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HCD fragmentation of glycated peptides

ABSTRACT: Protein glycation is a highly disease-relevant concentration-dependent non-enzymatic reaction of reducing sugars with amine groups of proteins to form early as well as advanced glycation products (AGEs). To complement our blood proteomics studies in diabetics, we here established higher-energy collisional dissociation (HCD) fragmentation on Orbitrap mass spectrometers for analyzing protein glycation. We evaluated parameters to most efficiently fragment and identify early glycation products on in-vitro glycated model proteins. We then applied our optimized workflow for glycation analysis of the entire HeLa proteome as well as for single-run analysis of undepleted and unenriched blood plasma and whole blood. We conclude that HCD fragmentation is well suited for analyzing glycated peptides when integrating the dominant neutral loss into the analysis and that single-run plasma proteomics measurements have great potential to diagnosing the diabetic status of patients.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Blood

SUBMITTER: Mario Oroshi

LAB HEAD: Matthias Mann

PROVIDER: PXD004182 | Pride | 2017-01-02

REPOSITORIES: Pride

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Project description:One of the hallmarks of Pseudomonas aeruginosa cystic fibrosis (CF) infection is very high-cell-density (HCD) replication in the lung, allowing this bacterium to induce virulence controlled by HCD quorum-sensing systems. However, the nutrient sources sustaining HCD replication in this chronic infection is largely unknown. Hence, understanding the nutrient factors contributing to HCD in the CF lung will yield new insights into the 'metabolic pathogenicity' and potential treatment of CF infections caused by P. aeruginosa. Herein, we performed microarray studies of P. aeruginosa directly isolated from the CF lung to demonstrate its metabolic capability and virulence in vivo. Our in vivo microarray data, confirmed by real-time reverse-transcription-PCR, indicated P. aeruginosa expressed several genes for virulence, drug-resistance, and utilization of multiple nutrient sources (lung surfactant lipids and amino acids) contributing to HCD replication. The data also indicates deregulation of several pathways, suggesting in vivo evolution by deregulation of a large portion of the transcriptome during chronic CF infection. To our knowledge, this is the first in vivo transcriptome of P. aeruginosa in a natural CF infection, and it indicates several important aspects of pathogenesis, drug-resistance, and nutrient-utilization never before observed in vivo. Experiment Overall Design: The purpose of the experiment was to observe which genes are upregulated in P. aeruginosa during chronic CF lung infection as compared to PAO1. All in vitro studies were grown in 1x M9 minimal media supplemented with 20 mM citrate and grown to mid-log phase prior to RNA isolation. The in vivo RNA was isolated directly from CF sputum samples after TRIzol treatment. Each in vitro sample (both for PAO1 and the CF sputum pool isolate) were processed individually and in triplicate. Two in vivo isolations from sputum were conducted from the same patient but two different sputum samples. After isolation of total RNA, samples were processed for microarrays (i.e. cDNA synthesis, fragmentation, labeling, etc) as recommended by Affymetrix, and processed on the GeneChip as recommended by Affymetrix.

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HCD fragmentation of glycated peptides

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