Dataset Information

OXIPEP: mass spectrometry strategy to characterize protein carbonylation.

ABSTRACT: Here we describe a peptide-centric approach (OXIPEP), where carbonylated residues within proteins are derivatized with biotin-hydrazide, enriched and identified by mass spectrometry. The strength of the method lies in an improved protocol for elution of biotinylated peptides from monomeric avidin resin using hot water (95C) and increased sensitivity achieved by reduction of sample losses during sample preparation and chromatographic steps. For the first time advanced bioinformatics analysis utilising diagnostic biotin fragment ions is used to facilitate semi-automatic in silico evaluation of biotin labelling and to increase the reliability of carbonyl peptide identification.

INSTRUMENT(S): LTQ Orbitrap, Q Exactive

ORGANISM(S): Homo Sapiens (human) Bos Taurus (bovine)

TISSUE(S): Blood Plasma

SUBMITTER: Adelina Rogowska-Wrzesinska

LAB HEAD: Ole N. Jensen

PROVIDER: PXD002966 | Pride | 2018-10-18

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	Diag.removed_QHF0006_semi_tryptic.msf	Msf
	Diag.removed_QHF0006_tryptic.msf	Msf
	Diag.removed_QHF0009_semi_tryptic.msf	Msf
	Diag.removed_QHF0009_tryptic.msf	Msf
	Diag.removed_QHF0012_semi_tryptic.msf	Msf

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Publications

A biotin enrichment strategy identifies novel carbonylated amino acids in proteins from human plasma.

Havelund Jesper F JF Wojdyla Katarzyna K Davies Michael J MJ Jensen Ole N ON Møller Ian Max IM Rogowska-Wrzesinska Adelina A

Journal of proteomics 20170104

Protein carbonylation is an irreversible protein oxidation correlated with oxidative stress, various diseases and ageing. Here we describe a peptide-centric approach for identification and characterisation of up to 14 different types of carbonylated amino acids in proteins. The modified residues are derivatised with biotin-hydrazide, enriched and characterised by tandem mass spectrometry. The strength of the method lies in an improved elution of biotinylated peptides from monomeric avidin resin ...[more]

PMID: 28062376

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Project description:Exposure to Paraquat and RNA interference knockdown of mitochondrial superoxide dismutase (Sod2) are known to result in significant lifespan reduction, locomotor dysfunction, and mitochondrial degeneration in Drosophila melanogaster. Both perturbations increase the flux of superoxide, a progenitor reactive oxygen species, but the molecular underpinnings of the resulting phenotypes are poorly understood. Improved understanding of such processes could lead to advances in the treatment of numerous age-related disorders. Superoxide toxicity can act through protein carbonylation. Analysis of carbonylated proteins is attractive since reactive carbonyl groups are not present in the twenty canonical amino acids and are amenable to labeling and enrichment strategies. Here, carbonylated proteins were labeled with biotin hydrazide and enriched on streptavidin-coated beads. On-bead digestion was used to release carbonylated protein peptides, with relative abundance ratios versus controls obtained using the iTRAQ mass spectrometry-based proteomics approach. While Paraquat exposure and Sod2 knockdown have similar phenotypes, differences in protein carbonylation were anticipated because Paraquat exposure was expected to increase the concentration of superoxide throughout the cell while Sod2 knockdown was only expected to raise the concentration of superoxide in the mitochondrial matrix. Paraquat exposure resulted in widespread increases in carbonylated protein relative abundance: the median Paraquat-exposed to control carbonylated protein relative abundance ratio was 1.53. For Sod2 knockdown, in contrast, the median carbonylated protein relative abundance ratios were 1.13 versus the RNA interference driver control and 1.05 versus the RNA interference transgene control. However, some proteins did show large increases in carbonylated protein relative abundance on Sod2 knockdown, most notably cytochrome c oxidase subunit Vb, possibly providing some indication of the molecular basis of the Sod2-knockdown phenotype.