Absolute protein quantification by LC-ICP-MS using MeCAT peptide labeling |
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Authors: | Esteban-Fernández Diego Scheler Christian Linscheid Michael W |
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Institution: | (1) Department of Chemistry, Humboldt-Universitaet zu Berlin, Brook-Taylor-Str. 2, 12489 Berlin, Germany;(2) Proteome Factory AG, Magnus-Str. 11, 12489 Berlin, Germany |
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Abstract: | Nowadays, the most common strategies used in quantitative proteomics are based on isotope-coded labeling followed by specific
molecule mass spectrometry. The implementation of inductively coupled plasma mass spectrometry (ICP-MS) for quantitative purposes
can solve important drawbacks such as lack of sensitivity, structure-dependent responses, or difficulties in absolute quantification.
Recently, lanthanide-containing labels as metal-coded affinity tag (MeCAT) reagents have been introduced, increasing the interest
and scope of elemental mass spectrometry techniques for quantitative proteomics. In this work one of the first methodologies
for absolute quantification of peptides and proteins using MeCAT labeling is presented. Liquid chromatography (LC) interfaced
to ICP-MS has been used to separate and quantify labeled peptides while LC coupled to electrospray ionization mass spectrometry
served for identification tasks. Synthetic-labeled peptides were used as standards to calibrate the response of the detector
with compounds as close as possible to the target species. External calibration was employed as a quantification technique.
The first step to apply this approach was MeCAT-Eu labeling and quantification by isotope dilution ICP-MS of the selected
peptides. The standards were mixed in different concentrations and subjected to reverse-phase chromatography before ICP-MS
detection to consider the column effect over the peptides. Thus, the prepared multi-peptide mix allowed a calibration curve
to be obtained in a single chromatographic run, correcting possible non-quantitative elutions of the peptides from the column.
The quantification strategy was successfully applied to other labeled peptides and to standard proteins such as digested lysozyme
and bovine serum albumin. |
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