Dataset Information

A novel tool, Multiple Marker Abundance Profiling, uses Selected Reaction Monitoring and data-dependent acquisition for rapid estimation of organelle abundance in subcellular samples.

ABSTRACT: Methods were investigated that would deliver rapid and semi-quantitative analysis of the subcellular composition of Arabidopsis samples, as the standard technique for this i.e. immunoblotting is difficult to quantify and relies on a very few protein targets, whose responses to developmental or environmental cues are often not known. Spectral counts from shotgun analysis of different Arabidopsis tissues, cells or growth stages were compared to selected reaction monitoring (SRM) analyses of the same samples. Results were further compared to a novel protein abundance scoring method, MASCP Gator scoring, described in this study for the first time. The latter is reliant on previously collated public proteomics data and so provides an estimate of protein abundance even in the absence of de-novo experimentation. This scoring method is presented as an online tool, Multiple Marker Abundance Profiling (MMAP), available at http:SUBA.live. This submission comprises the shotgun data from this study. SRM results are available in a separate submission: http://www.peptideatlas.org/PASS/PASS00906

INSTRUMENT(S): TripleTOF 5600, LTQ Orbitrap Elite

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Plant Cell, Flower, Cell Suspension Culture, Root, Rosette

SUBMITTER: Harriet Parsons

LAB HEAD: Joshua L Heazlewood

PROVIDER: PXD004576 | Pride | 2022-03-02

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	1M_interface (17_F094717_1Minterface).mzid_1M_interface_(17_F094717_1Minterface).pride.mgf.gz	Mzid
	1M_interface (17_F094717_1Minterface).pride.mztab.gz	Mztab
	1M_interface17_F094717_1Minterface.mzid.gz	Mzid
	1M_interface17_F094717_1Minterface.mzid_1M_interface_17_F094717_1Minterface.MGF	Mzid
	F094697_root.mzXML	Mzxml

Items per page:

1 - 5 of 85

Similar Datasets

Project description:Bile has gained increasing attention for its potential to reflect the overall health of the biliary system. Proteomic analysis of bile could deepen our understanding of the molecular pathophysiology of biliary disease and injury and provides a potential source of diagnostic biomarkers. With the emergence of normothermic machine perfusion (NMP) prior to liver transplantation, bile samples can be easily collected and analyzed. However, the unique composition of bile, including high concentrations of endogenous detergents and salts, can make application of proteomics challenging. In this study, we systematically evaluated a variety of available trypsin-digestion methods to optimize proteomics analysis of bile obtained from human NMP livers. Bile was collected from 12 human donor livers that were accepted for transplantation after NMP viability assessment. We performed tryptic digestion using 6 different methods: in-gel, in-solution, S-Trap, SMART, EasyPep, and filter-aided sample purification, with or without an additional precipitation step prior to digestion. Untargeted, data-independent acquisition proteomics was performed using an Exploris 480 mass spectrometer coupled with a FAIMS device. The methods were judged for total protein IDs, variation, and specific protein characteristics to determine the most optimal method. A total of 4650 unique proteins were identified across all samples and all methods. Methods involving precipitation identified substantially more proteins (4500 vs 3815, respectively). In-gel crude was the exception, identifying a comparable number of proteins to precipitated in-gel digest. We found minimal differences in the mass, cellular component, or hydrophobicity of identified proteins between methods. Intra-method variability was notably diverse, with in-gel, in-solution, and EasyPep outperforming other methods with considerably less variation. Age-related biological comparisons revealed significant disparities in protein abundances between methods, but younger donors consistently showed upregulation of metabolic-related processes compared to older donors. Older donors showed upregulation of immune response and cell cycle-related processes. The digestion method used for proteomic analysis of bile can heavily influence the results obtained. A method of protein purification appears to be essential for sufficient quality mass spectrometry runs and subsequent data. Sample processing speed, cost, cleanliness, and reproducibility should be carefully considered when selecting a protein digestion method for this difficult sample type.

Dataset Information

A novel tool, Multiple Marker Abundance Profiling, uses Selected Reaction Monitoring and data-dependent acquisition for rapid estimation of organelle abundance in subcellular samples.

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets