Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 2 de 2
Filter
Add more filters










Database
Language
Publication year range
1.
Proteomics Clin Appl ; 12(1)2018 01.
Article in English | MEDLINE | ID: mdl-28975736

ABSTRACT

PURPOSE: The objective of this study is to better understand factors governing the variability and sensitivity in SRM and PRM, compared to immunoassay. EXPERIMENTAL DESIGN: A 2D-LC-MS/MS-based SRM and PRM assay is developed for quantitative measurements of HSP90α in serum. Forty-three control sera are compared by SRM, PRM, and ELISA following the manufacturer's instructions. Serum samples are trypsin-digested and fractionated by strong cation exchange chromatography prior to SRM and PRM measurements. Analytical parameters such as linearity, LOD, LOQ, repeatability, and reproducibility of the SRM, PRM, and ELISA are determined. RESULTS: PRM data obtained by high-resolution MS correlate better with ELISA measurements than SRM data measured on a triple quadrupole mass spectrometer. While all three methods (SRM, PRM, and ELISA) are able to quantify HSP90α in serum at the ng mL-1 level, the use of PRM on a high-resolution mass spectrometer reduces variation and shows comparable sensitivity to immunoassay. CONCLUSIONS AND CLINICAL RELEVANCE: Using fractionation, it is possible to measure ng mL-1 levels of HSP90α in a reproducible, selective, and sensitive way using PRM in serum. This opens up the possibility to use PRM in a multiplexed way as an attractive alternative for immunoassays without the use of antibodies or comparable binders.


Subject(s)
HSP90 Heat-Shock Proteins/blood , Immunoassay/methods , Peptide Fragments/blood , Proteomics/methods , Tandem Mass Spectrometry/methods , Adult , Amino Acid Sequence , Chromatography, Liquid , Female , Humans , Limit of Detection , Proteolysis , Reproducibility of Results
2.
Rapid Commun Mass Spectrom ; 27(4): 546-52, 2013 Feb 28.
Article in English | MEDLINE | ID: mdl-23322661

ABSTRACT

RATIONALE: Cleavage of peptide bonds C-terminal to tyrosine and tryptophan after electrochemical oxidation may become a complementary approach to chemical and enzymatic cleavage. A chemical labeling approach specifically targeting reactive cleavage products is presented here and constitutes a promising first step towards the development of a new proteomics workflow. METHODS: Hexylamine was used to react with the spirolactone moieties generated after electrochemical oxidation and cleavage of tripeptides. The influence of pH and reaction time on the yield was determined and the excess of tagging reagent was optimized. Selective detection of the tagged cleavage products was achieved by precursor ion scanning in a triple quadrupole mass spectrometer. RESULTS: Optimal labeling was reached under aqueous conditions when working at pH 10 with a reaction time of 0.5 min. The excess of hexylamine over spirolactone groups can be significantly decreased by working under non-aqueous conditions in pure acetonitrile to prevent spirolactone hydrolysis. The specific formation of hexylamine-containing y(1) reporter ions generated by collision-induced dissociation (CID) tandem mass spectrometry (MS/MS) allows for selective detection by precursor ion scanning of the cleaved and labeled peptides. CONCLUSIONS: This work presents a method for selective labeling and detection of electrochemically cleaved Tyr- and Trp-containing peptides for which reaction conditions have been optimized with hexylamine as labeling agent. This workflow offers new possibilities for electrochemical oxidation, cleavage and labeling of peptides and proteins.


Subject(s)
Electrochemical Techniques/methods , Oligopeptides/analysis , Oligopeptides/chemistry , Tandem Mass Spectrometry/methods , Amines/chemistry , Hydrogen-Ion Concentration , Oxidation-Reduction , Spironolactone/chemistry , Tryptophan/chemistry , Tyrosine/chemistry
SELECTION OF CITATIONS
SEARCH DETAIL
...