Dataset Information

Characterization of disulfide linkages in proteins by 193 nm ultraviolet photodissociation (UVPD)

ABSTRACT: Inter-linked disulfide bonds connecting peptide chains are homolytically cleaved with 193 nm ultraviolet photodissociation (UVPD). Analysis of insulin demonstrates the ability for UVPD to cleave multiple disulfide bonds and provide sequence coverage of multiple peptide chains in the same MS/MS event. The information provided by UVPD of disulfide-bound peptides is comparable to information garnered from other high energy dissociation methods but requires an activation period an order of magnitude faster than these methods. This phenomenon was investigated, along with a partial reduction and alkylation sample preparation to determine disulfide connectivities of enzymatically digested proteins. The 4 disulfide bonds of lysozyme and the 19 disulfide bonds of serotransferrin were unambiguously characterized through non-reduced and partially reduced protein digestions.

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

SUBMITTER: Jennifer S. Brodbelt

PROVIDER: PXD009460 | JPOST Repository | Sat Apr 28 00:00:00 BST 2018

REPOSITORIES: jPOST

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Publications

Characterization of Disulfide Linkages in Proteins by 193 nm Ultraviolet Photodissociation (UVPD) Mass Spectrometry.

Quick M Montana MM Crittenden Christopher M CM Rosenberg Jake A JA Brodbelt Jennifer S JS

Analytical chemistry 20180628 14

Deciphering disulfide bond patterns in proteins remains a significant challenge. In the present study, interlinked disulfide bonds connecting peptide chains are homolytically cleaved with 193 nm ultraviolet photodissociation (UVPD). Analysis of insulin showcased the ability of UVPD to cleave multiple disulfide bonds and provide sequence coverage of the peptide chains in the same MS/MS event. For proteins containing more complex disulfide bonding patterns, an approach combining partial reduction ...[more]

PMID: 29902373

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Project description:Obesity-induced insulin resistance of the liver is characterised by increased gluconeogenesis, which contributes to elevated blood glucose levels in individuals with type 2 diabetes. Research into how fatty acids induce insulin resistance has commonly focused on the induction of insulin resistance. We hypothesise that by shifting focus to the reversal of an insulin resistant phenotype, novel insights can be made into the mechanisms by which insulin resistance can be overcome. Using global gene and lipid expression profiling, we aimed to identify biological pathways altered in parallel with restoration of palmitate-induced deregulation of glucose production using metformin and sodium salicylate. FAO hepatoma cells were treated with palmitate (0.075mM, 48h) with or without metformin (0.25mM) and sodium salicylate (2mM) in the final 24h of palmitate treatment, and effects on glucose production were determined. Microarray followed by gene set enrichment analysis was performed to investigate pathway regulation. A lipidomic analysis (HPLC-MS/MS) and measurement of secreted bile acids and cholesterol were performed. Reversal of palmitate-induced impairment of glucose production by metformin and sodium salicylate was characterised by down-regulated expression of metabolic pathways regulating acetyl-CoA to cholesterol and bile acid biosynthesis. Total levels of intracellular and secreted cholesterol and bile acids were not different between impaired and restored glucose production. Total intracellular levels of diacylgycerol, triacylglycerol and cholesterol esters increased with palmitate (impaired glucose production), however, these were not further altered with metformin and sodium salicylate (restored glucose production). Six individual lipid species containing 18:0 and 18:1 side-chains were reduced by metformin and sodium salicylate. Widespread lipid metabolism changes induced by the reversal of palmitate-induced deregulation of glucose production with metformin and sodium salicylate were identified. While cholesterol and bile acid levels remained unchanged, the flux through these pathways may in part explain these findings. The identification of lipid species containing 18:0 and 18:1 side chains being regulated alongside changes to glucose production may indicate potential mediators of glucose production and insulin resistance. Three-condition experiment, Vehicle, Palmitate (PA) and Palmitate (PA) + Metformin (Met) + Sodium Sailcylate (NaS) with biological replicates: 8 Vehicle, 20 PA and 20 PA+Met+NaS , independently grown and harvested. One replicate per array.

Dataset Information

Characterization of disulfide linkages in proteins by 193 nm ultraviolet photodissociation (UVPD)

Publications

Characterization of Disulfide Linkages in Proteins by 193 nm Ultraviolet Photodissociation (UVPD) Mass Spectrometry.

Similar Datasets

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