2D-LC as an on-line desalting tool allowing peptide identification directly from MS unfriendly HPLC methods

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Abstract

The increasing interest in peptides and proteins in pharmaceutical research and development has led to many challenges for the researchers tasked with characterizing and analyzing these larger molecules. Due to the more complicated impurity profile of peptides and proteins, multiple liquid chromatography techniques are often needed to achieve comprehensive analysis. However, many of these separation conditions require buffers, salts or additives that render them incompatible with mass spectrometry (MS) detection. Previous researchers have demonstrated proof of concept for the use of two dimensional liquid chromatography (2D-LC) to provide convenient second dimension online desalting of components purified in the first chromatographic dimension. In this paper, we evaluated the Agilent heart-cutting 2D-LC system connected with an Agilent Q-TOF mass spectrometer to address this frequently encountered analytical challenge. On this 2D-LC/MS system, fractions containing the compounds of interest are separated by the first dimension using an MS incompatible mobile phase, then sent to a second dimension HPLC method where fast desalting using an MS compatible mobile phase is performed prior to MS analysis. The system allows for fast and direct collection of MS information for chromatographic peaks eluted in MS incompatible mobile phases, without requiring difficult, time consuming and error-prone translation of chromatographic methods from MS incompatible to MS compatible eluents, or off-line fraction collection and preparation. Several examples showing the application of the approach to complex mixtures containing peptides with impurities and positional isomers are presented.

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