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PEPPI-SP3: Robust and Simple Sample Prefractionation Workflow for Top-Down Proteomics

ABSTRACT: In this study, we developed a complete, robust, and simple sample preparation workflow named PEPPI-SP3 for top-down proteomics by combining PEPPI-MS with the magnetic bead-based protein purification approach used in SP3 (single-pot, solid-phase-enhanced sample preparation), now one of the standard sample preparation methods in bottom-up proteomics.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Nobuaki Takemori

PROVIDER: PXD056413 | JPOST Repository | Thu Jan 23 00:00:00 GMT 2025

REPOSITORIES: jPOST

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			Action	DRS
	240720_DDA17_Slot1-76_1_3418.d.zip	Other
	240720_DDA1_Slot1-73_1_3415.d.zip	Other
	240720_DDA21_Slot1-77_1_3419.d.zip	Other
	240720_DDA25_Slot1-78_1_3420.d.zip	Other
	240720_DDA33_Slot1-79_1_3421.d.zip	Other

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Publications

Gel-Based Sample Fractionation with SP3-Purification for Top-Down Proteomics.

Takemori Ayako A Sugiyama Naoyuki N Kline Jake T JT Fornelli Luca L Takemori Nobuaki N

Journal of proteome research 20250122

Precise prefractionation of proteome samples is a potent method for realizing in-depth analysis in top-down proteomics. PEPPI-MS (Passively Eluting Proteins from Polyacrylamide gels as Intact species for MS), a gel-based sample fractionation method, enables high-resolution proteome fractionation based on molecular weight by highly efficient extraction of proteins from polyacrylamide gels after SDS-PAGE separation. Thereafter it is essential to effectively remove contaminants such as CBB and SDS ...[more]

PMID: 39841590

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PEPPI-SP3: Robust and Simple Sample Prefractionation Workflow for Top-Down Proteomics

Project description:Precise prefractionation of proteome samples is a potent method for realizing in depth analysis in top-down proteomics. PEPPI MS (Passively Eluting Proteins from Polyacrylamide gels as Intact species for MS), a gel-based sample fractionation method, enables high-resolution proteome fractionation based on molecular weight by highly efficient extraction of proteins from polyacrylamide gels after SDS PAGE separation. In this study, we developed a complete, robust, and simple sample preparation workflow named PEPPI SP3 for top down proteomics by combining PEPPI MS with the magnetic bead-based protein purification approach used in SP3.

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Influence of Different Sample Preparation Approaches on Proteoform Identification by Top-Down Proteomics

Project description:Top-down proteomics (TDP) has made significant advances in the past, and a plethora of sample preparation workflows have been developed. Here, we systematically investigated the influence of different sample preparation steps on proteoform and protein identifications, including cell lysis, reduction and alkylation, proteoform enrichment, purification, and fractionation. We found that all steps in sample preparation influence the subset of proteoforms identified, e.g., their number, confidence, physicochemical properties, and artificially generated modifications. The various sample preparations resulted in complementary identifications, significantly increasing the depth of the analysis. Overall, 15,089 proteoforms from 2,780 protein accessions of human Caco-2 cells were identified, providing the most comprehensive dataset of this cell line up to now. The results from our study can serve as a guideline for designing and adapting TDP sample preparation strategies to particular research questions.

2024-08-08 | PXD049969 | Pride

Combination of digital microfluidics and magnetic bead-based intact proteoform elution for top-down nanoproteomics of single C. elegans

Project description:While most nanoproteomics approaches for the analysis of low-input samples are based on bottom-up LC-MS workflows, top-down approaches enabling proteoform characterization are still underrepresented. Using mammalian cell proteomes, we here established a facile one-pot sample preparation protocol based on protein aggregation on magnetic beads and intact proteoform elution using 40% v/v formic acid. Performed on a digital microfluidics device, the workflow enabled sensitive analysis of single Caenorhabditis elegans nematodes, increasing the number of proteoform identifications compared to in-tube sample preparation by 46%. Label-free quantification of single nematodes grown under different conditions allowed to identify changes in abundance of proteoforms not distinguishable by bottom-up proteomics. The workflow presented here will help to elucidate cellular changes on the proteoform level in other samples of limited availability.

2023-06-19 | PXD039883 | Pride

Top-down/Bottom-up Mass Spectrometry Workflow Using Dissolvable Polyacrylamide Gels.

Project description:Biologists' preeminent toolbox for separating, analyzing, and visualizing proteins is SDS-PAGE, yet recovering the proteins embedded in these polyacrylamide media as intact species is a long-standing challenge for mass spectrometry. In conventional workflows, protein mixtures from crude biological samples are electrophoretically separated at high-resolution within N,N'-methylene-bis-acrylamide cross-linked polyacrylamide gels to reduce sample complexity and facilitate sensitive characterization. However, low protein recoveries, especially for high molecular weight proteins, often hinder characterization by mass spectrometry. We describe a workflow for top-down/bottom-up mass spectrometric analyses of proteins in polyacrylamide slab gels using dissolvable, bis-acryloylcystamine-cross-linked polyacrylamide, enabling high-resolution protein separations while recovering intact proteins over a broad size range efficiently. The inferior electrophoretic resolution long associated with reducible gels has been overcome, as demonstrated by SDS-PAGE of crude tissue extracts. This workflow elutes intact proteins efficiently, supporting MS and MS/MS from proteins resolved on biologists' preferred separation platform.

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Universal Solid-Phase Protein Preparation (USP3) for bottom-up and top-down proteomics

Project description:Selecting a sample preparation strategy for mass spectrometry-based proteomics is critical to the success of quantitative workflows. Here we present a universal, solid-phase protein preparation (USP3) method which is rapid, robust and scalable, facilitating high-throughput protein sample preparation for bottom-up and top-down mass spectrometry (MS) analysis.

2019-06-05 | PXD011169 | Pride

Robotic Top-down Intersphincteric Resection

Project description:The present study is to develop the novel robotic surgical technique and enhance the surgery quality for the treatment of distal rectal cancer.

| 67720 | ecrin-mdr-crc

A magnetic bead-based workflow for sensitive and streamlined cell surface proteomics

Project description:Cell-surface proteins represent an important class of molecules for therapeutic targeting and defining cellular phenotypes. However, their enrichment and detection via mass spectrometry-based proteomics remains challenging due to low abundance, posttranslational modifications, hydrophobic regions, and processing requirements. To improve the identification of cell-surface proteins via their corresponding N-linked glycopeptides (N-glycopeptides), we optimized a Cell-Surface Capture (CSC) workflow which incorporates magnetic bead-based processing. Using this approach, we evaluated labeling conditions (biotin tags and catalysts), enrichment specificity (streptavidin beads), missed cleavages (lysis buffers), non-enzymatic deamidation (digestion and de-glycosylation buffers), and data acquisition methods. Our findings support the use of alkoxyamine-PEG4-biotin plus 5-methoxy-anthranilic acid and streptavidin magnetic beads for maximal N-glycopeptide detection. Furthermore, single-pot solid-phased-enhanced sample-preparation (SP3) circumvented the need to isolate cell membranes by affording the use of strong detergents and chaotropes for protein extraction. Notably, with semi-automated processing, sample handling was simplified and between ~600-900 N-glycoproteins were identified from only 25-200µg of HeLa protein. Overall, the improved efficiency of the magnetic-based CSC workflow allowed us to identify both previously reported and novel N-glycosites with less material and high reproducibility, and should help advance the field of surfaceomics by providing insight in cellular phenotypes not previously documented.

2024-01-21 | PXD043678 | Pride

Ultrasensitive Top-Down Proteomics Analysis of Low Number of Mammalian Cells Using a Nanodroplet Sample Processing Platform

Project description:We developed an ultrasensitive top down platform by incorporating a microfluidic sample preparation system, termed nanoPOTS (Nanodroplet Processing in One pot for Trace Samples), into a top down workflow. We determined that a unique combination of a nonionic detergent DDM with urea as a protein extraction buffer can significantly improve both protein extraction efficiency and sample recovery. The nanoPOTS-based top down platform can reproducibly and quantitatively identify ~170 to ~620 proteoforms from ~70 to ~770 HeLa cells containing < ~100 ng total proteins. Data was searched with TopPIC and MSPathFinder.

2019-11-22 | MSV000084612 | MassIVE

Towards a universal sample preparation method for denaturing top-down proteomics of complex proteomes

Project description:A universal and standardized sample preparation method becomes vital for denaturing top-down proteomics (dTDP) to advance the scale and accuracy of proteoform delineation in complex biological systems. It needs to have high protein recovery, minimum bias, good reproducibility, and compatibility with downstream mass spectrometry (MS) analysis. Here we employed a lysis buffer containing sodium dodecyl sulfate (SDS) for extracting proteoforms from cells, and for the first time, compared membrane ultrafiltration (MU), chloroform-methanol precipitation (CMP), and single-spot solid-phase sample preparation using magnetic beads (SP3) for proteoform cleanup for dTDP. The MU method outperformed CMP and SP3 methods, resulting in high and reproducible protein recovery from both E. coli cell (59±3%) and human HepG2 cell (86±5%) samples without a significant bias. Single-shot capillary zone electrophoresis (CZE)-MS/MS analyses of the prepared E. coli and HepG2 cell samples using the MU method identified 821 and 516 proteoforms, respectively. Nearly 30% and 50% of the identified E. coli and HepG2 proteins are membrane proteins. CZE-MS/MS identified 94 histone proteoforms from the HepG2 sample with various post-translational modifications, including acetylation, methylation, and phosphorylation. Our results suggest that combining the SDS-based protein extraction and the MU-based protein cleanup could be a universal sample preparation method for dTDP.

2020-05-22 | PXD018248 | Pride

Fully automated sample processing and analysis workflow for low-input proteome profiling

Project description:Recent advances in sample preparation and analysis have enabled direct profiling of protein expression in single mammalian cells and other trace samples for characterization of cellular heterogeneity and high-resolution mapping of tissues. Several techniques used to prepare and analyze low-input samples employ custom fluidics for nanoliter sample processing and manual sample injection onto a specialized separation column. While effective, these highly specialized systems require significant expertise to fabricate and operate, which has greatly limited the implementation in most proteomics laboratories. Here we developed a fully automated platform termed autoPOTS (Automated Preparation in One pot for Trace Samples) that uses only commercially available instrumentation for sample processing and analysis. An unmodified, low-cost commercial robotic pipetting platform was evaluated and utilized for fully automated sample preparation. We used low-volume 384-well plates and periodically added water or buffer to the microwells to compensate for limited evaporation during sample incubation. Prepared samples were analyzed directly from the well plate with a commercial autosampler that was modified with a 10-port valve for compatibility with 30-µm-i.d. nanoLC columns. We used autoPOTS to analyze 1–500 HeLa cells and observed only a modest reduction in peptide coverage for 150 cells and a 24% reduction in coverage for single cells compared to our previously developed nanoPOTS platform. As a test of clinical feasibility, we used autoPOTS to identify an average of 1070 protein groups from ~130 fluorescence-activated cell sorted (FACS) B or T lymphocytes. The dataset here includes all the raw files of HeLa cells and lymphocytes. We anticipate that the simplicity and ease of implementation of autoPOTS will make it an attractive option for low-input and single-cell proteomics in many laboratories.

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