Project PXD001352

PRIDE Assigned Tags:
Biological Dataset

Summary

Title

Absolute protein quantification allows differentiation of cell specific metabolic routes and functions

Description

Total protein approach (TPA) is a proteomic method which allows calculation of concentrations of individual proteins and groups of functionally related proteins in any protein mixture without spike-in standards. Using the two step digestion filter aided sample preparation method and LC-MS/MS analysis we generated comprehensive quantitative datasets of mouse intestinal mucosa, liver, red muscle fibers, brain, and of human plasma, erythrocytes, and tumor cells lines. We show that the TPA based quantitative data reflect well-defined and specific physiological functions of different organs and cells, for example nutrient absorption and transport in intestine, amino acid catabolism and bile secretion in liver, and contraction of muscle fibers. Focusing on key metabolic processes we compared metabolic capacities in different tissues and cells. In addition, we demonstrate quantitative differences in the mitochondrial proteomes. Providing insight into the abundances of mitochondrial metabolite transporters we demonstrate that their titers are well tuned to cell specific metabolic requirements. This study provides for the first time a comprehensive overview of the protein hardware mediating metabolism in different mammalian organs and cells. The presented approach can be applied to any other system to study biological processes.

Sample Processing Protocol

The lysates were processed according to the MED-FASP protocol that was extended with nucleic acid digestion steps. Briefly, aliquots containing 50 ug total protein were mixed with 200 uL of 8 M urea in 0.1 M Tris/HCl, pH 8.5 in centrifugal ultrafiltration units with a nominal molecular weight cut off of 30,000 (Cat No. MRCF0R030, Millipore), and then centrifuged at 14,000 × g, 20°C, for 15 min. The eluates were discarded, 100 μL of UA was pipetted into the filtration unit, and the units were centrifuged again. Then 50 uL of 0.05 M iodoacetamide in UA was added to the filters, and samples were incubated in darkness for 20 min. Filters were washed twice with 100 uL of UA followed by two washes with 100 uL of 0.05M Tris/HCl pH 8.5. Proteins were digested in 40 uL 0.05M Tris/HCl pH 8.5 at 37°C for 18 h, using endoproteinase LysC, at the enzyme to protein ratio of 1:50. The released peptides were collected by centrifugation at 14,000 × g for 10 min followed by two washes with 0.05M Tris/HCl pH 8.5. After isolation of the peptides, the material remaining on the filter was digested with trypsin using the above conditions, except that the cleavage reaction was performed only for 2 h.

Data Processing Protocol

The MS data were analyzed within the software environment MaxQuant [version 1.2.6.20], using the Andromeda search engine. Proteins were identified by searching MS and MS/MS data of peptides against UniProtKB human or mouse database. The FDR threshold was derived by analyzing the decoy database. Carboamidomethylation of cysteines was set as fixed modification. The maximum false peptide discovery rate was specified as 0.01. Spectra were searched with K-specificity for LysC and K/R but not K/RP for trypsin.

Contact

Mario Oroshi, Proteomics
Jacek R Wisniewski, Dept. Proteomics and Signal Transduction Max-Planck Institute of Biochemistry ( lab head )

Submission Date

22/09/2014

Publication Date

15/12/2014

Cell Type

cell line cell

Instrument

Q Exactive

Software

Not available

Quantification

Not available

Experiment Type

Shotgun proteomics

Publication

    Wiśniewski JR, Koepsell H, Gizak A, Rakus D. Absolute protein quantification allows differentiation of cell specific metabolic routes and functions. Proteomics. 2014 Dec 5 PubMed: 25475432