Trapping efficiency of peptides on particle-packed and micro-pillar trap columns for proteomics analyses
Ontology highlight
ABSTRACT:
INSTRUMENT(S):
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): Permanent Cell Line Cell, Hela Cell
SUBMITTER: 
Goran Mitulović
LAB HEAD: Goran Mitulovic
PROVIDER: PXD052759 | Pride | 2026-02-18
REPOSITORIES: Pride
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| HeLa_200420_PF_1.raw | Raw | |||
| HeLa_200420_PF_2.raw | Raw | |||
| HeLa_200420_PF_3.raw | Raw | |||
| HeLa_200420_PF_4.raw | Raw | |||
| HeLa_200420_PF_5.raw | Raw |
Items per page: 5 1 - 5 of 13 |
Similar Datasets
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: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: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:Twenty microliter aliquots of BALF samples were dissolved in SDS-sample buffer and applied onto a 4-12% Nupage gel with MOPs running buffer. The run stopped after the samples migrated approximately ¼ distance into the gel. Each lane of the gel was sliced into smaller pieces, and subjected to destaining, reducing/alkylation, and in-gel trypsin digestion. The extracted peptides were applied for LC-MS/MS analysis using either a Thermo Orbitrap Fusion or a Thermo Orbitrap Fusion Lumos operated with an in-line Thermo nLC 1200 and an EASY-Spray ion source. Pepetides were separated using a 2 cm Pepmap 100 C18 trap column and a 25 cm Easy-spray Pepmap 100 C18 analytical column. MS/MS data acquisitions were operated at a 120,000 resolution (m/z 200) with a scan range of 350-1950 m/z and CID fragmentation.
2023-10-24 | PXD045119 | Pride
Project description:Nanoflow liquid chromatography-mass spectrometry is key to enabling in-depth proteome profiling of trace samples such as single cells, but these separations can lack robustness due to the use of narrow-bore columns that are susceptible to clogging. In the case of single-cell proteomics, offline cleanup steps are generally omitted to avoid losses to additional surfaces, and online solid-phase extraction/trap columns frequently provide the only opportunity to remove salts and insoluble debris before the sample is introduced to the analytical column. Trap columns are traditionally short, packed columns used to load and concentrate analytes at flow rates greater than those employed in analytical columns, and since these first encounter the uncleaned sample mixture, trap columns are also susceptible to clogging. We hypothesized that clogging could be avoided by using large-bore porous layer open tubular trap columns (PLOTrap). The low back pressure ensured that the PLOTraps could also serve as the sample loop, thus allowing sample cleanup and injection with a single 6-port valve. We found that PLOTraps could effectively remove debris to avoid column clogging. We also evaluated multiple stationary phases and PLOTrap diameters to optimize performance in terms of peak widths and sample loading capacities. Optimized PLOTraps were compared to conventional packed trap columns operated in forward and backflush modes, and were found to have similar chromatographic performance of backflushed traps while providing improved debris removal for robust analyses of trace samples.
2024-02-09 | PXD048565 | Pride
Project description:A comprehensive proteome map is essential to elucidate molecular pathways and protein functions. Although great improvements in sample preparation, instrumentation and data analysis already yield-ed impressive results, current studies suffer from a limited proteomic depth and dynamic range there-fore lacking low abundant or highly hydrophobic proteins. Here, we combine and benchmark advanced micro pillar array columns (µPAC) operated at nanoflow with Wide Window Acquisition (WWA) and the AI-based CHIMERYS search engine for data analysis to maximize chromatographic separation power, sensitivity and proteome coverage. Our data shows that µPAC columns clearly outperform classical packed bed columns boosting peptide IDs by up to 50% and protein IDs by up to 24%. Using the above-mentioned analysis platform, more than 10,000 proteins could be identified from a single 2 h gradient shotgun analysis for a triple proteome mix of human, yeast and E. coli digests. At high sample loads of 400 ng all three uPAC types yielded comparable number of protein identifications, whereas the 50cm neo column performed best when lower inputs of less than 200 ng were injected. Due to its unique architecture, the 5.5 cm brick column facilitates a highly meandering flow along the brick-shaped micropillars leading to an effective flow path length similar to the 50 cm neo column, while at the same time allowing high flow rates up to 2.5 µL/min. This enables to reduce overhead time by applying high flow rates during sample loading and column equilibration improving sample throughput to ~100 samples per day, while maintaining high protein ID numbers. Particularly for the single cell field, for which throughput is currently one of the most limiting factors, this column could present a valuable asset.
2024-02-13 | PXD039576 | Pride
Project description:Peptide separations that combine high sensitivity, robustness, peak capacity and throughput are essential for extending bottom-up proteomics to smaller samples including single cells. To this end, we have developed a multicolumn nanoLC system with accelerated offline gradient generation. One binary pump generates gradients in an accelerated manner to support multiple analytical columns, and a single trap column interfaces with all analytical columns to reduce required maintenance and simplify troubleshooting. A high degree of parallelization is possible, as one sample undergoes separation while the next sample plus its corresponding mobile phase gradient are transferred into the storage loop and a third sample is loaded into a sample loop. Selective offline elution from the trap column into the sample loop prevents salts and hydrophobic species from entering the analytical column, thus greatly enhancing column lifetime and system robustness. With this design, samples can be analyzed as fast as every 20 min at a flow rate of just 40 nL/min with close to 100% MS utilization time, and continuously for as long as several months without column replacement. We utilized the system to analyze the proteomes of single cells from a multiple myeloma cell line upon treatment with the immunomodulatory imide drug lenalidomide.
2025-05-06 | PXD049248 | Pride
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: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:Nanoflow chromatography remains the standard workflow for proteomics. Capillary flow chromatography applications are mainly limited to fast acquisitions, likely due to the absence of commercially available long (> 15 cm), 300 µm inner diameter columns. We present a methodology for packing 300 µm diameter columns of any desired length and systematically optimising several capLC parameters. Utilizing CSH chemistry, known for its ability to maintain excellent peak shapes even under high loading conditions, we systematically tested various bead sizes and column lengths. Specifically, we examined 30 cm columns packed with 1.7 µm beads, 55 cm columns with 2.5 µm beads, and 1 m columns using 3.5 µm beads. We also varied the gradient lengths from 30 minutes to 10 hours. Our results demonstrated outstanding performance with the 30 cm columns packed with 1.7 µm beads on a 2-hour gradient, achieving a median full width at half maximum of 7.4 seconds and a peak capacity of 530. We also show that the columns can handle high sample amounts in 0.5-8 µg range without loss of separation power. Finally, we systematically investigated the effect of gradient length and loading and demonstrate that we can quantify close to 9000 proteins and 124 thousand unique peptides on Exploris 240 system, analysing 4 µg of HeLa digest material on a three hour gradient. We demonstrate that the performance of the presented columns approaches or surpasses the most efficient commercially available columns.
2026-02-07 | PXD067260 | Pride