Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Modular bottom-up proteomic sample preparation for high-throughput applications

ABSTRACT: A collection of digital automated proteomic sample preparation protocols detail three optimized step-by-step methods to: (A) lyse Gram-negative bacteria and fungal cells; (B) quantify the amount of protein extracted; and (C) normalize the amount of protein and set up tryptic digestion. These protocols have been developed to facilitate rapid, low variance sample preparation of hundreds of samples, be easily implemented on widely-available Beckman-Coulter Biomek automated liquid handlers, and allow flexibility for future protocol development

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Pseudomonas Putida Rhodotorula Toruloides

SUBMITTER: Christopher Petzold

LAB HEAD: Christopher J. Petzold

PROVIDER: PXD029122 | Pride | 2022-02-14

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
	D7-Pp-R1.raw	Raw
	D7-Pp-R10.raw	Raw
	D7-Pp-R10__F022038_.mzid.gz	Mzid
	D7-Pp-R10__F022038_.mzid_D7-Pp-R10__F022038_.MGF	Mzid
	D7-Pp-R11.raw	Raw

Items per page:

1 - 5 of 564

Similar Datasets

30% O2 vs PS 10W/m2

Project description:Comparison of wild-type (2.4.1) R. sphaeroides gene expression in 30% O2 aerobic respiration vs. photosynthesis 10W/m2 light, to identify candidate photosynthesis genes. RNA preparation: pellet cells from exponential phase cultures using 5% phenol in ethanol stop solution; lysozyme + SDS to lyse; phenol/chloroform extraction + DNase treatment + RNeasy kit clean up. Manuscript in preparation. The below normalized data was generated using GeneSpring software with the following settings: A data: Data Transformation: set all values less than 0.01 to 0.01 Per Chip: normalize to 50th percentile, and then Per Gene: normalize to median B data: Data Transformation: set all values less than 0.01 to 0.01 Per Chip: normalize to 50th percentile Values from independent replicates were averaged Keywords = Rhodobacter sphaeroides Keywords = photosynthesis Keywords = purple bacteria Keywords: other

2005-04-01 | GSE2145 | GEO

Fully automated workflow for integrated sample digestion and Evotip loading enabling high-throughput clinical proteomics

Project description:Protein identification and quantification is an important tool for biomarker discovery. With the increased sensitivity and speed of modern mass spectrometers, sample-preparation remains a bottleneck for studying large cohorts. To address this issue, we prepared and evaluated a simple and efficient workflow on the Opentrons OT-2 (OT-2) robot that combines sample digestion, cleanup and Evotip loading in a fully automated manner, allowing the processing of up to 192 samples in 6 hours. Our results demonstrate a highly sensitive workflow yielding both reproducibility and stability even at low sample inputs. The workflow is optimized for minimal sample starting amount to reduce the costs for reagents needed for sample preparation, which is critical when analyzing large biological cohorts. Building on the digesting workflow, we incorporated an automated phosphopeptide enrichment step using magnetic Ti-IMAC beads. This allows for a fully automated proteome and phosphoproteome sample preparation in a single step with high sensitivity. Using the integrated workflow, we evaluated the effects of cancer immune therapy on the plasma proteome in metastatic melanoma patients.

2024-06-12 | PXD048325 | Pride

30% O2 vs PS 10W/m2

2010-06-10 | E-GEOD-2145 | biostudies-arrayexpress

ChipFilter: Microfluidic Based Comprehensive Sample Preparation Methodology for Metaproteomics

Project description:Here, we show that a ChipFilter microfluidic device coupled to LC-MS/MS can successfully be used for identification of microbial proteins. Using cultures of E. coli, B. subtilis and S. cerevisiae, we have shown that it is possible to directly lyse the cells and digest the proteins in the ChipFilter to allow higher number of proteins and peptides identification than standard protocols, even at low cell density. The peptides produced are overall longer after ChipFilter digestion but show no change in their degree of hydrophobicity. Analysis of a more complex mixture of 17 species from the gut microbiome showed that the ChipFilter preparation was able to identify and estimate the amount of 16 of these species.

2024-05-24 | PXD039581 | Pride

Automated sample preparation workflow for tandem mass tag-based proteomics

Project description:Although tandem mass tag (TMT)-based isobaric labeling has become a powerful technique for multiplexed protein quantitation, it has not been easy to automate the workflow for widespread adoption. This is because preparation of TMT labeled peptide samples involves multiple steps ranging from protein extraction, denaturation, reduction and alkylation to tryptic digestion, desalting, labeling with TMT reagents and cleanup, all of which require a high level of proficiency. The variability resulting from multiple processing steps is inherently problematic especially with large-scale studies such as clinical studies that involve hundreds of samples where reproducibility is critical for quantitation. Here, we sought to compare the performance of a recently introduced platform, AccelerOme, for automated proteomics workflows for TMT-labeling experiments with manual processing of samples. Cell pellets were prepared and subjected to a 16-plex experiment using the automated platform and a conventional manual protocol. Single shot LC-MS/MS analysis revealed a higher number of proteins and peptides identified using the automated platform. Efficiencies of tryptic digestion, alkylation and TMT labeling were similar both in manual and automated process. In addition, comparison of quantitation accuracy and precision showed similar performance in automated workflow compared to manual sample preparation. Overall, we demonstrated that the automated platform performs at a level similar to manual process in TMT-based proteomics. We expect that the automated workflow will increasingly replace manual work and be applied to large-scale TMT-baed studies providing robust results and high sample throughput.

2023-10-24 | PXD040788 | Pride

Rumen metaproteomics - Catching the tip of the iceberg - evaluation of sample preparation protocols for metaproteomic studies of the rumen microbiota

Project description:Metaproteomic studies of the rumen microbiota are challenged by the need of optimized sample preparation protocols in order to retrieve an enhanced amount of prokaryotic instead of plant and bovine derived cells before protein extraction and subsequent LC-MS/MS analysis. The present study evaluates three different protocols applied to the rumen microbiota either attached to plant fibres or present as planktonic cells. The findings of our work suggest the integration of cheesecloth-gauze filtration in sample preparation to achieve a better protein identification ratio.

2015-04-01 | PXD001526 | Pride

DIA based HCP detection in protein A purified IgG1

Project description:Detection and quantitation of host cell proteins in monoclonal antibody drug products using automated sample preparation and data independent acquisition LC-MS/MS

2021-05-26 | PXD020127 | Pride

Successive protein extraction using hydroxylamine to increase the depth of proteome coverage in fresh, forensic and archaeological bones

Project description:Proteomics is continually being applied to a wider range of applications, now including the analysis of archaeological samples and anatomical specimens, in particular, collagen-containing tissues such as bones and teeth. Here we present the application of a chemi-cal digestion-based proteomics sample preparation protocol to the analysis of fresh, anatomical and archaeological samples. We are describing and discussing two protocols, one using hydroxylamine as an additional step of the proteomic workflow, applied to the insoluble fraction, and another focusing directly on demineralized bones and teeth. We demonstrate the additional information that can be extracted using both protocols, including increasing the sequence coverage and number of peptides detected in modern and archaeological samples, and increasing the number of proteins identified in archaeological samples. Targeting research related to collagens or extracellular matrix proteins, the use of this protocol will open new insights considering both fresh and ancient mineral-ized samples.

2024-03-28 | PXD046969 | Pride

Assessment of automated trimethylsilyl derivatisation protocols for GC-MS-based untargeted metabolomic analysis of urine

Project description:GC-MS is a commonly used metabolomic platform for the analysis of urine. A key step in the preparation of samples for GC-MS is derivatisation, in particular, methoximation and trimethylsilylation. This paper presents an assessment of automated derivatisation protocols for GC-MS-based untargeted metabolomic analysis of rat urine. Automated batch and in-time (a sample ready for injection every 70 minutes) derivatisation protocols were tested using BSTFA and MSTFA. Principal component analysis determined differences based upon protocol tested (PC-1; 19%) and silylation reagent (PC-2; 17%) used. Of 249 compounds, 40 compounds were significantly different (P<0.05) based upon reagent and 154 compounds were significantly different (P<0.05) based upon protocol. A key outcome of this study was the demonstrated effects of derivatisation including reagent and protocol (i.e. reaction duration, temperature and mixing speed) on individual urinary metabolites. It is hoped that the current work will provide a reference on which to base future GC-MS-based untargeted and targeted metabolomic analyses of urine.

2015-09-28 | MTBLS171 | MetaboLights

Systematic assessment of next-generation sequencing for quantitative small RNA profiling: synthetic ratiometric pools

Project description:Small RNA-seq is increasingly being used for profiling of small RNAs. Quantitative characteristics of long RNA-seq have been extensively described, but small RNA-seq involves fundamentally different methods for library preparation, with distinct protocols and technical variations that have not been fully and systematically studied. Using common sets of reference samples, we evaluated the accuracy, reproducibility and bias of small RNA-seq library preparation for five distinct protocols and across nine different laboratories. As part of this larger study, we assessed reproducibility and accuracy of relative expression measurements using two pools of synthetic small RNA sequences, where subsets of the sRNAs vary in relative amount between pools A and B. The pools each contain 334 small RNAs, varying by 15 different ratios between pools A and B (10:1, 8:1, 5:1, 4:1, 3:1, 2:1, 1.5:1, 1:1, 1:1.5, 1:2, 1:3, 1:4 1:5, 1:8, 1:10). We find that although the sequencing bias varies extensively between protocols, the relative abundance measured between samples A and B is largely reproducible and accurate across labs and protocols. These results suggest that measurements of differential expression should be comparable across institutions and library preparation technologies.

2018-07-09 | GSE94585 | GEO

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