High-Throughput, Quantitative Analysis of Peptide-Exchanged MHCI Complexes by Native Mass Spectrometry.

Immune monitoring in cancer immunotherapy involves screening CD8+ T-cell responses against neoantigens, the tumor-specific peptides presented by Major histocompatibility complex Class I (MHCI) on the cell surface. High-throughput immune monitoring requires methods to produce and characterize small quantities of thousands of MHCI-peptide complexes that may be tested for a patient's T-cell response. MHCI synthesis has been achieved using a photocleavable peptide that is exchanged by the neoantigen; however, assays that measure peptide exchange currently disassemble the complex prior to analysis─precluding direct molecular characterization. Here, we use native mass spectrometry (MS) to profile intact recombinant MHCI complexes and directly measure peptide exchange. Coupled with size-exclusion chromatography or capillary-zone electrophoresis, the assay identified all tested human leukocyte antigen (HLA)/peptide combinations in the nanomole to picomole range with minimal run time, reconciling the synthetic and analytical requirements of MHCI-peptide screening with the downstream T-cell assays. We further show that the assay can be "multiplexed" by measuring exchange of multiple peptides simultaneously and also enables calculation of Vc50, a measure of gas-phase stability. Additionally, MHCI complexes were fragmented by top-down sequencing, demonstrating that the intact complex, peptide sequence, and their binding affinity can be determined in a single analysis. This screening tool for MHCI-neoantigen complexes represents a step toward the application of state-of-the-art MS technology in translational settings. Not only is this assay already informing on the viability of immunotherapy in practice, the platform also holds promise to inspire novel MS readouts for increasingly complex biomolecules used in the diagnosis and treatment of disease.

On-Chip Preconcentration Microchip Capillary Electrophoresis Based CE-PRM-LIVE for High-Throughput Selectivity Profiling of Deubiquitinase Inhibitors.

The family of deubiquitinases (DUBs) comprises ∼100 enzymes that cleave ubiquitin from substrate proteins and thereby regulate key aspects of human physiology. DUBs have recently emerged as disease-relevant and chemically tractable, although currently there are no approved DUB-targeting drugs and most preclinical small molecules are low-potency and/or multitargeted. We paired a novel capillary electrophoresis microchip containing an integrated, "on-chip" C18 bed (SPE-ZipChip) with a TMT version of our recently described PRM-LIVE acquisition scheme on a timsTOF Pro mass spectrometer to facilitate rapid activity-based protein profiling of DUB inhibitors. We demonstrate the ability of the SPE-ZipChip to improve proteome coverage of complex samples as well as the quantitation integrity of CE-PRM-LIVE for TMT labeled samples. These technologies provide a platform to accurately quantify competitive binding of covalent and reversible inhibitors in a multiplexed assay that spans 49 endogenous DUBs in less than 15 min.

Characterisation of the T-cell response to Ebola virus glycoprotein amongst survivors of the 2013-16 West Africa epidemic.

Zaire ebolavirus (EBOV) is a highly pathogenic filovirus which can result in Ebola virus disease (EVD); a serious medical condition that presents as flu like symptoms but then often leads to more serious or fatal outcomes. The 2013-16 West Africa epidemic saw an unparalleled number of cases. Here we show characterisation and identification of T cell epitopes in surviving patients from Guinea to the EBOV glycoprotein. We perform interferon gamma (IFNγ) ELISpot using a glycoprotein peptide library to identify T cell epitopes and determine the CD4+ or CD8+ T cell component response. Additionally, we generate data on the T cell phenotype and measure polyfunctional cytokine secretion by these antigen specific cells. We show candidate peptides able to elicit a T cell response in EBOV survivors and provide inferred human leukocyte antigen (HLA) allele restriction. This data informs on the long-term T cell response to Ebola virus disease and highlights potentially important immunodominant peptides.

Measurement of plasma HIV-1 RNA below the limit of quantification (<20 copies/mL) of commercial assays with the integrase HIV RNA single-copy assay.

BACKGROUND: Plasma HIV-1 RNA (viral load, VL) is measured routinely in HIV-infected persons with FDA-approved commercially available assays such as the Cobas-TaqMan HIV-1 Assay v2.0. This assay provides quantification of viremia ≥20 copies/mL. More sensitive methods, able to quantify low-level persistent viremia below the detection limit of commercially available assays, are needed to assess the impact of current HIV cure strategies on viremia. OBJECTIVES: The novel integrase HIV-1 RNA single-copy assay (iSCA) was evaluated for measurement of low-level persistent viremia in clinical trial samples (n = 151) from subjects participating in Gilead HIV clinical research. STUDY DESIGN: Paired plasma samples from HIV-1-infected patients treated with combination ART were assessed using both HIV-1 Cobas-TaqMan and iSCA; results from the two assays were compared. RESULTS: Paired Cobas-TaqMan/iSCA data were obtained for 151 HIV-infected adults. Most samples (117/151, 77%) had non-quantifiable Cobas-TaqMan result, either <20 copies/mL ("<20") or "Target Not Detected" (TND). All 117 non-quantified samples were quantified with iSCA and showed higher HIV-1 RNA levels in samples with <20 than TND Cobas-TaqMan results (p < 0.0001). CONCLUSIONS: In this large sample collection from virologically suppressed HIV-infected adults, use of iSCA led to quantification of low-level viremia below the limit of detection of the Cobas-TaqMan assay in all 117 previously non-quantifiable plasma samples. These data confirm the value of the iSCA as a helpful addition to the classical HIV VL assays and its potential for use in HIV cure studies to assess whether experimental interventions alter viremia.

Coupling Microchip Electrospray Ionization Devices with High Pressure Mass Spectrometry.

A microchip electrospray ionization source was coupled with high pressure mass spectrometry (HPMS). A continuous atmospheric inlet consisting of a stainless steel capillary and DC ion optics was designed to conduct ions into the mass spectrometer. Infusions of amino acids and peptides were performed and detected with a miniature cylindrical ion trap (mini-CIT)-based mass spectrometer operated at ≥1 Torr with air as the buffer gas. Detection of glycine and thymopentin (separately) demonstrated the mass range of the mini-CIT detector could span from m/z 75 to 681. A microchip capillary electrophoresis (CE) separation with mini-CIT detection was performed, and the results were compared with detection using a commercial instrument (Waters Synapt G2). Comparable separation efficiencies were observed with both mass spectrometers as detectors, with about 6 times better signal-to-noise observed on the Synapt G2. Comparison of mass spectra in the two systems reveals similar features observed, but with wider peak widths in the mini-CIT than on the Synapt G2 as expected due to high-pressure operation.

High resolution separations of charge variants and disulfide isomers of monoclonal antibodies and antibody drug conjugates using ultra-high voltage capillary electrophoresis with high electric field strength.

Ultra-high voltage capillary electrophoresis with high electric field strength has been applied to the separation of the charge variants, drug conjugates, and disulfide isomers of monoclonal antibodies. Samples composed of many closely related species are difficult to resolve and quantify using traditional analytical instrumentation. High performance instrumentation can often save considerable time and effort otherwise spent on extensive method development. Ideally, the resolution obtained for a given CE buffer system scales with the square root of the applied voltage. Currently available commercial CE instrumentation is limited to an applied voltage of approximately 30kV and a maximum electric field strength of 1kV/cm due to design limitations. The instrumentation described here is capable of safely applying potentials of at least 120kV with electric field strengths over 2000V/cm, potentially doubling the resolution of the best conventional CE buffer/capillary systems while decreasing analysis time in some applications. Separations of these complex mixtures using this new instrumentation demonstrate the potential of ultra-high voltage CE to identify the presence of previously unresolved components and to reduce analysis time for complex mixtures of antibody variants and drug conjugates.

Analysis of Hemoglobin Glycation Using Microfluidic CE-MS: A Rapid, Mass Spectrometry Compatible Method for Assessing Diabetes Management.

Diabetes has become a significant health problem worldwide with the rate of diagnosis increasing rapidly in recent years. Measurement of glycated blood proteins, particularly glycated hemoglobin (HbA1c), is an important diagnostic tool used to detect and manage the condition in patients. Described here is a method using microfluidic capillary electrophoresis with mass spectrometry detection (CE-MS) to assess hemoglobin glycation in whole blood lysate. Using denaturing conditions, the hemoglobin (Hb) tetramer dissociates into the alpha and beta subunits (α- and ß-Hb), which are then separated via CE directly coupled to MS detection. Nearly baseline resolution is achieved between α-Hb, ß-Hb, and glycated ß-Hb. A second glycated ß-Hb isomer that is partially resolved from ß-Hb is detected in extracted ion electropherograms for glycated ß-Hb. Glycation on α-Hb is also detected in the α-Hb mass spectrum. Additional modifications to the ß-Hb are detected, including acetylation and a +57 Da species that could be the addition of a glyoxal moiety. Patient blood samples were analyzed using the microfluidic CE-MS method and a clinically used immunoassay to measure HbA1c. The percentage of glycated α-Hb and ß-Hb was calculated from the microfluidic CE-MS data using peak areas generated from extracted ion electropherograms. The values for glycated ß-Hb were found to correlate well with the HbA1c levels derived in the clinic, giving a slope of 1.20 and an R(2) value of 0.99 on a correlation plot. Glycation of human serum albumin (HSA) can also be measured using this technique. It was observed that patients with elevated glycated Hb levels also had higher levels of HSA glycation. Interestingly, the sample with the highest HbA1c levels did not have the highest levels of glycated HSA. Because the lifetime of HSA is shorter than Hb, this could indicate a recent lapse in glycemic control for that patient. The ability to assess both Hb and HSA glycation has the potential to provide a more complete picture of a patient's glycemic control in the months leading up to blood collection. The results presented here demonstrate that the microfluidic CE-MS method is capable of rapidly assessing Hb and HSA glycation from low volumes of whole blood with minimal sample preparation and has the potential to provide more information in a single analysis step than current technologies.

Characterization of Intact Antibody Drug Conjugate Variants Using Microfluidic Capillary Electrophoresis-Mass Spectrometry.

In this work, we utilize capillary electrophoresis-mass spectrometry (CE-MS) in an integrated microfluidic platform to analyze an intact, lysine-linked antibody drug conjugate (ADC) in order to assess post translational modifications and drug load variants. The initial charge heterogeneity of the unconjugated IgG-2 monoclonal antibody (mAb) was assessed by separating intact charge variants. Three main charge variants were resolved in the CE dimension. These variants were attributed to pyroglutamic acid formation and decarboxylation on the primary structure of the mAb through characteristic mass shifts and changes in electrophoretic mobility. Additionally, glycoforms of the antibody charge variants were identified in the deconvoluted mass spectra. The observed glycoforms and their distribution compared favorably to a released N-glycan analysis performed on the mAb. After conjugation, the ADC was analyzed using the same microchip CE-MS method. The addition of a drug load resulted in a decrease in mobility and an increase in mass of 3145 Da. Five main species that differed in their respective drug-to-antibody ratios (DAR) were fully resolved in the CE separation, with each DAR displaying the same variant population observed on the unconjugated mAb. A DAR range of 0-4 was observed with an average of 1.7 drug loads. The DAR distribution generated from the microfluidic CE-MS data compared favorably to results from infusion-ESI-MS and imaging CE (iCE) analysis of the ADC, techniques commonly used for intact mAb and ADC characterization.

Utilizing Microchip Capillary Electrophoresis Electrospray Ionization for Hydrogen Exchange Mass Spectrometry.

Hydrogen exchange (HX) mass spectrometry (MS) of complex mixtures requires a fast, reproducible, and high peak capacity separation prior to MS detection. The current paradigm relies on liquid chromatography (LC) with fast gradients performed at low temperatures to minimize back exchange. Unfortunately, under these conditions, the efficiency of LC is limited due to resistance to mass transfer, reducing the capability to analyze complex samples. Capillary electrophoresis (CE), on the other hand, is not limited by resistance to mass transfer, enabling very rapid separations that are not adversely affected by low temperature. Previously, we have demonstrated an integrated microfluidic device coupling CE with electrospray ionization (ESI) capable of very rapid and high efficiency separations. In this work, we demonstrate the utility of this microchip CE-ESI device for HX MS. High speed CE-ESI of a bovine hemoglobin pepsin digestion was performed in 1 min with a peak capacity of 62 versus a similar LC separation performed in 7 min with peak capacity of 31. A room temperature CE method performed in 1.25 min provided similar deuterium retention as an 8.5 min LC method conducted at 0 °C. Separation of a complex mixture with CE was done with considerably better speed and nearly triple the peak capacity than the equivalent separation by LC. Overall, the results indicate the potential utility of microchip CE-ESI for HX MS.

Integrated microfluidic capillary electrophoresis-electrospray ionization devices with online MS detection for the separation and characterization of intact monoclonal antibody variants.

Here, we demonstrate an integrated microfluidic capillary electrophoresis-electrospray ionization (CE-ESI) device for the separation of intact monoclonal antibody charge variants with online mass spectrometric (MS) identification. The need for dynamic coating and zwitterionic background electrolyte (BGE) additives has been eliminated by utilizing surface chemistry within the device channels to control analyte adsorption and electroosmotic flow (EOF) while maintaining separation efficiency. The effectiveness of this strategy was illustrated with the separation of charge variants of Infliximab. Three major species corresponding to C-terminal lysine variants were separated with an average resolution of 0.80 and identified by mass difference. In addition to the lysine variants, masses were determined for minor acidic and basic species. The separation of these variants prior to MS analysis facilitated the identification of glycosylation patterns for each of the variants. The general applicability of this method was demonstrated by analyzing two additional monoclonal antibody species: an IgG2 antibody and an IgG1 antibody conjugate. The IgG2 proved to have similar modifications to Infliximab with lower relative abundances of the lysine variants. Analysis of the IgG1 drug conjugate further exemplified the advantages of MS detection; differences in the extent of antibody conjugation were detectable despite limited CE resolution. The CE-ESI-MS methodology described here is a rapid and generic strategy for the separation of intact mAb charge variants and facilitates the identification of variants through MS detection.

HIV latency. Specific HIV integration sites are linked to clonal expansion and persistence of infected cells.

The persistence of HIV-infected cells in individuals on suppressive combination antiretroviral therapy (cART) presents a major barrier for curing HIV infections. HIV integrates its DNA into many sites in the host genome; we identified 2410 integration sites in peripheral blood lymphocytes of five infected individuals on cART. About 40% of the integrations were in clonally expanded cells. Approximately 50% of the infected cells in one patient were from a single clone, and some clones persisted for many years. There were multiple independent integrations in several genes, including MKL2 and BACH2; many of these integrations were in clonally expanded cells. Our findings show that HIV integration sites can play a critical role in expansion and persistence of HIV-infected cells.

Chemical vapor deposition of aminopropyl silanes in microfluidic channels for highly efficient microchip capillary electrophoresis-electrospray ionization-mass spectrometry.

We describe a chemical vapor deposition (CVD) method for the surface modification of glass microfluidic devices designed to perform electrophoretic separations of cationic species. The microfluidic channel surfaces were modified using aminopropyl silane reagents. Coating homogeneity was inferred by precise measurement of the separation efficiency and electroosmotic mobility for multiple microfluidic devices. Devices coated with (3-aminopropyl)di-isopropylethoxysilane (APDIPES) yielded near diffusion-limited separations and exhibited little change in electroosmotic mobility between pH 2.8 and pH 7.5. We further evaluated the temporal stability of both APDIPES and (3-aminopropyl)triethoxysilane (APTES) coatings when stored for a total of 1 week under vacuum at 4 °C or filled with pH 2.8 background electrolyte at room temperature. Measurements of electroosmotic flow (EOF) and separation efficiency during this time confirmed that both coatings were stable under both conditions. Microfluidic devices with a 23 cm long, serpentine electrophoretic separation channel and integrated nanoelectrospray ionization emitter were CVD coated with APDIPES and used for capillary electrophoresis (CE)-electrospray ionization (ESI)-mass spectrometry (MS) of peptides and proteins. Peptide separations were fast and highly efficient, yielding theoretical plate counts over 600,000 and a peak capacity of 64 in less than 90 s. Intact protein separations using these devices yielded Gaussian peak profiles with separation efficiencies between 100,000 and 400,000 theoretical plates.

Hybrid capillary/microfluidic system for comprehensive online liquid chromatography-capillary electrophoresis-electrospray ionization-mass spectrometry.

A hybrid multidimensional separation system was made by coupling capillary liquid chromatography (LC) to a microfluidic device. The microfluidic device integrated flow splitting, capillary electrophoresis (CE), electroosmotic pumping, and electrospray ionization (ESI) emitter functional elements. The system was used with a time-of-flight mass spectrometer for comprehensive online LC-CE-MS of proteolytic digests. Analysis of a complex mixture of peptides yielded a peak capacity of approximately 1400 in 50 min. Three replicate runs demonstrated mean reproducibility for LC retention and CE migration times of 0.32% and 0.75% relative standard deviation (RSD), respectively. The same LC-CE-MS method was also used to characterize the N-linked glycosylation of a monoclonal antibody. Glycopeptides from two different N-linked glycosylation sites were separated from all other tryptic peptides and identified using MS data. The relative amounts of each glycoform and total site occupancy were quantified using LC-CE-MS data.

Monolithic integration of two-dimensional liquid chromatography-capillary electrophoresis and electrospray ionization on a microfluidic device.

A microfluidic device capable of two-dimensional reversed-phase liquid chromatography-capillary electrophoresis with integrated electrospray ionization (LC-CE-ESI) for mass spectrometry (MS)-based proteomic applications is described. Traditional instrumentation was used for the LC sample injection and delivery of the LC mobile phase. The glass microfabricated device incorporated a sample-trapping region and an LC channel packed with reversed-phase particles. Rapid electrokinetic injections of the LC effluent into the CE dimension were performed at a cross-channel intersection. The CE separation channel terminated at a corner of the square device, which functioned as an integrated electrospray tip. In addition to LC-CE-ESI, this device was used for LC-ESI without any instrumental modifications. To evaluate the system, LC-MS and LC-CE-MS analyses of protein digests were performed and compared.

Prevalence of XMRV nucleic acid and antibody in HIV-1-Infected men and in men at risk for HIV-1 Infection.

Xenotropic MLV-Related Virus (XMRV) was recently reported to be associated with prostate cancer and chronic fatigue syndrome (CFS). Infection was also reported in 3.7% of healthy individuals. These highly reported frequencies of infection prompted concerns about the possibility of a new, widespread retroviral epidemic. The Multicenter AIDS Cohort Study (MACS) provides an opportunity to assess the prevalence of XMRV infection and its association with HIV-1 infection among men who have sex with men. Reliable detection of XMRV infection requires the application of multiple diagnostic methods, including detection of human antibodies to XMRV and detection of XMRV nucleic acid. We, therefore, tested 332 patient plasma and PBMC samples obtained from recent visits in a subset of patients in the MACS cohort for XMRV antibodies using Abbott prototype ARCHITECT chemiluminescent immunoassays (CMIAs) and for XMRV RNA and proviral DNA using a XMRV single-copy qPCR assay (X-SCA). Although 9 of 332 (2.7%) samples showed low positive reactivity against a single antigen in the CMIA, none of these samples or matched controls were positive for plasma XMRV RNA or PBMC XMRV DNA by X-SCA. Thus, we found no evidence of XMRV infection among men in the MACS regardless of HIV-1 serostatus.

Nucleic Acid, Antibody, and Virus Culture Methods to Detect Xenotropic MLV-Related Virus in Human Blood Samples.

The MLV-related retrovirus, XMRV, was recently identified and reported to be associated with both prostate cancer and chronic fatigue syndrome. At the National Cancer Institute-Frederick, MD (NCI-Frederick), we developed highly sensitive methods to detect XMRV nucleic acids, antibodies, and replication competent virus. Analysis of XMRV-spiked samples and/or specimens from two pigtail macaques experimentally inoculated with 22Rv1 cell-derived XMRV confirmed the ability of the assays used to detect XMRV RNA and DNA, and culture isolatable virus when present, along with XMRV reactive antibody responses. Using these assays, we did not detect evidence of XMRV in blood samples (N = 134) or prostate specimens (N = 19) from two independent cohorts of patients with prostate cancer. Previous studies detected XMRV in prostate tissues. In the present study, we primarily investigated the levels of XMRV in blood plasma samples collected from patients with prostate cancer. These results demonstrate that while XMRV-related assays developed at the NCI-Frederick can readily measure XMRV nucleic acids, antibodies, and replication competent virus, no evidence of XMRV was found in the blood of patients with prostate cancer.

Short-course raltegravir intensification does not reduce persistent low-level viremia in patients with HIV-1 suppression during receipt of combination antiretroviral therapy.

BACKGROUND: Combination antiretroviral therapy suppresses but does not eradicate human immunodeficiency virus type 1 (HIV-1) in infected persons, and low-level viremia can be detected despite years of suppressive antiretroviral therapy. Short-course (28-day) intensification of standard antiretroviral combination therapy is a useful approach to determine whether complete rounds of HIV-1 replication in rapidly cycling cells contribute to persistent viremia. We investigated whether intensification with the integrase inhibitor raltegravir decreases plasma HIV-1 RNA levels in patients receiving suppressive antiretroviral therapy. METHODS: Subjects (n = 10) with long-term HIV-1 suppression receiving combination antiretroviral regimens had their regimens intensified for 4 weeks with raltegravir. Plasma HIV-1 RNA level was determined before, during, and after the 4-week intensification period, using a sensitive assay (limit of detection, 0.2 copies of HIV-1 RNA/mL of plasma). A 4-week intensification course was chosen to investigate potential HIV-1 replication in cells with relatively short (approximately 1-14-day) half-lives. RESULTS: There was no evidence in any subject of a decline in HIV-1 RNA level during the period of raltegravir intensification or of rebound after discontinuation. Median levels of HIV-1 RNA before (0.17 log10 copies/mL), during (0.04 log10 copies/mL), and after (0.04 log10 copies/mL) raltegravir intensification were not significantly different (P > .1 for all comparisons in parametric analyses). High-performance liquid chromatography and mass spectroscopy experiments confirmed that therapeutic levels of raltegravir were achieved in plasma during intensification. CONCLUSIONS: Intensification of antiretroviral therapy with a potent HIV-1 integrase inhibitor did not decrease persistent viremia in subjects receiving suppressive regimens, indicating that rapidly cycling cells infected with HIV-1 were not present. Eradication of HIV-1 from infected persons will require new therapeutic approaches. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT00618371.

Integrated microfluidic device for automated single cell analysis using electrophoretic separation and electrospray ionization mass spectrometry.

A microfabricated fluidic device was developed for the automated real-time analysis of individual cells using capillary electrophoresis (CE) and electrospray ionization-mass spectrometry (ESI-MS). The microfluidic structure incorporates a means for rapid lysis of single cells within a free solution electrophoresis channel, where cellular constituents were separated, and an integrated electrospray emitter for ionization of separated components. The eluent was characterized using mass spectrometry. Human erythrocytes were used as a model system for this study. In this monolithically integrated device, cell lysis occurs at a channel intersection using a combination of rapid buffer exchange and an increase in electric field strength. An electroosmotic pump is incorporated at the end of the electrophoretic separation channel to direct eluent to the integrated electrospray emitter. The dissociated heme group and the alpha and beta subunits of hemoglobin from individual erythrocytes were detected as cells continuously flowed through the device. The average analysis throughput was approximately 12 cells per minute, demonstrating the potential of this method for high-throughput single cell analysis.

Treatment intensification does not reduce residual HIV-1 viremia in patients on highly active antiretroviral therapy.

In HIV-1-infected individuals on currently recommended antiretroviral therapy (ART), viremia is reduced to <50 copies of HIV-1 RNA per milliliter, but low-level residual viremia appears to persist over the lifetimes of most infected individuals. There is controversy over whether the residual viremia results from ongoing cycles of viral replication. To address this question, we conducted 2 prospective studies to assess the effect of ART intensification with an additional potent drug on residual viremia in 9 HIV-1-infected individuals on successful ART. By using an HIV-1 RNA assay with single-copy sensitivity, we found that levels of viremia were not reduced by ART intensification with any of 3 different antiretroviral drugs (efavirenz, lopinavir/ritonavir, or atazanavir/ritonavir). The lack of response was not associated with the presence of drug-resistant virus or suboptimal drug concentrations. Our results suggest that residual viremia is not the product of ongoing, complete cycles of viral replication, but rather of virus output from stable reservoirs of infection.

Capillary-based instrument for the simultaneous measurement of solution viscosity and solute diffusion coefficient at pressures up to 2000 bar and implications for ultrahigh pressure liquid chromatography.

An instrument based on the Poiseuille flow principle capable of measuring solution viscosities at high pressures has been modified to observe UV-absorbent analytes in order to allow for the simultaneous measurement of analyte diffusivity. A capillary time-of-flight (CTOF) instrument was used to measure the viscosity of acetonitrile-water mixtures in all decade volume percent increments and the corresponding diffusion coefficients of small aromatic molecules in these solvent mixtures from atmospheric pressure to 2000 bar (approximately 30,000 psi) at 25 degrees C. The instrument works by utilizing a relatively small pressure drop (<100 bar) across a fused-silica capillary which has both the inlet and outlet pressurized so that the average column pressure can be significantly elevated (up to 2000 bar). Measurements with this instrument agree with high-pressure viscosity data collected previously using a CTOF viscometer, as well as with literature values obtained with falling-body viscometers of the Bridgman design. It has been further determined that, for the small molecules included in this study, trends in solute diffusivity with respect to pressure follow the predictions of the Stokes-Einstein equation when the solvent viscosity is corrected as a function of pressure. Because the instrument described herein determines viscosity and diffusivity independently, the effect of pressure on analyte hydrodynamic radius can also be monitored. An analysis of ultrahigh pressure liquid chromatography (UHPLC) data was performed using the pressure-corrected diffusion coefficient of hydroquinone to demonstrate the effect of this phenomenon on the analysis of chromatographic performance.