CEEPC/IPM/CMSC - Abstrakt prezentace

(CEEPC/IPM/CMSC 2022 - ThP-11)
Maximizing peptide yield in biological sample preparation for LC-MS analysis

Hynek Mácha 1,2 *, Rutuja Patil 1,2, Syed Moin Uddin 3, L'udovít Škultéty 2, Milan Raška 3, Vladimír Havlíček 1,2

  1. Department of Analytical Chemistry, Faculty of Science, Palacký University, Olomouc
  2. Institute of Microbiology of the Czech Academy of Sciences, Vídeňská 1083, Prague
  3. Department of Immunology, Faculty of Medicine and Dentistry, Palacký University, Olomouc

Abstrakt

The mass spectrometric (MS) analysis of peptides and proteins has become an important tool for understanding biological processes. Due to variability in the chemical nature of the peptides, its accurate and sensitive quantitation is not routine. In this work, we report the development and optimization of peptide extraction protocol using solid-phase extraction (SPE) for liquid chromatography (LC)-MS analysis. A set of four peptides with different structures in the 1–3.5 kDa range (concentration of 100 ng/mL) were selected and analyzed using C18-LC connected to Fourier transform ion cyclotron resonance MS. Initially, suitable sample solution and acid modifiers were tuned; after which 40% acetonitrile with 1% formic acid was selected as an initial sample solvent. Based on it, a suitable SPE protocol for peptides extraction was optimized. Later, we compared the peptide extraction by HLB and Sep-Pak SPEs and molecular weight cut-off filters (MWCO) (Amicon, Microcon). MWCO returned recovery from 0 % to 10 %, whereas SPE (Sep-Pak) with developed protocol had recovery from 60 % to 95 %. Further, we used 6M urea with 4% TFA (1:1) in a protein denaturation step for human cell culture samples. The application of the pre-treatment step had a beneficial effect in reducing the non-specific binding of peptides. Selected conditions were applied on cell culture samples wherein recoveries between 60 % to 550 % were obtained for spiked peptides. Our results showed significantly different peptide recovery for organic and acid modifiers and extraction approaches tested, which was in contrast to routinely used protocols. The optimized peptide extraction protocol will now be used in mapping the antimicrobial peptides in human neutrophils.

* Korespondující autor: macha.hynek12@gmail.com

Poděkování:

The authors gratefully acknowledge the support from the Doctoral Student Grant Competition 2021 (DSGC-2021-0144).


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