Daniel R. Knapp

Microfabricated PDMS multichannel emitter for electrospray ionization mass spectrometry

A novel microfabricated multichannel emitter for electrospray ionization mass spectrometry (ESI-MS) was implemented with polydimethylsiloxane (PDMS) using a soft lithography technique. The emitters are formed as electrospray tips along a thin membrane on the edge of the device with channels of 100 μm × 30 μm dimensions. The electrospray performance of the PDMS emitters for a single channel device and a four channel device interfaced with a time-of-flight mass spectrometer was evaluated for detecting the molecular weight of reference peptides (angiotensin I and bradykinin). The emitters were durable at the flow rate of 1–20 μL min−1 for more than 30 h of continuous electrospray with limit of detection of 1 μM (S/N 18). This microfabrication method for a PDMS multichannel emitter as an integral part of a microfluidic device will facilitate development of more complex microfluidic analysis systems using ESI-MS.

11-cis-Retinal Reduces Constitutive Opsin Phosphorylation and Improves Quantum Catch in Retinoid-deficient Mouse Rod Photoreceptors

Rpe65 −/− mice produce minimal amounts of 11-cis-retinal, the ligand necessary for the formation of photosensitive visual pigments. Therefore, the apoprotein opsin in these animals has not been exposed to its normal ligand. The Rpe65 −/− mice contain less than 0.1% of wild type levels of rhodopsin. Mass spectrometric analysis of opsin from Rpe65 −/− mice revealed unusually high levels of phosphorylation in dark-adapted mice but no other structural alterations. Single flash and flicker electroretinograms (ERGs) from 1-month-old animals showed trace rod function but no cone response. B-wave kinetics of the single-flash ERG are comparable with those of dark-adapted wild type mice containing a full compliment of rhodopsin. Application (intraperitoneal injection) of 11-cis-retinal to Rpe65 −/−mice increased the rod ERG signal, increased levels of rhodopsin, and decreased opsin phosphorylation. Therefore, exogenous 11-cis-retinal improves photoreceptor function by regenerating rhodopsin and removes constitutive opsin phosphorylation. Our results indicate that opsin, which has not been exposed to 11-cis-retinal, does not generate the activity generally associated with the bleached apoprotein.

Light-actuated high pressure-resisting microvalve for on-chip flow control based on thermo-responsive nanostructured polymer

A simple light-actuated microvalve using a quartz halogen illuminator with tungsten filament was introduced to manipulate flow path effectively in micro-total analysis systems, which reduces system complexity and the need for on-chip integration. The microvalve device in cyclic olefin copolymer (COC) microchip functions very well based on the thermo-responsive polymer, poly(N-isopropylacrylamide) (PNIPAAm), whose pressure-tolerance can be tuned by changing the mechanical strength of polymer monolith inside the microchannel with the choice of suitable amount of monomer and crosslinker. The response time and pressure resistance of the valve can be optimized by the tetrahydrofuran composition in the polymerization mixture as well. Very importantly, the microvalve can withstand the leakage pressure up to around 1350 psi, and its opening and closing response time is only 4.0 and 6.2 s respectively. Microchips with such valves will be very useful in drug delivery, chemical analysis and proteomic analysis.

Miniaturized multichannel electrospray ionization emitters on poly(dimethylsiloxane) microfluidic devices

A multichannel electrospray ionization (ESI) emitter was fabricated as part of a poly(dimethylsiloxane) (PDMS) microfluidic device using a three‐layer photoresist process which also produces a self‐alignment system to make a bonding between the top and bottom PDMS parts. The prototype device (2 cm high×5 cm wide×5 cm long) had 16‐channels (30 νm wide×50 νm deep) with emitters of 1 mm length and 60° point angle. The PDMS emitter tips enabled interfacing the device to ESI‐mass spectrometry; a stable electrospray from the tips was performed with limits of detection under 1 νM for reference peptides (adrenocorticotropic hormone fragment 1–17, angiotensin I and III).

Use of O-p-Nitrobenzyl-N, N′-Diisopropylisourea as a Chromogenic Reagent for Liquid Chromatographic Analysis of Carboxylic Acids

Abstract O-p-Nitrobenzyl-N, N′-diisopropylisourea was shown to react under relatively mild conditions with carboxylic acids to yield the p-nitrobenzyl esters as chromogenic derivatives useful for liquid chromatographic analysis. The p-nitrobenzyl ester of stearic acid was detectable down to the picomole range.

Inhibition of platelet thromboxane synthesis by 7-(1-imidazolyl) heptanoic acid: dissociation from inhibition of aggregation.

Abstract The effects of 7-(1-imidazolyl)heptanoic acid (7-IHA), a thromboxane synthetase inhibitor, and indomethacin on platelet aggregation and immunoreactive thromboxane B2 (iTXB2) synthesis were studied in human platelet rich plasma. The ID50 for inhibition of arachidonic acid induced iTXB2 synthesis by 7-IHA was 1.9 μM but the ID50 for inhibition of platelet aggregation was estimated to be 1.35 mM. 7-IHA (0.5 mM) inhibited arachidonic acid induced iTXB2 synthesis to values less than that obtained in the presence of indomethacin (10 μM); but did not inhibit aggregation, while indomethacin completely inhibited platelet aggregation. 7-IHA had no effect on platelet fatty acid cyclooxygenase; did not promote synergism with ADP or arachidonic acid; arachidonic acid plus ADP or stable PGH2 analogs. We conclude: 1) that 7-IHA is a specific inhibitor of platelet thromboxane synthesis which at the same concentrations does not inhibit arachidonic acid induced platelet aggregation and 2) that inhibition of platelet TXA2 synthesis by thromboxane synthetase inhibitors may not necessarily inhibit arachidonic acid induced platelet aggregation.

Matrix-Free LDI Mass Spectrometry Platform Using Patterned Nanostructured Gold Thin Film

A novel matrix-free LDI MS platform using a thin film of patterned nanostructured gold, capped with methyl- and carboxy-terminated self-assembled monolayers (SAMs) is presented. Calibration on the matrix-free LDI surface was performed using a peptide standard mixture available for MALDI analysis. MS analysis for limit of detection was performed using angiotensin I peptide. Peptide fragments from standard protein digests of bovine serum albumin, bovine catalase, and bovine lactoperoxidase were used to carry out peptide mass fingerprinting analysis. Sequence coverage of each protein digest and the number of detected peptide fragments were compared with conventional MALDI MS on a standard MALDI plate. Versatility of the nanostructured gold LDI substrate is illustrated by performing MS analysis on a protein digest using different enzymes and by small molecule MS analysis.

Microfabrication of polydimethylsiloxane electrospray ionization emitters

Microfabricated polydimethylsiloxane (PDMS) emitters for electrospray ionization mass spectrometry (ESI-MS) were implemented as tips along the edge of the PDMS device by three methods which utilize soft lithography processes. These microfabrication methods for producing PDMS emitters as an integral part of a microfluidic device will facilitate development of more complex microfluidic analysis systems using ESI-MS.

Matrix-assisted laser desorption mass spectrometry of rhodopsin and bacteriorhodopsin

Matrix-assisted laser desorption ionization (MALDI) mass spectrometry has been used to obtain accurate molecular weight information for the integral membrane proteins bacteriorhodopsin and bovine rhodopsin desorbed from solubilized membrane preparations. Mass differences in the molecular weights measured for bleached and unbleached bacteriorhodopsin and rhodopsin indicate the removal of the retinal chromophores upon bleaching. The MALDI technique was also successful for determination of the major cleavage products obtained upon treatment of membrane bound rhodopsin with endoproteinase Asp-N and thermolysin. Our results indicate that the MALDI method is a useful means of obtaining accurate molecular weight information on hydrophobic proteins isolated in their native membranes.

Shotgun Proteomic Analysis of Cerebrospinal Fluid Using Off-Gel Electrophoresis as the First Dimension Separation

Shotgun proteomic analysis usually employs multidimensional separations with the first dimension most commonly being strong cation exchange (SCX) liquid chromatography (LC). SCX-LC is necessarily a serial process for preparation of multiple samples. Here, we apply a newly available tool, off-gel electrophoresis (OGE), for first-dimension separation of peptide mixtures from digests of cerebrospinal fluid (CSF), a complex and low total protein-containing sample. OGE first-dimension fractionation enabled identification of a total of 156 unique proteins compared to 115 identified in previous work using first-dimension SCX fractionation. OGE can be used to process multiple samples unattended with easy retrieval of the separated fractions. Thus, shotgun analysis using OGE as the first-dimension separation offers a significant advantage both in terms of sample throughput as well as increased numbers of identified proteins.