MALDI-MS Analysis of Peptide Libraries Expands the Scope of Substrates for Farnesyltransferase

Protein farnesylation is a post-translational modification where a 15-carbon farnesyl isoprenoid is appended to the C-terminal end of a protein by farnesyltransferase (FTase). This modification typically causes proteins to associate with the membrane and allows them to participate in signaling pathw...

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Autores principales:	Garrett L. Schey, Peter H. Buttery, Emily R. Hildebrandt, Sadie X. Novak, Walter K. Schmidt, James L. Hougland, Mark D. Distefano
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	protein prenylation enzymology MALDI libraries Biology (General) QH301-705.5 Chemistry QD1-999
Acceso en línea:	https://doaj.org/article/273def10be134692b1140cbbd0a3aa91
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spelling	oai:doaj.org-article:273def10be134692b1140cbbd0a3aa912021-11-11T17:26:27ZMALDI-MS Analysis of Peptide Libraries Expands the Scope of Substrates for Farnesyltransferase10.3390/ijms2221120421422-00671661-6596https://doaj.org/article/273def10be134692b1140cbbd0a3aa912021-11-01T00:00:00Zhttps://www.mdpi.com/1422-0067/22/21/12042https://doaj.org/toc/1661-6596https://doaj.org/toc/1422-0067Protein farnesylation is a post-translational modification where a 15-carbon farnesyl isoprenoid is appended to the C-terminal end of a protein by farnesyltransferase (FTase). This modification typically causes proteins to associate with the membrane and allows them to participate in signaling pathways. In the canonical understanding of FTase, the isoprenoids are attached to the cysteine residue of a four-amino-acid CaaX box sequence. However, recent work has shown that five-amino-acid sequences can be recognized, including the pentapeptide CMIIM. This paper describes a new systematic approach to discover novel peptide substrates for FTase by combining the combinatorial power of solid-phase peptide synthesis (SPPS) with the ease of matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS). The workflow consists of synthesizing focused libraries containing 10–20 sequences obtained by randomizing a synthetic peptide at a single position. Incubation of the library with FTase and farnesyl pyrophosphate (FPP) followed by mass spectrometric analysis allows the enzymatic products to be clearly resolved from starting peptides due to the increase in mass that occurs upon farnesylation. Using this method, 30 hits were obtained from a series of libraries containing a total of 80 members. Eight of the above peptides were selected for further evaluation, reflecting a mixture that represented a sampling of diverse substrate space. Six of these sequences were found to be bona fide substrates for FTase, with several meeting or surpassing the in vitro efficiency of the benchmark sequence CMIIM. Experiments in yeast demonstrated that proteins bearing these sequences can be efficiently farnesylated within live cells. Additionally, a bioinformatics search showed that a variety of pentapeptide CaaaX sequences can be found in the mammalian genome, and several of these sequences display excellent farnesylation in vitro and in yeast cells, suggesting that the number of farnesylated proteins within mammalian cells may be larger than previously thought.Garrett L. ScheyPeter H. ButteryEmily R. HildebrandtSadie X. NovakWalter K. SchmidtJames L. HouglandMark D. DistefanoMDPI AGarticleprotein prenylationenzymologyMALDI librariesBiology (General)QH301-705.5ChemistryQD1-999ENInternational Journal of Molecular Sciences, Vol 22, Iss 12042, p 12042 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	protein prenylation enzymology MALDI libraries Biology (General) QH301-705.5 Chemistry QD1-999
spellingShingle	protein prenylation enzymology MALDI libraries Biology (General) QH301-705.5 Chemistry QD1-999 Garrett L. Schey Peter H. Buttery Emily R. Hildebrandt Sadie X. Novak Walter K. Schmidt James L. Hougland Mark D. Distefano MALDI-MS Analysis of Peptide Libraries Expands the Scope of Substrates for Farnesyltransferase
description	Protein farnesylation is a post-translational modification where a 15-carbon farnesyl isoprenoid is appended to the C-terminal end of a protein by farnesyltransferase (FTase). This modification typically causes proteins to associate with the membrane and allows them to participate in signaling pathways. In the canonical understanding of FTase, the isoprenoids are attached to the cysteine residue of a four-amino-acid CaaX box sequence. However, recent work has shown that five-amino-acid sequences can be recognized, including the pentapeptide CMIIM. This paper describes a new systematic approach to discover novel peptide substrates for FTase by combining the combinatorial power of solid-phase peptide synthesis (SPPS) with the ease of matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS). The workflow consists of synthesizing focused libraries containing 10–20 sequences obtained by randomizing a synthetic peptide at a single position. Incubation of the library with FTase and farnesyl pyrophosphate (FPP) followed by mass spectrometric analysis allows the enzymatic products to be clearly resolved from starting peptides due to the increase in mass that occurs upon farnesylation. Using this method, 30 hits were obtained from a series of libraries containing a total of 80 members. Eight of the above peptides were selected for further evaluation, reflecting a mixture that represented a sampling of diverse substrate space. Six of these sequences were found to be bona fide substrates for FTase, with several meeting or surpassing the in vitro efficiency of the benchmark sequence CMIIM. Experiments in yeast demonstrated that proteins bearing these sequences can be efficiently farnesylated within live cells. Additionally, a bioinformatics search showed that a variety of pentapeptide CaaaX sequences can be found in the mammalian genome, and several of these sequences display excellent farnesylation in vitro and in yeast cells, suggesting that the number of farnesylated proteins within mammalian cells may be larger than previously thought.
format	article
author	Garrett L. Schey Peter H. Buttery Emily R. Hildebrandt Sadie X. Novak Walter K. Schmidt James L. Hougland Mark D. Distefano
author_facet	Garrett L. Schey Peter H. Buttery Emily R. Hildebrandt Sadie X. Novak Walter K. Schmidt James L. Hougland Mark D. Distefano
author_sort	Garrett L. Schey
title	MALDI-MS Analysis of Peptide Libraries Expands the Scope of Substrates for Farnesyltransferase
title_short	MALDI-MS Analysis of Peptide Libraries Expands the Scope of Substrates for Farnesyltransferase
title_full	MALDI-MS Analysis of Peptide Libraries Expands the Scope of Substrates for Farnesyltransferase
title_fullStr	MALDI-MS Analysis of Peptide Libraries Expands the Scope of Substrates for Farnesyltransferase
title_full_unstemmed	MALDI-MS Analysis of Peptide Libraries Expands the Scope of Substrates for Farnesyltransferase
title_sort	maldi-ms analysis of peptide libraries expands the scope of substrates for farnesyltransferase
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/273def10be134692b1140cbbd0a3aa91
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MALDI-MS Analysis of Peptide Libraries Expands the Scope of Substrates for Farnesyltransferase

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