Molecular Characterization of Organic Aerosols Using Nanospray-Desorption/Electrospray Ionization-Mass Spectrometry

Nanospray desorption electrospray ionization (nano-DESI) combined with high-resolution mass spectrometry (HR-MS) is a promising approach for the detailed, molecular-level chemical characterization of atmospheric organic aerosols (OA) collected in laboratory and field experiments. The nano-DESI technique possesses distinct advantages of technical simplicity, enhanced sensitivity, and signal stability. In nano-DESI, analyte is desorbed into a solvent bridge formed between two capillaries and the analysis surface, which enables fast and efficient characterization of OA collected on substrates without sample preparation. Stable signals achieved using nano-DESI make it possible to obtain high-quality HR-MS data both for laboratory-generated and field-collected OA using only a small amount of material (<10 ng). Furthermore, nano-DESI enables the efficient detection of chemically labile compounds in OA, which is important for understanding chemical aging phenomena

Entropy Is the Major Driving Force for Fragmentation of Proteins and Protein−Ligand Complexes in the Gas Phase

This paper presents a critical analysis of Arrhenius parameters for gas-phase fragmentation of proteins and protein−ligand complexes reported in the literature. We demonstrate that there is a surprisingly strong correlation between the Arrhenius activation energy (Ea) and the preexponential factor (A). This correlation becomes extremely important for reactions characterized by very high or very low values of A. This correlation is a direct consequence of the relative change in the spacing between vibrational levels of the reactant and the transition state for reaction. Converting the Arrhenius activation energy into the threshold energy for the reaction using Tolman's theorem reveals the true magnitude of the correlation between molecular complexity and stability. Tolman's correction factor (ΔEcorr) increases linearly with log(A) from 3 kcal/mol for log(A) = 16.2 to 36.4 kcal/mol for log(A) = 39.2. Threshold energies extracted from the Arrhenius activation parameters for 56 different reactions are the same within the experimental error bars, while the preexponential factors differ by many orders of magnitude. This indicates that activation entropy is the major driving force for dissociation of proteins and protein−ligand complexes in the gas phase

Quantitative Extraction and Mass Spectrometry Analysis at a Single-Cell Level

Quantitative live cell mass spectrometry analysis at a subcellular level requires the precisely controlled extraction of subpicoliter volumes of material from the cell, sensitive analysis of the extracted analytes, and their accurate quantification without prior separation. In this study, we demonstrate that localized electroosmotic extraction provides a direct path to addressing this challenge. Specifically, we demonstrate quantitative mass spectrometry analysis of biomolecules in picoliter volumes extracted from live cells. Electroosmotic extraction was performed using two electrodes and a finely pulled nanopipette with tip diameter of <1 μm containing a hydrophobic electrolyte compatible with mass spectrometry analysis. The electroosmotic drag was used to drive analytes out of the cell into the nanopipette. Analyte molecules extracted both from solutions and cell samples were analyzed using nanoelectrospray ionization (nanoESI) directly from the nanopipette into a mass spectrometer. More than 50 metabolites including sugars and flavonoids were detected in positive mode in 2−5 pL volumes of the cytoplasmic material extracted from Allium cepa. Quantification of the extracted glucose was performed using sequential extraction of a known volume of the aqueous solution containing glucose-<i>d</i>₂ standard of known concentration. We found that the ratio of the signal of glucose to glucose-<i>d</i>₂ increased linearly with glucose concentration. This observation indicates that the approach developed in this study enables quantitative analysis of small volumes of metabolites extracted from cells. Furthermore, we observed efficient separation of hydrophilic and hydrophobic analytes through partitioning into the aqueous and hydrophobic electrolyte phase, respectively, which provides additional important information on the molecular properties of extracted metabolites

Quantitative Extraction and Mass Spectrometry Analysis at a Single-Cell Level

Quantitative live cell mass spectrometry analysis at a subcellular level requires the precisely controlled extraction of subpicoliter volumes of material from the cell, sensitive analysis of the extracted analytes, and their accurate quantification without prior separation. In this study, we demonstrate that localized electroosmotic extraction provides a direct path to addressing this challenge. Specifically, we demonstrate quantitative mass spectrometry analysis of biomolecules in picoliter volumes extracted from live cells. Electroosmotic extraction was performed using two electrodes and a finely pulled nanopipette with tip diameter of <1 μm containing a hydrophobic electrolyte compatible with mass spectrometry analysis. The electroosmotic drag was used to drive analytes out of the cell into the nanopipette. Analyte molecules extracted both from solutions and cell samples were analyzed using nanoelectrospray ionization (nanoESI) directly from the nanopipette into a mass spectrometer. More than 50 metabolites including sugars and flavonoids were detected in positive mode in 2−5 pL volumes of the cytoplasmic material extracted from Allium cepa. Quantification of the extracted glucose was performed using sequential extraction of a known volume of the aqueous solution containing glucose-<i>d</i>₂ standard of known concentration. We found that the ratio of the signal of glucose to glucose-<i>d</i>₂ increased linearly with glucose concentration. This observation indicates that the approach developed in this study enables quantitative analysis of small volumes of metabolites extracted from cells. Furthermore, we observed efficient separation of hydrophilic and hydrophobic analytes through partitioning into the aqueous and hydrophobic electrolyte phase, respectively, which provides additional important information on the molecular properties of extracted metabolites

Quantitative Extraction and Mass Spectrometry Analysis at a Single-Cell Level

Quantitative live cell mass spectrometry analysis at a subcellular level requires the precisely controlled extraction of subpicoliter volumes of material from the cell, sensitive analysis of the extracted analytes, and their accurate quantification without prior separation. In this study, we demonstrate that localized electroosmotic extraction provides a direct path to addressing this challenge. Specifically, we demonstrate quantitative mass spectrometry analysis of biomolecules in picoliter volumes extracted from live cells. Electroosmotic extraction was performed using two electrodes and a finely pulled nanopipette with tip diameter of <1 μm containing a hydrophobic electrolyte compatible with mass spectrometry analysis. The electroosmotic drag was used to drive analytes out of the cell into the nanopipette. Analyte molecules extracted both from solutions and cell samples were analyzed using nanoelectrospray ionization (nanoESI) directly from the nanopipette into a mass spectrometer. More than 50 metabolites including sugars and flavonoids were detected in positive mode in 2−5 pL volumes of the cytoplasmic material extracted from Allium cepa. Quantification of the extracted glucose was performed using sequential extraction of a known volume of the aqueous solution containing glucose-<i>d</i>₂ standard of known concentration. We found that the ratio of the signal of glucose to glucose-<i>d</i>₂ increased linearly with glucose concentration. This observation indicates that the approach developed in this study enables quantitative analysis of small volumes of metabolites extracted from cells. Furthermore, we observed efficient separation of hydrophilic and hydrophobic analytes through partitioning into the aqueous and hydrophobic electrolyte phase, respectively, which provides additional important information on the molecular properties of extracted metabolites

Isolation, Characterization of an Intermediate in an Oxygen Atom-Transfer Reaction, and the Determination of the Bond Dissociation Energy

The synthesis and structure of a phosphine oxide-bound intermediate molecule originating from a dioxo−molybdenum(VI) complex is described. The loss of phosphine oxide has been followed by surface-induced dissociation mass spectrometry that gave the bond dissociation energy of 29.5 (± 3.5) kcal/mol. Considering the bond dissociation energy for a MoO bond to be 100 kcal/mol, this value suggests that the primary driving force for the reaction is the formation of the intermediate complex

Covalent Immobilization of Peptides on Self-Assembled Monolayer Surfaces Using Soft-Landing of Mass-Selected Ions

Controlled immobilization of peptides onto N-hydroxysuccinimidyl ester functionalized self-assembled monolayer (SAM) surfaces using reactive landing of mass-selected ions has been demonstrated. Surface characterization using time-of-flight secondary ion mass spectrometry and grazing-incidence infrared reflection−absorption spectroscopy confirmed highly efficient covalent linkage through an amide bond formed between the SAM and the free amino group of the lysine side chain. Reactive landing opens a new way for controlled preparation of peptide arrays

Nitrogen-Containing Organic Compounds and Oligomers in Secondary Organic Aerosol Formed by Photooxidation of Isoprene

Electrospray ionization high-resolution mass spectrometry (ESI HR-MS) was used to probe molecular structures of oligomers in secondary organic aerosol (SOA) generated in laboratory experiments on isoprene photooxidation at low- and high-NOx conditions. Approximately 80–90% of the observed products are oligomers and up to 33% by number are nitrogen-containing organic compounds (NOC). We observe oligomers with maximum 8 monomer units in length. Tandem mass spectrometry (MSn) confirms NOC compounds are organic nitrates and elucidates plausible chemical building blocks contributing to oligomer formation. Most organic nitrates are comprised of methylglyceric acid units. Other important multifunctional C2–C5 monomer units are identified including methylglyoxal, hydroxyacetone, hydroxyacetic acid, and glycolaldehyde. Although the molar fraction of NOC in the high-NOx SOA is high, the majority of the NOC oligomers contain only one nitrate moiety resulting in a low average N:C ratio of 0.019. Average O:C ratios of the detected SOA compounds are 0.54 under the low-NOx conditions and 0.83 under the high-NOx conditions. Our results underscore the importance of isoprene photooxidation as a source of NOC in organic particulate matter