CEEPC/IPM/CMSC - Abstrakt prezentace

(CEEPC/IPM/CMSC 2022 - ThO-04)
Non-Immunoaffinity Extraction of Intact Proteins from Biological Fluids and Their Analysis by Liquid Chromatography – Triple Quadrupole Mass Spectrometry

Katarína Maráková 1 *, Shannon L. Thomas 2, Beatriz J. Renner 2, Kevin A. Schug 2

  1. Farmaceutická fakulta, Univerzita Komenského v Bratislave
  2. University of Texas at Arlington, Arlington, TX, United States


One of the crucial steps in quantitation of intact proteins from complex biological matrices is, except their reliable analysis, also sample preparation to achieve sufficient specificity and sensitivity. Commonly used immunoaffinity-based methods are characterized by their superior selectivity, although this can be a drawback if simultaneous analysis of multiple different proteins from a single sample is required. In our work, we developed non-immunoaffinity sample preparation based on a generally widely affordable microelution solid phase extraction for eleven model intact proteins (5.5 – 29 kDa) with various isoelectric points (4.5 - 11.3). Extracted intact proteins were analysed by reversed-phase liquid chromatography coupled with a triple quadrupole mass spectrometer operated in a multiple reaction monitoring (MRM) mode. Reversed-phase separations were performed on the Restek wide-pore Viva C4 column, as mobile phases served water and acetonitrile acidified by 0.1% difluoroacetic acid and 0.2% formic acid. The best recoveries for most of the selected proteins were obtained by using the HLB stationary phase. 1% trifluoroacetic acid and 0.2% Triton X-100 were used as efficient pretreatment reagents to release interactions between the proteins and biological matrix. Multiple sample loading was found out to be essential to obtain recoveries >65% in urine for all targeted proteins (up to 30kDa) and >50% in serum/plasma for most of the proteins. Limits of quantitation in biological matrices were in the range 2 - 1200 ng/mL, corresponding to 0.23 - 97.6 nM.

* Korespondující autor: marakova@fpharm.uniba.sk


The research stay of Dr. Maráková at the University of Texas at Arlington was supported by the Fulbright Scholarship Program. This work was also partially supported by the Scientific Grant Agency of the Ministry of Education, Science, Research and Sport of the Slovak Republic under the project VEGA 1/0483/20.

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