Dataset Information

PyQms - partial labeled proteome gold standard

ABSTRACT: " The novel mass spectrometry quantitation tool pyQms can calculate isotope patterns for molecules that have any isotope enrichment or combinations of, e.g. 13C or 15N and allows partially labeled molecules to be quantified as well, as observed in pulse or pulse-chase experiments where enrichment levels change dynamically. In order to test how pyQms performs on such a task, we created a gold standard (GS) data set of partially labeled proteomes, which allows evaluation of algorithms detecting partial labeld isotopologues against the ground truth against 5 mixed samples. Cultures of Chlamydomonas reinhardtii were grown on medium containing 20, 40, 60, 80 and 100% 15N. Since partially labeled peptides will not produce interpretable fragmentation spectra, each of the partially labeled proteomes were mixed with an unlabeled (14N) or fully labeled (15N) proteome, yielding 5 mixed samples (0|100, 20|100, 40|100, 0|60 and 0|80). " (Modified from manuscript)

INSTRUMENT(S): Q Exactive

ORGANISM(S): Chlamydomonas Reinhardtii

TISSUE(S): Photosynthetic Cell

SUBMITTER: Johannes Leufken

LAB HEAD: Dr. Christian Fufezan

PROVIDER: PXD003236 | Pride | 2017-08-10

REPOSITORIES: Pride

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Publications

pyQms enables universal and accurate quantification of mass spectrometry data.

Leufken Johannes J Niehues Anna A Sarin L Peter LP Wessel Florian F Hippler Michael M Leidel Sebastian A SA Fufezan Christian C

Molecular & cellular proteomics : MCP 20170720 10

Quantitative mass spectrometry (MS) is a key technique in many research areas (1), including proteomics, metabolomics, glycomics, and lipidomics. Because all of the corresponding molecules can be described by chemical formulas, universal quantification tools are highly desirable. Here, we present pyQms, an open-source software for accurate quantification of all types of molecules measurable by MS. pyQms uses isotope pattern matching that offers an accurate quality assessment of all quantificatio ...[more]

PMID: 28729385

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Project description:To ensure that the pool of precursor amino acids utilized for protein synthesis is completely labeled prior to the first labeling time-point, we conducted an isotopomer analysis. After adaption to media, cells were plated at a density of 500,000 cells per 10 cm plate. 8 days after plating, the confluent quiescent cultures were switched to 15N labeling medium. Cells were collected after 0h, 6h, 1d, 2d, 4d of labeling, washed with PBS and cell pellets were frozen prior to further analysis. 15N labeling medium was made from “Cell free” Amino Acid Mix (20AA, U-15N, 96-98%)(Cambridge Isotope Laboratories) and supplemented with 15% dialyzed fetal bovine serum (Thermo Scientific), 100U/mL penicillin, 100U/mL streptomycin. 1g is used for making 702mL labeling medium. The final amino acids concentrations in 15N labeling medium are as following: Alanine: 0.1140 g/L, Arginine: 0.0499 g/L, Asparagine: 0.0940 g/L, Aspartic acid: 0.1353 g/L, Cystine: 0.01140 g/L, Glutamic acid: 0.1852 g/L, Glutamine: 0.0869 g/L, Glycine: 0.0698 g/L, Histidine: 0.0157 g/L, Isoleucine: 0.0698 g/L, Leucine: 0.1211 g/L, Lysine: 0.0527 g/L, Methionine: 0.0228 g/L, Phenylalanine: 0.0541 g/L, Proline: 0.0299 g/L, Serine: 0.0541 g/L, Threonine: 0.0741 g/L, Tryptophan: 0.0456 g/L, Tyrosine: 0.0613g/L, Valine: 0.0798 g/L. LC-MS/MS and quantitative analysis of isotopic envelopes and 15N incorporation of peptides was conducted as described in (Zhang et al., 2014). The data indicate that when exposed to fully 15N-labeled media (where all 20 natural amino acids are isotopically labeled), the fraction of proteins that are synthesized within the first day are almost fully labeled. Thus, the extent of fractional labeling is almost exclusively influenced by the kinetics of protein turnover rather than amino acid uptake and recycling within the cell. Table of files Cells Time points Labeling Fractionation Raw Data Filenames Wildtype 0d, 1d,2d,4d,7d13C 6Lys, 6Arg& 15N No 0d.raw 1d.raw 2d.raw 4d.raw 7d.raw

Dataset Information

PyQms - partial labeled proteome gold standard

Publications

pyQms enables universal and accurate quantification of mass spectrometry data.

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