Peptide labeling with lanthanide-NHS-ester-DOTA investigated by nano-HPLC

International audienceLabeling of peptides and proteins using chelators binding metal ions has become a novel approach for quantitative proteomics in recent years. The aim of this work was the optimization of a new method for peptide derivatization with lanthanide labels (holmium, lutetium, and thulium) followed by nano high performance liquid chromatography (nano-HPLC) separation with UV detection. Matrix-assisted laser desorption ionization-mass spectrometry (MALDI MS) was used to confirm the derivatization and to identify the derivatized peptides. Peptides were labeled with the three different lanthanide metals using a bifunctional DOTA-based (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) reagent. The results demonstrate that the derivatization reaction using the chelating reagent DOTA-NHS-ester was effective for single peptides and peptide mixtures. Furthermore, an efficient pre-cleaning method was applied by nano-HPLC using a C-18 trap column for elimination of the excess of labeling reagent. The application of the optimized method to label peptides in a Cytochrome C digest delivered comparable results to those obtained with model peptides. © 2014 Elsevier B.V

Evaluation and standardization of different purification procedures for fish bile and liver metallothionein quantification by spectrophotometry and SDS-PAGE analyses

International audienceFish bile metallothioneins (MT) have been recently reported as biomarkers for environmental metal contamination; however, no studies regarding standardizations for their purification are available. Therefore, different procedures (varying centrifugation times and heat-treatment temperatures) and reducing agents (DTT, β-mercaptoethanol and TCEP) were applied to purify MT isolated from fish (Oreochromis niloticus) bile and liver. Liver was also analyzed, since these two organs are intrinsically connected and show the same trend regarding MT expression. Spectrophotometrical analyses were used to quantify the resulting MT samples, and SDS-PAGE gels were used to qualitatively assess the different procedure results. Each procedure was then statistically evaluated and a multivariate statistical analysis was then applied. A response surface methodology was also applied for bile samples, in order to further evaluate the responses for this matrix. Heat treatment effectively removes most undesired proteins from the samples, however results indicate that temperatures above 70 °C are not efficient since they also remove MTs from both bile and liver samples. Our results also indicate that the centrifugation times described in the literature can be decreased in order to analyze more samples in the same timeframe, of importance in environmental monitoring contexts where samples are usually numerous. In an environmental context, biliary MT was lower than liver MT, as expected, since liver accumulates MT with slower detoxification rates than bile, which is released from the gallbladder during feeding, and then diluted by water. Therefore, bile MT seems to be more adequate in environmental monitoring scopes regarding recent exposure to xenobiotics that may affect the proteomic and metalloproteomic expression of this biological matrix