Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

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

ABSTRACT: 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.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Caenorhabditis Elegans

TISSUE(S): Whole Body

SUBMITTER: Andreas Tholey

LAB HEAD: Andreas Tholey

PROVIDER: PXD039883 | Pride | 2023-06-19

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
	030322_BS_single_worm_TD-SP3-40-FA_SDS_C4_4CVs_15ul_180min_R2.pdResult	Other
	030322_BS_single_worm_TD-SP3-40-FA_SDS_C4_4CVs_15ul_180min_R2.raw	Raw
	030322_BS_single_worm_TD-SP3-40-FA_SDS_C4_4CVs_15ul_180min_R4.pdResult	Other
	030322_BS_single_worm_TD-SP3-40-FA_SDS_C4_4CVs_15ul_180min_R4.raw	Raw
	030322_BS_single_worm_TD-SP3-40-FA_SDS_C4_4CVs_15ul_180min_R5.pdResult	Other

Items per page:

1 - 5 of 87

Similar Datasets

Miniaturized sample preparation on a digital microfluidics device for sensitive bottom-up microproteomics of mammalian cells using magnetic beads and mass spectrometry-compatible surfactants

Project description:We used digital microfluidics (DMF) for the sample preparation of approximately 100 mammalian cells, and subsequent bottom-up proteome analysis by LC-MS. This comprised optimization of cell lysis conditions for DMF, the development of detergent-buffer systems, and adaptation of the single-pot, solid-phase-enhanced sample preparation (SP3) approach on-chip. Application of the methodology to the proteome analysis of Jurkat T cells led to the identification of up to 2,500 proteins from approximately 500 cells, and up to 1,200 proteins from approximately 100 cells.

2019-10-07 | PXD014158 | Pride

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

Improved identification of proteoforms using FAIMS with internal CV stepping in top-down proteomics

Project description:In top-down (TD) proteomics, fractionation prior to mass spectrometric (MS) analysis is a crucial step for the high confidence identification of proteoforms and increased sample depth. In addition to liquid phase separations, gas-phase fractionation strategies such as field asymmetric ion mobility spectrometry (FAIMS) have been shown to be highly beneficial in TD proteomics. However, the need for multiple injections using different compensation voltages (CV) leads to a huge increase in measurement time and the amount of sample required. Therefore, we here investigated for the first time the use of internal CV stepping for single shot TD analysis, i.e., the application of multiple CVs per acquisition. In addition, MS parameters were optimized for the individual CVs since different CVs target certain mass ranges. For example, small proteoforms identified mainly with lower CVs can be identified with a lower resolution and number of microscans than larger proteins identified mainly with higher CVs. We investigated the optimal combination and number of CVs for different gradient lengths and validated the optimized settings with the low molecular weight proteome of CaCo 2 cells obtained by different sample preparation techniques. Compared to measurements without FAIMS, both the number of identified protein groups (+60-94%) and proteoforms (+46-127%), and their confidence were significantly increased, while the measurement time remained identical. In total, we identified 684 protein groups and 2,675 proteoforms from CaCo-2 cells in less than 24 hours using the optimized multi-CV method.

2022-02-28 | PXD029792 | Pride

Positional proteomics reveals differences in N-terminal proteoform stability

Project description:To understand the impact of alternative translation initiation on a proteome, we performed the first study on protein turnover using positional proteomics and ribosome profiling to distinguish between N-terminal proteoforms of individual genes. Overall, we monitored the stability of 1,941 human N-terminal proteoforms, including 147 N-terminal proteoform pairs that originate from alternative translation initiation, alternative splicing or incomplete processing of the initiator methionine. Ribosome profiling of lactimidomycin and cycloheximide treated human Jurkat T-lymphocytes

2015-10-23 | E-GEOD-74279 | biostudies-arrayexpress

GeLC-FAIMS-MS Workflow for In-Depth Middle-Down Proteomics

Project description:Middle-down proteomics is an analytical approach whereby protein samples are digested with proteases such as Glu-C to generate large peptides (>3 kDa) that are then analyzed by mass spectrometry. This method is useful for characterizing high-molecular-weight proteins that are difficult to detect by top-down proteomics, in which intact proteins are analyzed by mass spectrometry. In this study, we applied GeLC-FAIMS-MS, a multidimensional separation workflow that combines gel-based prefractionation with LC-FAIMS Orbitrap mass spectrometry, for deep middle-down proteomics. Middle-down peptides obtained under the condition of optimized limited Glu-C digestion were first size-fractionated by polyacrylamide gel electrophoresis, followed by C4 reversed-phase liquid chromatography separation and additional ion mobility fractionation, resulting in a significant increase in peptide length detectable by mass spectrometry. In addition to global analysis, the GeLC-FAIMS concept can also be applied to targeted middle-down proteomics, where only proteins in the desired molecular weight range are gel-fractionated and their Glu-C digestion products are analyzed, as demonstrated by targeted analysis of integrins in exosomes. In-depth middle-down proteomics achieved by global and targeted GeLC-FAIMS-MS allows the exploration of proteoform information not covered by conventional top-down proteomics via increase in the number of detectable proteoforms and improvement in sequence coverage.

2024-11-20 | PXD041358 | Pride

Microproteomics workflows for quantitative proteome analysis of single Caenorhabditis elegans nematodes

Project description:Miniaturization of sample preparation including omissible manual sample handling steps is key for reproducible nanoproteomics as material is often restricted to only hundreds of cells or single model organisms. Here, we demonstrate a highly sensitive digital microfluidics (DMF)-based sample preparation workflow making use of single-pot solid-phase enhanced sample preparation (SP3) in combination with high-field asymmetric-waveform ion mobility spectrometry (FAIMS), and fast and sensitive ion trap detection on an Orbitrap tribrid MS system. Compared to a manual in-tube SP3-supported sample preparation, the number of peptides identified, the achievable standard deviations between replicates, and the number of proteins identified as differentially abundant were markedly increased. We identified up to 5,000 proteins from single nematodes. Moreover, label-free quantification of protein changes in single Caenorhabditis elegans treated with a heat stimulus yielded 45 differentially abundant proteins when compared to the untreated control, demonstrating the potential of this technology for low-input proteomics studies.

2022-08-12 | PXD033143 | Pride

Isobaric peptide labeling on digital microfluidics for quantitative proteomics of low cell numbers is facilitated by low critical micellar concentration detergents

Project description:We developed protocols for isobaric TMT labeling on digital microfluidics (DMF) devices enabling the quantitative proteome analysis of approximately 25 mammalian cells per channel by subsequent bottom-up proteome analysis by LC-MS. This comprised identification of a compatible detergent for digestion, on-chip labeling, and LC-MS. Application of the method in a TMT 6-plex sample analysis enabled the relative quantification 648 proteins (2 unique peptides for quantification) from a total of 125 cell equivalents.

2022-02-28 | PXD023226 | Pride

Qualitative Analysis of short open reading frame encoded proteins from Methanosarcina mazei under stress conditions via bottom up and top down proteomics

Project description:In the present work we have analysed Methanosarcina mazei grown under different stressor conditions via combined top-down and bottom-up proteomic workflows to determine which SEP are differenitally present in responce to stress conditions

2024-09-24 | PXD055745 | Pride

PROTEOFORMER 2.0: MS validation

Project description:PROTEOFORMER is a pipeline for generating a search space of candidate translation products based on ribosome profiling data. The pipeline was published in 2014, but since then, several features have been added. For this project, the PROTEOFORMER pipeline 2.0 was tested on human HCT116 and Jurkat cell ribosome profiling data with all its new features and then compared to raw MS data of both cell lines, validating its use as a proteogenomic tool. In this study, results of 3 proteoform calling methods were combined (classical PROTEOFORMER proteoform calling, PRICE and SPECtre). Afterwards, results were eventually combined with the canonical or splicing-included version of human UniProt. The different described combinations were exported as a FASTA file and used as search space for searching matching MS data.

2019-05-03 | PXD011353 | Pride

Application of Microdroplet PCR for Large-Scale Targeted Bisulfite Sequencing

Project description:Cytosine methylation of DNA CpG dinucleotides in gene promoters is an epigenetic modification that regulates gene transcription. While many methods exist to interrogate methylation states, no current methods offer large-scale, targeted, single CpG resolution. We report an approach combining bisulfite treatment followed by RainDance microdroplet PCR with next-generation sequencing to assay the methylation state of 50 genes in the regions 1 kb upstream and downstream of their transcription start sites. Wildtype and hypermethylated Jurkat DNA (New Englad Biolabs) was treated with bisulfite to convert all unmethylated cytosines to uracil. Following bisulfite treatment, targeted amplification was carried out using a custom primer library and microdroplet PCR. PCR product was sheared to 200 bp and ligated to sequencing adapters following standard protocols. Sequencing was conducted with single-end 100 bp reads on an Illumina GAIIx for wild type Jurkat DNA or Jurkat CpG DNA with a single sample per lane.

2011-06-25 | E-GEOD-30179 | biostudies-arrayexpress

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data