scholarly journals Analysis of 8000 proteins and reduced carry over significantly increase the throughput of single-shot proteomics

2021 ◽  
Author(s):  
Karel Stejskal ◽  
Jeff Op de Beeck ◽  
Manuel Matzinger ◽  
Gerhard Duernberger ◽  
Oleksandr Boychenko ◽  
...  
Keyword(s):  
Packed Bed ◽  
Reversed Phase ◽  
Single Shot ◽  
Quality Of Data ◽  
Proteome Coverage ◽  
Critical Dimensions ◽  
Pillar Array ◽  
Capillary Tubing ◽  
Carry Over ◽  
Separation Columns

In the field of LC-MS based proteomics, increases in sampling depth and proteome coverage have mainly been accomplished by rapid advances in mass spectrometer technology. The comprehensiveness and quality of data that can be generated do however also depend on the performance provided by nano liquid chromatography (nanoLC) separations. Proper selection of reversed-phase separation columns can be of paramount importance to provide the MS instrument with peptides at the highest possible concentration and separated at the highest possible resolution. As an alternative to traditional packed bed LC column technology that uses beads packed into capillary tubing, we present a novel LC column format based on photolithographic definition and Deep Reactive Ion Etching (DRIE) into silicon wafers. With a next generation pillar array column designed for universal use in bottom-up proteomics, the critical dimensions of the stationary phase support structures have been reduced by a factor of 2 to provide further increases in separation power. To demonstrate the potential for single-shot proteomics workflows, we report on a series of optimization and benchmarking experiments where we combine LC separation on a new generation of pillar array columns using Vanquish Neo UHPLC with fast Orbitrap Tribrid MS data-dependent acquisition (DDA) and High-Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS). In addition to providing superior proteome coverage, robust operation over more than 1 month with a single nanoESI emitter and reduction of the column related sample carry over are additional figures of merit that can help improve proteome research sensitivity, productivity and standardization.

scholarly journals A well-ordered nanoflow LC-MS/MS approach for proteome profiling using 200 cm long micro pillar array columns

2018 ◽  
Author(s):  
Jeff Op De Beeck ◽  
Jarne Pauwels ◽  
Natalie Van Landuyt ◽  
Paul Jacobs ◽  
Wim De Malsche ◽  
...  
Keyword(s):  
High Resolution Mass Spectrometry ◽  
Hek293t Cell ◽  
Packed Bed ◽  
High Reproducibility ◽  
Proteome Coverage ◽  
Pillar Array ◽  
Order Of Magnitude ◽  
New Type ◽  
Peak Widths ◽  
Resolution Mass

ABSTRACTIn bottom-up proteomics, capillaries up to 75 cm long with internal diameters of 50 to 100 µm packed with sub-2-µm C18-functionalized particles are routinely used in combination with high-resolution mass spectrometry. Unlike such conventional liquid chromatography (LC) columns, micro pillar array columns (µPAC™) are fabricated using micromachining technology, resulting in perfectly ordered chromatographic separation beds, leading to a minimized analyte dispersion while column permeability is increased by one order of magnitude. This allows using very long columns (up to 200 cm) at only a fraction of the pressure needed to operate packed bed columns. To validate µPAC™ column performances, different amounts of tryptic digests of HEK293T cell lysates were prepared and separated using a 200 cm µPAC™ column or a 40 cm long conventional column. Using an Orbitrap Elite instrument, on average 25% more proteins were identified with the µPAC™ column. Moreover, the rate at which the peak width increases with gradient time is much lower on the µPAC™ column. For a 10-hour long gradient, average peak widths below 0.5 min were observed, resulting in consistent identification of over 5,000 proteins. Combining long solvent gradients and this new type of LC column, substantial improvements in proteome coverage could be obtained. Finally, we demonstrated high reproducibility and durability of the µPAC™ column. Data are available via ProteomeXchange with identifiers PXD011547 and PXD013235.

Comparison of particulate pyrite autotrophic denitrification (PPAD) and sulfur oxidizing denitrification (SOD) for treatment of nitrified wastewater

2016 ◽  
Vol 75 (1) ◽  
pp. 239-246 ◽  
Author(s):  
Shuang Tong ◽  
Laura C. Rodriguez-Gonzalez ◽  
Chuanping Feng ◽  
Sarina J. Ergas
Keyword(s):  
Packed Bed ◽  
Product Formation ◽  
Electron Donors ◽  
Autotrophic Denitrification ◽  
Reduced Sulfur Compounds ◽  
N Removal ◽  
Sulfur Oxidizing ◽  
Reduced Sulfur ◽  
Carry Over ◽  
Set Up

The use of reduced sulfur compounds as electron donors for biological denitrification has the potential to reduce chemical and sludge disposal costs as well as carry-over of organic carbon to the effluent that often occurs with heterotrophic denitrification. Although a number of prior studies have evaluated sulfur oxidizing denitrification (SOD), no prior studies have evaluated particulate pyrite autotrophic denitrification (PPAD) in continuous flow systems. Bench-scale upflow packed bed reactors (PBRs) were set up to compare denitrification rates, by-product production and alkalinity consumption of PPAD and SOD. At an empty bed contact time of 2.9 h, average NO3−-N removal efficiencies were 39.7% and 99.9% for PPAD and SOD, respectively. Although lower denitrification rates were observed with PPAD than SOD, lower alkalinity consumption and reduced sulfur by-product formation (SO42−, S2− and SO32− plus S2O32−) were observed with PPAD. Furthermore, higher denitrification rates and lower by-product production was observed for SOD than in prior studies, possibly due to the media composition, which included sand and oyster shells. The results show that both pyrite and elemental sulfur can be used as electron donors for wastewater denitrification in PBRs.

Pillar array columns for peptide separations in nanoscale reversed-phase chromatography

2019 ◽  
Vol 1603 ◽  
pp. 426-432 ◽  
Author(s):  
Gábor Tóth ◽  
Tanja Panić-Janković ◽  
Goran Mitulović
Keyword(s):  
Reversed Phase ◽  
Reversed Phase Chromatography ◽  
Pillar Array ◽  
Peptide Separations

Experimental study of the retention properties of a cyclo olefin polymer pillar array column in reversed-phase mode

2010 ◽  
Vol 33 (21) ◽  
pp. 3313-3318 ◽  
Author(s):  
Xavi Illa ◽  
Wim De Malsche ◽  
Han Gardeniers ◽  
Gert Desmet ◽  
Albert Romano-Rodríguez
Keyword(s):  
Experimental Study ◽  
Reversed Phase ◽  
Pillar Array

Factors Affecting Degradation of MSMA in Soil

Weed Science ◽  
1986 ◽  
Vol 34 (5) ◽  
pp. 781-787 ◽  
Author(s):  
Khalid H. Akkari ◽  
Robert E. Frans ◽  
Terry L. Lavy
Keyword(s):  
Soil Water ◽  
High Performance ◽  
Graphite Furnace ◽  
Field Capacity ◽  
Reversed Phase ◽  
Rate Of Change ◽  
Factors Affecting ◽  
Use Patterns ◽  
Cacodylic Acid ◽  
Carry Over

The effects of herbicide concentration, soil water content, and temperature on the fate and degradation of the monosodium salt of methylarsonic acid (MSMA) were evaluated in four soils. Available arsenicals were extracted, purified, and separated using reversed-phase high-performance liquid chromatography (HPLC). Residual MSMA was determined with a graphite furnace atomic absorption spectrophotometer at 193.7 nm. Initial loss of MSMA was rapid, and degradation followed first-order kinetics. The rate of change of the rate constant was temperature dependent only at soil water contents less than field capacity. MSMA dissipation was significantly faster in finer textured soils with continuous flooding and under controlled laboratory conditions (<180 days) than under field (ca. 350 days) conditions. Also, in finer textured soils, degradation of MSMA resulted in significant increases in arsenate and cacodylic acid (dimethyl arsinic acid) over native levels under flooded conditions. However, MSMA treatments contributed only a small fraction to total soil arsenic which dissipated to original concentrations by 120 days. The results indicate that under present recommended use patterns it is highly unlikely that MSMA will accumulate in the environment or carry over from one growing season to the next.

Linewidth measurement using the low voltage SEM

1986 ◽  
Vol 44 ◽  
pp. 652-653
Author(s):  
M.G. Rosenfield
Keyword(s):  
Electron Microscope ◽  
Scanning Electron Microscope ◽  
Integrated Circuits ◽  
Secondary Electron ◽  
Low Voltage ◽  
Fabrication Process ◽  
Critical Dimensions ◽  
Linewidth Measurement ◽  
Threshold Technique ◽  
Scanning Electron

Minimum feature sizes in experimental integrated circuits are approaching 0.5 μm and below. During the fabrication process it is usually necessary to be able to non-destructively measure the critical dimensions in resist and after the various process steps. This can be accomplished using the low voltage SEM. Submicron linewidth measurement is typically done by manually measuring the SEM micrographs. Since it is desirable to make as many measurements as possible in the shortest period of time, it is important that this technique be automated.Linewidth measurement using the scanning electron microscope is not well understood. The basic intent is to measure the size of a structure from the secondary electron signal generated by that structure. Thus, it is important to understand how the actual dimension of the line being measured relates to the secondary electron signal. Since different features generate different signals, the same method of relating linewidth to signal cannot be used. For example, the peak to peak method may be used to accurately measure the linewidth of an isolated resist line; but, a threshold technique may be required for an isolated space in resist.

The use of high-resolution FESEM to study solid-state phase transformations and reactions

1994 ◽  
Vol 52 ◽  
pp. 1028-1029
Author(s):  
P. G. Kotula ◽  
D. D. Erickson ◽  
C. B. Carter
Keyword(s):  
Solid State ◽  
High Resolution ◽  
Phase Transformations ◽  
High Efficiency ◽  
Sol Gel ◽  
Quantitative Information ◽  
Solid State Reactions ◽  
Critical Dimensions ◽  
Backscattered Electrons ◽  
Backscattered Electron

High-resolution field-emission-gun scanning electron microscopy (FESEM) has recently emerged as an extremely powerful method for characterizing the micro- or nanostructure of materials. The development of high efficiency backscattered-electron detectors has increased the resolution attainable with backscattered-electrons to almost that attainable with secondary-electrons. This increased resolution allows backscattered-electron imaging to be utilized to study materials once possible only by TEM. In addition to providing quantitative information, such as critical dimensions, SEM is more statistically representative. That is, the amount of material that can be sampled with SEM for a given measurement is many orders of magnitude greater than that with TEM.In the present work, a Hitachi S-900 FESEM (operating at 5kV) equipped with a high-resolution backscattered electron detector, has been used to study the α-Fe2O3 enhanced or seeded solid-state phase transformations of sol-gel alumina and solid-state reactions in the NiO/α-Al2O3 system. In both cases, a thin-film cross-section approach has been developed to facilitate the investigation. Specifically, the FESEM allows transformed- or reaction-layer thicknesses along interfaces that are millimeters in length to be measured with a resolution of better than 10nm.

Methods to Monitor and Improve the Performance of Specimen Holders for Transmission Electron Cryomicroscopy

1996 ◽  
Vol 54 ◽  
pp. 454-455
Author(s):  
B. L. Armbruster ◽  
B. Kraus ◽  
M. Pan
Keyword(s):  
Electron Microscopy ◽  
Electron Beam ◽  
Electron Beam Irradiation ◽  
Beam Irradiation ◽  
Quality Of Data ◽  
Electron Cryomicroscopy ◽  
Thermal Equilibration ◽  
Transmission Electron ◽  
Electron Microscopes

One goal in electron microscopy of biological specimens is to improve the quality of data to equal the resolution capabilities of modem transmission electron microscopes. Radiation damage and beam- induced movement caused by charging of the sample, low image contrast at high resolution, and sensitivity to external vibration and drift in side entry specimen holders limit the effective resolution one can achieve. Several methods have been developed to address these limitations: cryomethods are widely employed to preserve and stabilize specimens against some of the adverse effects of the vacuum and electron beam irradiation, spot-scan imaging reduces charging and associated beam-induced movement, and energy-filtered imaging removes the “fog” caused by inelastic scattering of electrons which is particularly pronounced in thick specimens.Although most cryoholders can easily achieve a 3.4Å resolution specification, information perpendicular to the goniometer axis may be degraded due to vibration. Absolute drift after mechanical and thermal equilibration as well as drift after movement of a holder may cause loss of resolution in any direction.

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