Characterization of Electron Transfer Dissociation in the Orbitrap Velos HCD Cell

Electron transfer dissociation (ETD) is commonly employed in ion traps utilizing rf fields that facilitate efficient electron transfer reactions. Here, we explore performing ETD in the HCD collision cell on an Orbitrap Velos instrument by applying a static DC gradient axially to the rods. This gradient enables simultaneous three dimensional, charge sign independent, trapping of cations and anions, initiating electron transfer reactions in the center of the HCD cell where oppositely charged ions clouds overlap. Here, we evaluate this mode of operation for a number of tryptic peptide populations and the top-down sequence analysis of ubiquitin. Our preliminary data show that performing ETD in the HCD cell provides similar fragmentation as ion trap-ETD but requires further optimization to match performance of ion trap-ETD.      

Stabilization of Large Adsorbates by Rotational Entropy: A Time‐Resolved Variable‐Temperature STM Study

Investigating the dynamics in an adlayer of the oligopyridine derivative 2‐phenyl‐4,6‐bis(6‐(pyridine‐2‐yl)‐4‐(pyridine‐4‐yl)pyridine‐2‐yl)pyrimidine (2,4′‐BTP) on Ag(111) by fast scanning tunneling microscopy (video‐STM), we found that rotating 2,4′‐BTP adsorbates coexist in a two‐dimensional (2D) liquid phase (β‐phase) in a dynamic equilibrium with static adsorbate molecules. Furthermore, exchange between an ordered phase (α‐phase) and β‐phase leads to fluctuations of the domain boundary on a time scale of seconds. Quantitative evaluation of the temperature‐dependent equilibrium between rotating and static adsorbates, evaluated from a large number of STM images, gains insight into energetic and entropic stabilization and underlines that the rotating adsorbate molecules are stabilized by an entropy contribution, which is compatible with that derived by using statistical mechanics. The general validity of the concept of entropic stabilization of rotating admolecules, favoring rotation already at room temperature, is tested for other typical small, mid‐size and large adsorbates.     

SPE HG-AAS method for the determination of inorganic arsenic in rice—results from method validation studies and a survey on rice products

The present paper describes the development, validation and application of a method for inorganic arsenic (iAs) determination in rice samples. The separation of iAs from organoarsenic compounds was done by off-line solid-phase extraction (SPE) followed by hydride generation atomic absorption spectrometry (HG-AAS) detection. This approach was earlier developed for seafood samples (Rasmussen et al., Anal Bioanal Chem 403:2825–2834, 2012) and has in the present work been tailored for rice products and further optimised for a higher sample throughput and a lower detection limit. Water bath heating (90 °C, 60 min) of samples with dilute HNO₃ and H₂O₂ solubilised and oxidised all iAs to arsenate (As^V). Loading of buffered sample extracts (pH 6?±?1) followed by selective elution of arsenate from a strong anion exchange SPE cartridge enabled the selective iAs quantification by HG-AAS, measuring total arsenic (As) in the SPE eluate. The in-house validation gave mean recoveries of 101–106 % for spiked rice samples and in two reference samples. The limit of detection was 0.02 mg kg^?1, and repeatability and intra-laboratory reproducibility were less than 6 and 9 %, respectively. The SPE HG-AAS method produced similar results compared to parallel high-performance liquid chromatography coupled to inductively coupled plasma mass spectrometry (ICP-MS) analysis. The SPE separation step was tested collaboratively, where the laboratories (N?=?10) used either HG-AAS or ICP-MS for iAs determination in a wholemeal rice powder. The trial gave satisfactory results (HorRat value of 1.6) and did not reveal significant difference (t test, p?>?0.05) between HG-AAS and ICP-MS quantification. The iAs concentration in 36 rice samples purchased on the Danish retail market varied (0.03–0.60 mg kg^?1), with the highest concentration found in a red rice sample.      

Multidimensional nano-HPLC coupled with tandem mass spectrometry for analyzing biotinylated proteins

Multidimensional high-performance liquid chromatography (HPLC) is a key method in shotgun proteomics approaches for analyzing highly complex protein mixtures by complementary chromatographic separation principles. Here, we describe an integrated 3D-nano-HPLC/nano-electrospray ionization quadrupole time-of-flight mass spectrometry system that allows an enzymatic digestion of proteins followed by an enrichment and subsequent separation of the created peptide mixtures. The online 3D-nano-HPLC system is composed of a monolithic trypsin reactor in the first dimension, a monolithic affinity column with immobilized monomeric avidin in the second dimension, and a reversed phase C18 HPLC-Chip in the third dimension that is coupled to a nano-ESI-Q-TOF mass spectrometer. The 3D-LC/MS setup is exemplified for the identification of biotinylated proteins from a simple protein mixture. Additionally, we describe an online 2D-nano-HPLC/nano-ESI-LTQ-Orbitrap-MS/MS setup for the enrichment, separation, and identification of cross-linked, biotinylated species from chemical cross-linking of cytochrome c and a calmodulin/peptide complex using a novel trifunctional cross-linker with two amine-reactive groups and a biotin label.

Simple approach to study biomolecule adsorption in polymeric microfluidic channels

Herein a simple analytical method is presented for the characterization of biomolecule adsorption on cyclo olefin polymer (COP, trade name: Zeonor^®) substrates which are widely used in microfluidic lab-on-a-chip devices. These Zeonor^® substrates do not possess native functional groups for specific reactions with biomolecules. Therefore, depending on the application, such substrates must be functionalized by surface chemistry methods to either enhance or suppress biomolecular adsorption. This work demonstrates a microfluidic method for evaluating the adsorption of antibodies and oligonucleotides surfaces. The method uses centrifugal microfluidic flow-through chips and can easily be implemented using common equipment such as a spin coater. The working principle is very simple. The user adds 40 L of the solution containing the sample to the starting side of a microfluidic channel, where it is moved through by centrifugal force. Some molecules are adsorbed in the channel. The sample is then collected at the other end in a small reservoir and the biomolecule concentration is measured. As a pilot application, we characterized the adsorption of goat anti-human IgG and a 20-mer DNA on Zeonor^®, and on three types of functionalized Zeonor: 3-aminopropyltriethoxysilane (APTES) modified surface with mainly positive charge, negatively charged surface with immobilized bovine serum albumin (BSA), and neutral, hydrogel-like film with polyethylene glycol (PEG) characteristics. This simple analytical approach adds to the fundamental understanding of the interaction forces in real, microfluidic systems. This method provides a straightforward and rapid way to screen surface compositions and chemistry, and relate these to their effects on the sensitivity and resistance to non-specific binding of bioassays using them. In an additional set of experiments, the surface area of the channels in this universal microfluidic chip was increased by precision milling of microscale trenches. This modified surface was then coated with APTES and tested for its potential to serve as a unique protein dilution feature.     

At-line bioprocess monitoring by immunoassay with rotationally controlled serial siphoning and integrated supercritical angle fluorescence optics

In this paper we report a centrifugal microfluidic “lab-on-a-disc” system for at-line monitoring of human immunoglobulin G (hIgG) in a typical bioprocess environment. The novelty of this device is the combination of a heterogeneous sandwich immunoassay on a serial siphon-enabled microfluidic disc with automated sequential reagent delivery and surface-confined supercritical angle fluorescence (SAF)-based detection. The device, which is compact, easy-to-use and inexpensive, enables rapid detection of hIgG from a bioprocess sample. This was achieved with, an injection moulded SAF lens that was functionalized with aminopropyltriethoxysilane (APTES) using plasma enhanced chemical vapour deposition (PECVD) for the immobilization of protein A, and a hybrid integration with a microfluidic disc substrate. Advanced flow control, including the time-sequenced release of on-board liquid reagents, was implemented by serial siphoning with ancillary capillary stops. The concentration of surfactant in each assay reagent was optimized to ensure proper functioning of the siphon-based flow control. The entire automated microfluidic assay process is completed in less than 30 min. The developed prototype system was used to accurately measure industrial bioprocess samples that contained 10 mg mL⁻¹ of hIgG.     

Synthesis of New N‐(5‐Oxo‐2,5‐dihydro)pyrrol‐3‐yl Glycines and N‐(5‐Oxo‐2,5‐dihydro)pyrrol‐3‐yl Glycines Esters

Following our efforts towards the synthesis of new potential inhibitors of Xanthine Dehydrogenase (XDH), we describe here a general method for the preparation of N-(5-oxo-2,5-dihydro)pyrrol-3-yl glycines and N-(5-oxo-2,5-dihydro)pyrrol-3-yl glycine esters from glycine ethyl ester hydrochloride and various 4-hydroxy-5-oxo-2,5-dihydro-1H-pyrrole-3-carboxylic acid esters and carbonitriles.     

Highly Enantioselective Hydrogenation of β‐Alkyl and β‐(ω‐Chloroalkyl) Substituted β‐Keto Esters

Highly enantioselective hydrogenation of β‐alkyl and β‐(ω‐chloroalkyl) substituted β‐keto esters was achieved with Ru catalysts based on chiral diphosphines in EtOH at 50°C under 50‐bar initial hydrogen pressure, affording the corresponding β‐hydroxy esters in >98% ee.