Dataset Information

13C6-Lysine-labelled mouse serum N-terminomics

ABSTRACT: To investigate the putative lifetime of proteolytically cleaved protein fragments in mouse serum, wildtype mice were fed with 13C6-Lysine for 1 or 2 weeks and the incorporation of 13C6-Lysine into proteins/protein fragments was investigated to achieve lifetime estimations for the fragment(s') generation.

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Blood Serum

SUBMITTER: Fatih Demir

LAB HEAD: Markus Rinschen

PROVIDER: PXD051273 | Pride | 2025-09-12

REPOSITORIES: Pride

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			Action	DRS
	2024-01-17-decoys-isoforms-contam-UP000000589.fas	Other
	230309-13CLys-pulseChase_Serum-HUNTER-DIA_8PT_0w-R2.raw	Raw
	230309-13CLys-pulseChase_Serum-HUNTER-DIA_8PT_0w-R2_FS_-45V.raw	Raw
	230309-13CLys-pulseChase_Serum-HUNTER-DIA_8PT_0w-R2_FS_-65V.raw	Raw
	230309-13CLys-pulseChase_Serum-HUNTER-DIA_8PT_1w-R2.raw	Raw

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Project description:Hypertension and kidney disease, two related, common, and severe disease entities have been repeatedly associated with genomic variants and metabolic alterations of lysine metabolism. Here, we developed a stable isotope labeling strategy compatible with untargeted metabolomics acquisition to investigate the physiology and molecular spectrum of lysine’s metabolic fate in vivo. Mice received 13C6 labeled lysine through the diet over two months to track more than 100 lysine metabolites across various organs and body fluids. Globally, lysine reacts rapidly with molecules of the central carbon metabolism, as opposed to slow or incorporation into proteins and metabolization into acylcarnitines. Lysine metabolism is accelerated in the rat model of the Dahl salt-sensitive hypertension and kidney damage, chiefly through N-alpha-mediated degradation. Here, lysine administration completely diminished the development of salt-sensitive hypertension and kidney injury. Administration of lysine leads to diuresis, even further acceleration of 13C6 lysine conjugate formation, and inhibition of albumin uptake, thereby protecting from nephron injury and metabolic stress. Lysine conjugates with malonyl-CoA to form a novel metabolite N-malonyl-lysine, to deplete malonyl-CoA from fatty acid synthesis. This process occurs at the expense of protein malonylation. In hypertensive rats with kidney damage, lysine molecules were excreted as fructoselysine, saccharopine and Nε-acetyllysine, via the urine, leading to an overall depletion of central carbon metabolites from the organism and kidney. Consistent with findings in the salt-sensitive rat, lysine challenge of patients with mild kidney damage inhibited tubular albumin uptake, increased lysine conjugate formation in the urine, and reduced TCA cycle metabolites, in contrast to kidney-healthy volunteers. In conclusion, comprehensive lysine isotope tracing mapped an accelerated lysine metabolism in hypertension, and further, lysine administration induced kidney protection in kidney disease.

Project description:Protein extraction and proteolytic digestion were performed using a Filter-Assisted Sample Preparation (FASP) protocol. In case of phosphopeptide enrichment immobilized Fe(III) affinity chromatography (Fe-IMAC) was performed. The peptides were fractionated using an HPLC system fitted with an SCX column. 10 sample datasets were used: 1. CRC_N: Normal tissue samples were obtained from 20 colorectal cancer patients, no quantification.Phosphopeptide enrichment was performed. 2. CRC_T: Human colorectal cancer tissue samples were obtained from 20 patients, no quantification. Phosphopeptide enrichment was performed. 3. CRC_iTRAQ: Human colorectal cancer tissue samples were obtained from 24 patients, iTRAQ quantification. 4. CRC_phospho_iTRAQ:Human colorectal cancer tissue samples were obtained from 24 patients, iTRAQ quantification.Phosphopeptide enrichment was performed. 5. HCT116_iTRAQ: Human colon cancer cell HCT116, iTRAQ quantification. 6. HCT116_phospho_iTRAQ: Human colon cancer cell HCT116, phosphopeptide enrichment, iTRAQ quantification. 7. SW_SILAC_HL: Human colon cancer cell SW480 and SW620, SW480 and SW620 were grown in SILAC media, containing l-arginine (Arg0) and l-lysine (Lys0) (SW480; light), or 13C615N4-l-arginine (Arg10) and 13C6-l-Lysine (Lys6) (SW620; heavy). 8. SW_SILAC_LH: SW480 and SW620 were grown in SILAC media, containing l-arginine (Arg0) and l-lysine (Lys0) (SW620; light), or 13C615N4-l-arginine (Arg10) and 13C6-l-Lysine (Lys6) (SW480; heavy). 9. SW_phospho_SILAC_HL: SW480 and SW620 were grown in SILAC media, containing l-arginine (Arg0) and l-lysine (Lys0) (SW480; light), or 13C615N4-l-arginine (Arg10) and 13C6-l-Lysine (Lys6) (SW620; heavy). Phosphopeptide enrichment was performed. 10. SW_phospho_SILAC_LH: SW480 and SW620 were grown in SILAC media, containing l-arginine (Arg0) and l-lysine (Lys0) (SW620; light), or 13C615N4-l-arginine (Arg10) and 13C6-l-Lysine (Lys6) (SW480; heavy). Phosphopeptide enrichment was performed. For creation of xml and mgf files, Proteome Discoverer 1.3 and Mascot v2.3 software were used.

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13C6-Lysine-labelled mouse serum N-terminomics

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