A protocol for robust discovery proteomics using nano liquid chromatography using pillar-array column technology and high-resolution mass spectrometry with data- independent acquisition
Ontology highlight
ABSTRACT:
INSTRUMENT(S):
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): Hela Cell
SUBMITTER: 
Dániel Papp
LAB HEAD: Sebastiaan Eeltink
PROVIDER: PXD069993 | Pride | 2026-01-13
REPOSITORIES: Pride
Dataset's files
Items per page: 5 1 - 5 of 70 |
Similar Datasets
Project description:Capitalizing on the massive increase in sample concentrations which are produced by extremely low elution volumes, nano-LC-ESI-MS/MS is currently the most sensitive analytical technology for the comprehensive characterization of complex protein samples. However, despite tremendous technological improvements made in the production and the packing of monodisperse spherical particles for nano-flow HPLC, current state-of-the-art systems still suffer from limits in operation at the maximum potential of the technology. With the recent introduction of the µPAC system, which provides perfectly ordered micro-pillar array based chromatographic support materials, completely new chromatographic concepts for optimization towards the needs of ultra-sensitive proteomics become available. Here we report on a series of benchmarking experiments comparing the performance of a commercially available 50 cm micro-pillar array column to a widely used nano-flow HPLC column for the proteomics analysis of 10 ng tryptic HeLa cell digest. Comparative analysis of LC-MS/MS-data corroborated that micro-pillar array cartridges provide outstanding chromatographic performance and excellent retention time stability, which substantially increase sensitivity in the analysis of low-input proteomics samples, and thus repeatedly yielded almost twice as many unique peptide and unique protein group identifications when compared to conventional nano-flow HPLC columns.
2019-10-23 | PXD014124 | Pride
Project description:To understand the basic biological properties of supporting cells of the mammalian inner ear, we examined the transcriptome of adult pillar and Deiters’ cells. Morphologically distinct pillar and Deiters’ cells were isolated from the organ of Corti from adult CBA/J mice. One thousand pillar and Deiters’ cells were separately collected for each biological replicate, using the suction pipette technique. RNA sequencing of three biological replicates, each with two technical repeats, was performed. The resulting sequenced reads were mapped. Comparisons between pillar cells and Deiters’ cells allow identification of enriched genes, as well as differentially expressed genes that result in cellular specialization. Our dataset provides an extensive resource for understanding the molecular mechanisms underlying morphology, function, and pathology of adult mouse pillar and Deiters’ cells.
2018-08-03 | GSE111347 | GEO
Project description:In the light of the ongoing single-cell revolution, scientific disciplines are combining forces with the ultimate goal to retrieve as much relevant data as possible from trace amounts of biological material. For single cell proteomics, this implies optimizing the entire workflow from initial cell isolation down to sample preparation, LC separation, MS/MS data acquisition and data analysis. To demonstrate the potential for single cell and limited sample proteomics, we report on a series of benchmarking experiments where we combine LC separation on a new generation of micro pillar array columns with state-of-the-art Orbitrap MS/MS detection and FAIMS. As compared to the currently commercially available pillar array columns, this dedicated limited sample column has a reduced cross section (factor of 2) and micro pillar dimensions that have been further downscaled (also by a factor of 2; 2.5 µm instead of 5 µm diameter), resulting in improved chromatography at reduced void times. A dilution series ranging from 5 to 0.05 ng/µL was prepared using trace amounts of PEG in the sample solvent to reduce adsorptive effects in the autosampler vial, sample stability up to 24h after dilution was demonstrated. Comparative processing of the MS/MS data with different database search algorithms (with and without second search feature activated and with and without rescoring based on fragmentation pattern prediction) pointed out the benefits of using Sequest HT together with INFERYS when analyzing samples at a concentration below 1 ng/µL. On average (data from quadruplicate runs), we were able to successfully identify 2855 unique protein groups from just 1 ng of HeLa lysate and this using a 60 min non-linear LC solvent gradient at a flow rate of 250 nL/min, hereby increasing detection sensitivity as compared to a previous contribution by a factor well above 10 (2436 proteins identified from 10 ng of HeLa lysate in 2019). By successfully identifying 1486 and 2210 proteins (average values, quadruplicates) from as little as 250 and 500 pg of HeLa lysate respectively, we demonstrate outstanding sensitivity with great promise for use in limited sample proteomics workflows.
2021-06-28 | PXD024017 | Pride
Project description:Chromatographic column selection can impact proteomic profiling, yet comparative studies remain limited. Here, we evaluate the performance of a conventional flame-pulled Accucore resin-packed capillary column and a microfabricated pillar array column (μPAC) in a sample multiplexed global proteome profiling experiment using six human cell lines prepared in triplicate as a TMTpro18-plex. Overall, the chromatography columns exhibited comparable performance. Specifically, the number and overlap of quantified peptides, as well as proteins, was similar between columns. Principal component and hierarchical clustering analyses highlighted reproducible patterns of cell line organization, while correlation analyses showed high replicate consistency across column formats. Similarity, analytical parameters like XCorr scores, signal-to-noise ratio, and peak resolution showed consistency. These findings demonstrate the potential for using robust, standardized microfluidic columns, such as μPAC, in lieu of traditional pull-tipped capillary columns without sacrificing depth or quantitative accuracy. Key advantages of μPAC include its ease of use and durability in a uniform format, although this advantage does come at a higher cost. This comparative analysis offers valuable insights into column selection for TMT-based quantitative proteomics.
2025-11-24 | PXD063964 | Pride
Project description:Nanoflow liquid chromatography capable of delivering consistent and reliable results across extensive sample sets is essential for the advancement of mass spectrometry-based proteomics. Micro-Pillar Array Columns (μPACs) represent a significant breakthrough, offering durability and performance stability. Here, we evaluate the robustness of μPACs by comparing a column used continuously for over 16 months with more than 7,000 injections (μPAC1) to a nearly new column (μPAC2). Analysis of two TMT-labeled yeast TKO standards (TKOpro10u, a 10-plex unit-resolved standard; and TKOpro12, a 12-plex isotopolog-inclusive standard) showed that μPAC1 and μPAC2 yielded comparable numbers of unique peptides and proteins, exhibited similar reproducibility, and delivered equivalent chromatographic and spectral quality. Notably, these data showed that μPAC1 maintained high performance with minimal degradation of data quality, highlighting the exceptional durability of μPAC technology. These findings underscore that μPAC could contribute to reduced workflow disruptions in high-throughput analytical workflows, notably in proteomics workflows using mass spectrometry.
2025-11-24 | PXD067649 | Pride
Project description:The effective separation of complex peptide mixtures is a cornerstone of mass spectrometry-based proteomics analysis that enhances the accuracy and depth of proteomic analyses. Here we compare datasets collected of whole cell tryptic peptides which were fractionated by either conventional flame-pulled, C18 packed bed microcapillary columns or microfabricated pillar array columns (μPAC). Sixteen samples from four yeast strains (Δmet6, Δpfk2, Δura2, and wildtype) were analyzed in quadruplicate using data-independent acquisition (DIA). Each column enabled the quantification of >4,700 protein, with >95.4% overlap between column formats. The μPAC showed higher MS1 and MS2 signal intensities, while maintaining similar peptide characteristics as the capillary column. The capillary column favored slightly longer and more hydrophobic peptides. Both columns achieved high data completeness at the protein level (>95%) and reproducible quantification, with μPAC offering slight improvements. Principal component analysis and correlation analysis confirmed the capture of yeast strain-specific differences, with hierarchical clustering prioritizing strain over column effects. Protein quantification validated gene knockouts in both column formats, demonstrating similar accuracy of quantification. These findings highlight the μPAC as a standardized and robust alternative to capillary columns in proteomic analysis.
2025-11-18 | PXD063991 | Pride
Project description:Disease diagnostics requires detection and quantification of nano-sized bioparticles including DNA, proteins, viruses, and exosomes. Here, a fluorescent label-free method for sensitive detection of bioparticles is explored using a pillar array with micrometer-sized features in a deterministic lateral displacement (DLD) device. The method relies on measuring changes in size and/or electrostatic charges of 1 µm polymer beads due to the capture of target bioparticles on the surface. These changes can be sensitively detected through the lateral displacement of the beads in the DLD array, wherein the lateral shifts in the output translates to a quantitative measurement of bioparticles bound to the bead. The detection of albumin protein and nano-sized polymer vesicles with a concentration as low as 10 ng mL-1 (150 pM) and 3.75 μg mL-1, respectively, is demonstrated. This label-free method holds potential for point-of-care diagnostics, as it is low-cost, fast, sensitive, and only requires a standard laboratory microscope for detection.
| S-EPMC5871788 | biostudies-literature
Project description:Reversed-phase chromatography is the most common technique for separation of tryptic peptides. In this manuscript, we describe the optimization of sample loading and separation parameters for a novel micro machined column and provide a detailed description on the performance and reproducibility of this separation system.
2019-07-18 | PXD013158 | Pride
Project description:Gold nanorods (GNRs) are one of the most promising biomaterial choices for the photothermal activation of neurons due to their relative biocompatibility, unique photothermal properties, and broad optical tunability through their synthetic shape control. While photothermal stimulation using randomly accumulated GNRs successfully demonstrates the potential treatment of functional neural disorders by modulating the neuronal activities using localized heating, there are limited demonstrations to translate this new concept into large-arrayed neural stimulations. In this paper, we report an arrayed PDMS micropillar platform in which GNRs are embedded as pixel-like, arrayed photothermal stimulators at the tips of the pillars. The proposed platform will be able to localize GNRs at predetermined pillar positions and create thermal stimulations using near-infrared (NIR) light. This will address the limitations of randomly distributed GNR-based approaches. Furthermore, a flexible PDMS pillar structure will create intimate interfaces on target cells. By characterizing the spatiotemporal temperature change in the platform with rhodamine B dye, we have shown that the localized temperature can be optically modulated within 4°C, which is in the range of temperature variation required for neuromodulation using NIR light. We envision that our proposed platform has the potential to be applied as a photothermal, neuronal stimulation interface with high spatiotemporal resolution.
| S-EPMC11449256 | biostudies-literature
Project description:Predicting individual radiotherapy (RT) response is valuable in managing head and neck squamous cell carcinoma (HNSCC). We assessed the feasibility of our novel 3D culture platform to measure radioresponse using patient-derived cells (PDCs) from HNSCC patients. Cells from the FaDu line and tumor samples from 39 HNSCC patients were cultivated serially in MatrigelTM on a 3D pillar/well array culture system. The 3D tumor models were exposed to 0 to 8 Gy of radiation dose, and the radioresponse index (RTauc, area under the dose-response curve) was measured quantitatively with Calcein AM staining of live tumor cells. Calcein AM fluorescence showed reduced density and the number of FaDu colonies as radiation increased, implying a dose-dependent effect on cell viability in the 3D pillar/well culture system. 3D tumor models using PDCs were established successfully from 39 HNSCC patient tumor samples, maintaining original genomic and pathological characteristics. These 3D tumor models were exposed to ionizing radiation on a 3D pillar/well array, with a mean period of 12 days from tumor harvest to the measurement of RTauc. The RTauc of all PDCs varied from 3.5 to 9.4, and the lower 40th percentile (Z-score = -0.26) was considered a good radioresponse group with a threshold RTauc of 4.6. The good radioresponse group showed fewer adverse features than others. As of the last follow-up, recurrence-free survival was better in the good radioresponse group (p = 0.037). 3D pillar/well array platforms using PDC could rapidly quantify radioresponse index in patients with HNSCC, showing potential as a novel prognosticator.
| S-EPMC9294182 | biostudies-literature