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Probing label-free intracellular quantification of free peptide by MALDI-ToF mass spectrometry

Probing label-free intracellular quantification of free peptide by MALDI-ToF mass spectrometry
Probing label-free intracellular quantification of free peptide by MALDI-ToF mass spectrometry

Cell-penetrating peptides are promising reagents for gene and drug delivery. They can efficiently traverse the plasma membrane and deliver various cargo materials ranging from genes to nanoparticles. The functional efficiency of cargo often depends on the completeness of intracellular peptide uptake, which can be measured, but its quantification remains largely inconclusive. Existing approaches rely on the use of radioactive and fluorescent labels or tags which allow colorimetric, fluorescent or spectrometric detection, but lack the ability to detect free peptide. Herein we describe a generic label- and tag-free method to measure the concentration of internalised peptide by matrix-assisted laser desorption/ionisation time of flight mass spectrometry. Quantification is preceded by two-dimensional chromatography and is performed at benign temperatures for the lysates of human dermal fibroblasts transfected with cell penetrating peptides in free form. Isotopically labelled peptides of the same structure are used as internal standards to enable accurate determination of concentration of the recovered free peptide. The method offers a minimalistic approach for intracellular quantification, which can be used as a correlative measure for fluorescence-based imaging methods.

Cell penetrating peptides, Gene therapy, Mass spectrometry, Transfection
1046-2023
331-337
Rakowska, Paulina D.
73bf2145-e2fa-46ee-9ad9-f74c80d5a369
Lamarre, Baptiste
f269271f-1c0b-4d1e-b0c3-4fd15f795bb8
Ryadnov, Maxim G.
5bd99202-02e9-42e6-b56f-300852222d06
Rakowska, Paulina D.
73bf2145-e2fa-46ee-9ad9-f74c80d5a369
Lamarre, Baptiste
f269271f-1c0b-4d1e-b0c3-4fd15f795bb8
Ryadnov, Maxim G.
5bd99202-02e9-42e6-b56f-300852222d06

Rakowska, Paulina D., Lamarre, Baptiste and Ryadnov, Maxim G. (2014) Probing label-free intracellular quantification of free peptide by MALDI-ToF mass spectrometry. Methods, 68 (2), 331-337. (doi:10.1016/j.ymeth.2014.03.012).

Record type: Article

Abstract

Cell-penetrating peptides are promising reagents for gene and drug delivery. They can efficiently traverse the plasma membrane and deliver various cargo materials ranging from genes to nanoparticles. The functional efficiency of cargo often depends on the completeness of intracellular peptide uptake, which can be measured, but its quantification remains largely inconclusive. Existing approaches rely on the use of radioactive and fluorescent labels or tags which allow colorimetric, fluorescent or spectrometric detection, but lack the ability to detect free peptide. Herein we describe a generic label- and tag-free method to measure the concentration of internalised peptide by matrix-assisted laser desorption/ionisation time of flight mass spectrometry. Quantification is preceded by two-dimensional chromatography and is performed at benign temperatures for the lysates of human dermal fibroblasts transfected with cell penetrating peptides in free form. Isotopically labelled peptides of the same structure are used as internal standards to enable accurate determination of concentration of the recovered free peptide. The method offers a minimalistic approach for intracellular quantification, which can be used as a correlative measure for fluorescence-based imaging methods.

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More information

Accepted/In Press date: 3 March 2013
e-pub ahead of print date: 20 March 2014
Additional Information: Funding Information: We acknowledge funding from the United Kingdom’s Department of Business, Innovation and Skills and from the European Community’s Seventh Framework Programme , ERA-NET Plus , under Grant Agreement No. 217257 .
Keywords: Cell penetrating peptides, Gene therapy, Mass spectrometry, Transfection

Identifiers

Local EPrints ID: 485934
URI: http://eprints.soton.ac.uk/id/eprint/485934
ISSN: 1046-2023
PURE UUID: 512edbed-305f-4c59-abc1-a588dc0cb14b
ORCID for Paulina D. Rakowska: ORCID iD orcid.org/0000-0002-3710-8395

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Date deposited: 04 Jan 2024 04:56
Last modified: 06 Jun 2024 02:09

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Contributors

Author: Baptiste Lamarre
Author: Maxim G. Ryadnov

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