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Pine Island Glacier and Thwaites Glacier in the Amundsen Sea Embayment are among the fastest changing outlet glaciers in West Antarctica with large consequences for global sea level. Yet, assessing how much and how fast both glaciers will weaken if these changes continue remains a major uncertainty as many of the processes that control their ice shelf weakening and grounding line retreat are not well understood. Here, we combine multisource satellite imagery with modeling to uncover the rapid development of damage areas in the shear zones of Pine Island and Thwaites ice shelves. These damage areas consist of highly crevassed areas and open fractures and are first signs that the shear zones of both ice shelves have structurally weakened over the past decade. Idealized model results reveal moreover that the damage initiates a feedback process where initial ice shelf weakening triggers the development of damage in their shear zones, which results in further speedup, shearing, and weakening, hence promoting additional damage development. This damage feedback potentially preconditions these ice shelves for disintegration and enhances grounding line retreat. The results of this study suggest that damage feedback processes are key to future ice shelf stability, grounding line retreat, and sea level contributions from Antarctica. Moreover, they underline the need for incorporating these feedback processes, which are currently not accounted for in most ice sheet models, to improve sea level rise projections. [ABSTRACT FROM AUTHOR]

We present a reconstruction of historical (1950–2014) surface mass balance (SMB) of the Greenland ice sheet (GrIS) using a high-resolution regional climate model (RACMO2; ∼11 km) to dynamically downscale the climate of the Community Earth System Model version 2 (CESM2; ∼111 km). After further statistical downscaling to 1 km spatial resolution, evaluation using in situ SMB measurements and remotely sensed GrIS mass change shows good agreement. Comparison with an ensemble of previously conducted RACMO2 simulations forced by climate reanalysis demonstrates that the current product realistically represents the long-term average and variability of individual SMB components and captures the recent increase in meltwater runoff that accelerated GrIS mass loss. This means that, for the first time, climate forcing from an Earth system model (CESM2), which assimilates no observations, can be used without additional corrections to reconstruct the historical GrIS SMB and its recent decline that initiated mass loss in the 1990s. This paves the way for attribution studies of future GrIS mass loss projections and contribution to sea level rise. [ABSTRACT FROM AUTHOR]

The present paper discusses practical aspects of prototyping of microfluidic chips using cyclic olefin copolymer as substrate and the application in high-performance liquid chromatography. The developed chips feature a 60 mm long straight separation channel with circular cross section (500 μm i.d.) that was created using a micromilling robot. To irreversibly seal the top and bottom chip substrates, a solvent-vapor-assisted bonding approach was optimized, allowing to approximate the ideal circular channel geometry. Four different approaches to establish the micro-to-macro interface were pursued. The average burst pressure of the microfluidic chips in combination with an encasing holder was established at 38 MPa and the maximum burst pressure was 47 MPa, which is believed to be the highest ever report for these polymer-based microfluidic chips. Porous polymer monolithic frits were synthesized in-situ via UV-initiated polymerization and their locations were spatially controlled by the application of a photomask. Next, high-pressure slurry packing was performed to introduce 3 μm silica reversed-phase particles as the stationary phase in the separation channel. Finally, the application of the chip technology is demonstrated for the separation of alkyl phenones in gradient mode yielding baseline peak widths of 6 s by applying a steep gradient of 1.8 min at a flow rate of 10 μL/min. [ABSTRACT FROM AUTHOR]

Sample preparation is a labor-intensive and time-consuming procedure, especially for the bioanalysis of small-volume samples with low-abundant analytes. To minimize losses and dilution, sample preparation should ideally be hyphenated to downstream on-line analysis such as liquid chromatography-mass spectrometry (LC-MS). In this study, an automated three-phase electro-extraction (EE) method coupled to machine vision was developed, integrated with a robotic autosampler hyphenated to LC-MS. Eight model compounds, i.e. amitriptyline, clemastine, clomipramine, haloperidol, loperamide, propranolol, oxeladin, and verapamil were utilized for the optimization and evaluation of the automated EE setup. The stability of automated EE was evaluated by monitoring the acceptor droplet size by machine vision and recording the current during EE. A Design of Experiment approach (Box-Behnken design) was utilized to optimize the critical parameters of the EE method, i.e., the ratio of formic acid in the sample to acceptor phase, extraction voltage, and extraction time. The developed quadratic models showed good fitness (p < 0.001, R2 > 0.95). Automated EE could be achieved in less than 2 min with enrichment factors (EF) up to 387 and extraction recoveries (ER) up to 97% for academic samples. Finally, the optimized EE method was successfully applied to both spiked human urine and plasma samples with low-concentration (50 ng mL−1) analytes and a low starting sample volume of 20 μL of plasma and urine in 10-fold diluted samples. The developed automated EE setup is easy to operate, provides a fast extraction method for analytes from volume-limited biological samples, and is hyphenated with on-line LC-MS analysis. Therefore, this method can provide fast and automated sample preparation to solve bottlenecks in high-throughput bioanalysis workflows. [Display omitted] • An automated EE setup was integrated with a robotic autosampler coupled to LC-MS. • The stability of EE process was assessed by machine vision and current monitoring. • High enrichment factors up to 387 of the automated three-phase EE were achieved. • Limits of detection were as low as 3.2 pg mL-1 in plasma samples. [ABSTRACT FROM AUTHOR]

Satellite gravimetry is a unique measurement technique for observing mass transport processes in the Earth system on a global scale, providing essential indicators of both subtle and dramatic global change. Although past and current satellite gravity missions have achieved spectacular science results, due to their limited spatial and temporal resolution as well as limited length of the available time series numerous important questions are still unresolved. Therefore, it is important to move from current demonstration capabilities to sustained observation of the Earth's gravity field. In an international initiative performed under the umbrella of the International Union of Geodesy and Geophysics, consensus on the science and user needs for a future satellite gravity observing system has been derived by an international panel of scientists representing the main fields of application, i.e., continental hydrology, cryosphere, ocean, atmosphere and solid Earth. In this paper the main results and findings of this initiative are summarized. The required target performance in terms of equivalent water height has been identified as 5 cm for monthly fields and 0.5 cm/year for long-term trends at a spatial resolution of 150 km. The benefits to meet the main scientific and societal objectives are investigated, and the added value is demonstrated for selected case studies covering the main fields of application. The resulting consolidated view on the required performance of a future sustained satellite gravity observing system represents a solid basis for the definition of technological and mission requirements, and is a prerequisite for mission design studies of future mission concepts and constellations. [ABSTRACT FROM AUTHOR]

The impact of initial conditions relative to external forcings in decadal integrations from an ensemble of state-of-the-art prediction models has been assessed using specifically designed sensitivity experiments (SWAP experiments). They consist of two sets of 10-yr-long ensemble hindcasts for two initial dates in 1965 and 1995 using either the external forcings from the "correct" decades or swapping the forcings between the two decades. By comparing the two sets of integrations, the impact of external forcing versus initial conditions on the predictability over multiannual time scales was estimated as the function of lead time of the hindcast. It was found that over time scales longer than about 1 yr, the predictability of sea surface temperatures (SSTs) on a global scale arises mainly from the external forcing. However, the correct initialization has a longer impact on SST predictability over specific regions such as the North Atlantic, the northwestern Pacific, and the Southern Ocean. The impact of initialization is even longer and extends to wider regions when below-surface ocean variables are considered. For the western and eastern tropical Atlantic, the impact of initialization for the 700-m heat content (HTC700) extends to as much as 9 years for some of the models considered. In all models the impact of initial conditions on the predictability of the Atlantic meridional overturning circulation (AMOC) is dominant for the first 5 years. [ABSTRACT FROM AUTHOR]

A multi-model analysis of Atlantic multidecadal variability is performed with the following aims: to investigate the similarities to observations; to assess the strength and relative importance of the different elements of the mechanism proposed by Delworth et al. (J Clim 6:1993-2011, 1993) (hereafter D93) among coupled general circulation models (CGCMs); and to relate model differences to mean systematic error. The analysis is performed with long control simulations from ten CGCMs, with lengths ranging between 500 and 3600 years. In most models the variations of sea surface temperature (SST) averaged over North Atlantic show considerable power on multidecadal time scales, but with different periodicity. The SST variations are largest in the mid-latitude region, consistent with the short instrumental record. Despite large differences in model configurations, we find quite some consistency among the models in terms of processes. In eight of the ten models the mid-latitude SST variations are significantly correlated with fluctuations in the Atlantic meridional overturning circulation (AMOC), suggesting a link to northward heat transport changes. Consistent with this link, the three models with the weakest AMOC have the largest cold SST bias in the North Atlantic. There is no linear relationship on decadal timescales between AMOC and North Atlantic Oscillation in the models. Analysis of the key elements of the D93 mechanisms revealed the following: Most models present strong evidence that high-latitude winter mixing precede AMOC changes. However, the regions of wintertime convection differ among models. In most models salinity-induced density anomalies in the convective region tend to lead AMOC, while temperature-induced density anomalies lead AMOC only in one model. However, analysis shows that salinity may play an overly important role in most models, because of cold temperature biases in their relevant convective regions. In most models subpolar gyre variations tend to lead AMOC changes, and this relation is strong in more than half of the models. [ABSTRACT FROM AUTHOR]

The modeling of the surface mass balance (SMB) of the Greenland Ice Sheet (GIS) requires high-resolution models in order to capture the observed large gradients in the steep marginal areas. Until now, global climate models have not been considered suitable to model ice sheet SMB owing to model biases and insufficient resolution. This study analyzes the GIS SMB simulated for the period 1850-2005 by the Community Earth System Model (CESM), which includes a new ice sheet component with multiple elevation classes for SMB calculations. The model is evaluated against observational data and output from the regional model Regional Atmospheric Climate Model version 2 (RACMO2). Because of a lack of major climate biases, a sophisticated calculation of snow processes (including surface albedo evolution) and an adequate downscaling technique, CESM is able to realistically simulate GIS surface climate and SMB. CESM SMB agrees reasonably well with in situ data from 475 locations ( r = 0.80) and output from RACMO2 ( r = 0.79). The simulated mean SMB for 1960-2005 is 359 ± 120 Gt yr−1 in the range of estimates from regional climate models. The simulated seasonal mass variability is comparable with mass observations from the Gravity Recovery and Climate Experiment (GRACE), with synchronous annual maximum (May) and minimum (August-September) and similar amplitudes of the seasonal cycle. CESM is able to simulate the bands of precipitation maxima along the southeast and northwest margins, but absolute precipitation rates are underestimated along the southeastern margin and overestimated in the high interior. The model correctly simulates the major ablation areas. Total refreezing represents 35% of the available liquid water (the sum of rain and melt). [ABSTRACT FROM AUTHOR]

Glaciers distinct from the Greenland and Antarctic Ice Sheets are losing large amounts of water to the world's oceans. However, estimates of their contribution to sea level rise disagree. We provide a consensus estimate by standardizing existing, and creating new, mass-budget estimates from satellite gravimetry and altimetry and from local glaciological records. In many regions, local measurements are more negative than satellite-based estimates. All regions lost mass during 2003-2009, with the largest losses from Arctic Canada, Alaska, coastal Greenland, the southern Andes, and high-mountain Asia, but there was little loss from glaciers in Antarctica. Over this period, the global mass budget was -259 ± 28 gigatons per year, equivalent to the combined loss from both ice sheets and accounting for 29 ± 13% of the observed sea level rise. [ABSTRACT FROM AUTHOR]

Lipid nanoparticle (LNP) remains the most advanced platform for messenger RNA (mRNA) delivery. To date, mRNA LNPs synthesis is mostly performed by mixing lipids and mRNA with microfluidics. In this study, a cost-effective microfluidic setup for synthesizing mRNA LNPs is developed. It allows to fine-tune the LNPs characteristics without compromising LNP properties. It is compared with a commercial device (NanoAssemblr) and ethanol injection and the influence of manufacturing conditions on the performance of mRNA LNPs is investigated. LNPs prepared by ethanol injection exhibit broader size distributions and more inhomogeneous internal structure (e.g., bleb-like substructures), while other LNPs show uniform structure with dense cores. Small angel X-ray scattering (SAXS) data indicate a tighter interaction between mRNA and lipids within LNPs synthesized by custom device, compared to LNPs produced by NanoAssemblr. Interestingly, the better transfection efficiency of polysarcosine (pSar)-modified LNPs correlates with a higher surface roughness than that of PEGylated ones. The manufacturing approach, however, shows modest influence on mRNA expression in vivo. In summary, the home-developed cost-effective microfluidic device can synthesize LNPs and represents a potent alternative to NanoAssemblr. The preparation methods show notable effect on LNPs' structure but a minor influence on mRNA delivery in vitro and in vivo., (© 2024 The Author(s). Advanced Healthcare Materials published by Wiley‐VCH GmbH.)

Antarctic ice sheet (AIS) mass loss is predominantly driven by increased solid ice discharge, but its variability is governed by surface processes. Snowfall fluctuations control the surface mass balance (SMB) of the grounded AIS, while meltwater ponding can trigger ice shelf collapse potentially accelerating discharge. Surface processes are essential to quantify AIS mass change, but remain poorly represented in climate models typically running at 25-100 km resolution. Here we present SMB and surface melt products statistically downscaled to 2 km resolution for the contemporary climate (1979-2021) and low, moderate and high-end warming scenarios until 2100. We show that statistical downscaling modestly enhances contemporary SMB (3%), which is sufficient to reconcile modelled and satellite mass change. Furthermore, melt strongly increases (46%), notably near the grounding line, in better agreement with in-situ and satellite records. The melt increase persists by 2100 in all warming scenarios, revealing higher surface melt rates than previously estimated., (© 2023. The Author(s).)

With the development of highly sensitive bioanalytical techniques, the volume of samples necessary for accurate analysis has reduced. Microsampling, the process of obtaining small amounts of blood, has thus gained popularity as it offers minimal-invasiveness, reduced logistical costs and biohazard risks while simultaneously showing increased sample stability and a potential for the decentralization of the approach and at-home self-sampling. Although the benefits of microsampling have been recognised, its adoption in clinical practice has been slow. Several microsampling technologies and devices are currently available and employed in research studies for various biomedical applications. This review provides an overview of the state-of-the-art in microsampling technology with a focus on the latest developments and advancements in the field of microsampling. Research published in the year 2022, including studies (i) developing strategies for the quantitation of analytes in microsamples and (ii) bridging and comparing the interchangeability between matrices and choice of technology for a given application, is reviewed to assess the advantages, challenges and limitations of the current state of microsampling. Successful implementation of microsampling in routine clinical care requires continued efforts for standardization and harmonization. Microsampling has been shown to facilitate data-rich studies and a patient-centric approach to healthcare and is foreseen to play a central role in the future digital revolution of healthcare through continuous monitoring to improve the quality of life., Competing Interests: The authors have declared no conflict of interest., (© 2023 The Authors. Analytical Science Advances published by Wiley‐VCH GmbH.)

Glaciers distinct from the Greenland and Antarctic ice sheets are currently losing mass rapidly with direct and severe impacts on the habitability of some regions on Earth as glacier meltwater contributes to sea-level rise and alters regional water resources in arid regions. In this review, we present the different techniques developed during the last two decades to measure glacier mass change from space: digital elevation model (DEM) differencing from stereo-imagery and synthetic aperture radar interferometry, laser and radar altimetry and space gravimetry. We illustrate their respective strengths and weaknesses to survey the mass change of a large Arctic ice body, the Vatnajökull Ice Cap (Iceland) and for the steep glaciers of the Everest area (Himalaya). For entire regions, mass change estimates sometimes disagree when a similar technique is applied by different research groups. At global scale, these discrepancies result in mass change estimates varying by 20%-30%. Our review confirms the need for more thorough inter-comparison studies to understand the origin of these differences and to better constrain regional to global glacier mass changes and, ultimately, past and future glacier contribution to sea-level rise., (© 2023 IOP Publishing Ltd.)

High-throughput analysis in fields such as industrial biotechnology, combinatorial chemistry, and life sciences is becoming increasingly important. Nuclear magnetic resonance (NMR) spectroscopy is a powerful technique providing exhaustive molecular information on complex samples. Flow NMR in particular is a cost- and time-efficient method for large screenings. In this study, we have developed a novel 3.0 mm inner diameter polychlorotrifluoroethylene (PCTFE) flow cell for a segmented-flow analysis (SFA) - NMR automated platform. The platform uses FC-72 fluorinated oil and fluoropolymer components to achieve a fully fluorinated flow path. Samples were repeatably transferred from 96-deepwell plates to the flow cell by displacing a fixed volume of oil, with a transfer time of 42 s. 1 H spectra were acquired fully automated with 500 and 600 MHz NMR spectrometers. The spectral performance of the novel PCTFE cell was equal to that of commercial glass cells. Peak area repeatability was excellent with a relative standard deviation of 0.1-0.5% for standard samples, and carryover was below 0.2% without intermediate washing. The sample temperature was conditioned by using a thermostated transfer line in order to reduce the equilibration time in the probe and increase the throughput. Finally, analysis of urine samples demonstrated the applicability of this platform for screening complex matrices.

Biodegradable polymeric materials may eventually replace biostable materials for medical applications, including therapeutic devices, scaffolds for tissue engineering, and drug-delivery vehicles. To further develop such materials, a more fundamental understanding is necessary to correlate parameters including chemical-composition distribution within a macromolecular structure with the final properties of the material, including particle-size. A wide variety of analytical techniques have been applied for the characterization of polymer materials, including hyphenated techniques such as comprehensive two-dimensional liquid chromatography (LC × LC). In this context, we have investigated enzymatic degradation of polyester-based nanoparticles, both in-solution and by the use of an immobilized-enzyme reactor (IMER). We have demonstrated for the first time the implementation of such an IMER in a size-exclusion chromatography system for on-line degradation and subsequent analysis of the polymer degradation products. The effect of residence times ranging from 12 s to 4 min on polymer degradation was assessed. IMER-assisted degradation is much faster compared to in-solution degradation, which requires several hours to days, and opens the possibility to use such reactors in LC × LC modulation interfaces., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A critical step in the bottom-up characterization of proteomes is the conversion of proteins to peptides, by means of endoprotease digestion. Nowadays this method typically uses overnight digestion and as such represents a considerable bottleneck for high-throughput analysis. This report describes protein digestion using an immobilized-enzyme reactor (IMER), which enables accelerated digestion times that are completed within seconds to minutes. For rapid digestion to occur, a cyclic-olefin-copolymer microfluidic reactor was constructed containing trypsin immobilized on a polymer monolithic material through a 2-vinyl-4,4-dimethylazlactone linker. The IMER was applied for the rapid offline digestion of both singular protein standards and a complex protein mixture prior to liquid chromatography-electrospray ionisation-tandem mass spectrometry (LC-ESI-MS/MS) analysis. The effects of protein concentration and residence time in the IMER were assessed for protein standards of varying molecular weight between 11 and 240kDa. Compared to traditional in-solution digestion, IMER-facilitated protein digestion at room temperature for 5min yielded similar results in terms of sequence coverage and number of identified peptides. Good repeatability was demonstrated with a relative standard deviation of 6% for protein-sequence coverage. The potential of the IMER was also demonstrated for a complex protein mixture in the analysis of dried blood spots. Compared to a traditional workflow a similar number of proteins could be identified, while reducing the total analysis time from 22.5h to 4h and importantly omitting the sample-pre-treatment steps (denaturation, reduction, and alkylation). The identified proteins from two workflows showed similar distributions in terms of molecular weight and hydrophobic character., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

We present spatiotemporal mass balance trends for the Antarctic Ice Sheet from a statistical inversion of satellite altimetry, gravimetry, and elastic-corrected GPS data for the period 2003-2013. Our method simultaneously determines annual trends in ice dynamics, surface mass balance anomalies, and a time-invariant solution for glacio-isostatic adjustment while remaining largely independent of forward models. We establish that over the period 2003-2013, Antarctica has been losing mass at a rate of -84 ± 22 Gt yr -1 , with a sustained negative mean trend of dynamic imbalance of -111 ± 13 Gt yr -1 . West Antarctica is the largest contributor with -112 ± 10 Gt yr -1 , mainly triggered by high thinning rates of glaciers draining into the Amundsen Sea Embayment. The Antarctic Peninsula has experienced a dramatic increase in mass loss in the last decade, with a mean rate of -28 ± 7 Gt yr -1 and significantly higher values for the most recent years following the destabilization of the Southern Antarctic Peninsula around 2010. The total mass loss is partly compensated by a significant mass gain of 56 ± 18 Gt yr -1 in East Antarctica due to a positive trend of surface mass balance anomalies.

The gradient-performance limits of capillary ion chromatography have been assessed at maximum system pressure (34.5 MPa) using capillary columns packed with 4.1 μm macroporous anion-exchange particles coated with 65 nm positively-charged nanobeads. In analogy to the van-Deemter curve, the gradient performance was assessed applying different flow rates, while decreasing the gradient time inversely proportional to the increase in flow rate in order to maintain the same retention properties. The gradient kinetic-performance limits were determined at maximum system pressure, applying tG/t0=5, 10, and 20. In addition, the effect of retention on peak width was assessed in gradient mode for mono-, di-, and trivalent inorganic anions. The peak width of late-eluting ions can be significantly reduced by using concave gradient, resulting in better detection sensitivity. A signal enhancement factor of 8 was measured for a late-eluting ion when applying a concave instead of a linear gradient. For the analysis of a complex anion mixture, a coupled column with a total length of 1.05 m was operated at the kinetic-performance limit applying a linear 250 min gradient (tG/t0=10). The peak capacity varied between 200 and 380 depending on analyte retention, and hence on charge and size of the ion., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A microfluidic membrane suppressor has been constructed to suppress ions of alkaline mobile-phases via an acid-base reaction across a sulfonated poly(tetrafluoroethylene)-based membrane and was evaluated for anion-exchange separations using conductivity detection. The membrane was clamped between two chip substrates, accommodating rectangular microchannels for the eluent and regenerant flow, respectively. Additionally, a clamp-on chip holder has been constructed which allows the alignment and stacking of different chip modules. The response and efficacy of the microfluidic chip suppressor was assessed for a wide range of eluent (KOH) concentrations, using 127 and 183μm thick membranes, while optimizing the flow rate and concentration of the regenerant solution (H2SO4). The optimal operating eluent flow rate was determined at 5μL/min, corresponding to the optimal van-Deemter flow velocity of commercially-available column technology, i.e. a 0.4mm i.d.×250mm long column packed with 7.5μm anion-exchange particles. When equilibrated at 10mM KOH, a 99% decrease in conductivity signal could be obtained within 5min when applying 10mM H2SO4 regenerant at 75μL/min. A background signal as low as 1.2μS/cm was obtained, which equals the performance of a commercially-available electrolytic hollow-fiber suppressor. When increasing the temperature of the membrane suppressor from 15 to 20°C, ion suppression was significantly improved allowing the application of 75mM KOH. The applicability of the chip suppressor has been demonstrated with an isocratic baseline separation of a mixture of seven inorganic ions, yielding plate numbers between 5300 and 10,600 and with a gradient separation of a complex ion mixture., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Thermal analysis and SEM were employed to gain insights in the different stages of morphology development and the thermal properties of polymer-monolithic stationary phases. The studied system was a thermally initiated free-radical copolymerization reaction at 70°C of styrene and divinylbenzene in the presence of tetrahydrofuran and 1-decanol. The key events in the early stages of morphology development are initiation, chain growth, branching, and cyclization, leading to microgel particles. Interparticle reactions through pendant vinyl groups lead to the formation of microgel clusters. The rapid increase in molecular weight and cross-link density of the microgel clusters causes a reaction-induced phase separation, and the formation of a macroscopic network of interconnected globules was observed (macrogelation) at around 45 min. After 3 h or 65% conversion, a space-filling macroporous monolithic network was observed. Afterwards, mainly growth of existing globules takes place, reducing the macropore size. The porogen ratio affects the timing of the reaction-induced phase separation, strongly influencing the morphology of the polymer material. The use of a mixture of divinylbenzene isomers yielded a monolithic material that is less cross-linked at the surface compared to the central part of the polymer backbone due to copolymerization-composition drift. The less cross-linked outer layer starts devitrifying at 100°C., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The present study concerns the application of visualization methods, i.e. coomassie-brilliant-blue-R staining (CBB-R), silver-nitrate staining, and fluorescamine labeling, and subsequent MALDI-MS analysis of intact proteins and peptides on the surface of flat-bed monoliths, intended for spatial two-dimensional chromatographic separations. The use of 100-μm thick macroporous poly(butyl methacrylate-co-ethylene dimethacrylate) flat-bed monoliths renders a fixation step obsolete, so that CBB-R and silver-nitrate staining and destaining could be achieved in 10-15 min as opposed to up to 24h, as is typical on 2D-PAGE gels. The detection limits remained comparable. The compatibility of the monolithic layer with subsequent MALDI-MS analysis of individual proteins and peptide spots was investigated with regards to mass accuracy, mass precision, resolution, and signal intensity. When comparing results from MALDI-MS analysis of proteins and peptides on a flat-bed monolith to results obtained directly on stainless-steel target plates, significant losses in mass precision, signal intensity, and an increased variation in resolution were observed. In addition, a loss in signal intensity up to two orders of magnitude was observed when using monolithic layers. After CCB-R and silver-nitrate staining and destaining to disrupt the protein-dye complexes no MALDI spectra with significant S/N ratios could be achieved. After fluorescamine labeling heterogeneous signals were observed, which resulted from a distribution in the number of fluorescence-labeled lysine groups and from the presence of labeled derivatives that had undergone condensation reactions., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The separation of intact proteins, including protein isoforms arising from various amino-acid modifications, employing a poly(styrene-co-divinylbenzene) monolithic capillary column in high-performance liquid chromatography coupled on-line to a time-of-flight mass spectrometer (MS) is described. Using a 250 mm × 0.2 mm monolithic capillary column high-sensitivity separations yielding peak capacities of >600 were achieved with a 2h linear gradient and formic acid added in the mobile phase as ion-pairing agent. The combination of high-resolution chromatography with high-accuracy MS allowed to distinguish protein isoforms that differ only in their oxidation and biotinylation state allowing the separation between structural isoforms. Finally, the potential to separate proteins isoforms due to glycosylation is discussed., (Copyright © 2011 Elsevier B.V. All rights reserved.)