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Generating high quality libraries for DIA MS with empirically corrected peptide predictions.

ABSTRACT: Data-independent acquisition approaches typically rely on experiment-specific spectrum libraries, requiring offline fractionation and tens to hundreds of injections. We demonstrate a library generation workflow that leverages fragmentation and retention time prediction to build libraries containing every peptide in a proteome, and then refines those libraries with empirical data. Our method specifically enables rapid, experiment-specific library generation for non-model organisms, which we demonstrate using the malaria parasite Plasmodium falciparum, and non-canonical databases, which we show by detecting missense variants in HeLa.

SUBMITTER: Searle BC

PROVIDER: S-EPMC7096433 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Generating high quality libraries for DIA MS with empirically corrected peptide predictions.

Searle Brian C BC Swearingen Kristian E KE Barnes Christopher A CA Schmidt Tobias T Gessulat Siegfried S Küster Bernhard B Wilhelm Mathias M

Nature communications 20200325 1

Data-independent acquisition approaches typically rely on experiment-specific spectrum libraries, requiring offline fractionation and tens to hundreds of injections. We demonstrate a library generation workflow that leverages fragmentation and retention time prediction to build libraries containing every peptide in a proteome, and then refines those libraries with empirical data. Our method specifically enables rapid, experiment-specific library generation for non-model organisms, which we demon ...[more]

PMID: 32214105

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Project 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.

Dataset Information

Generating high quality libraries for DIA MS with empirically corrected peptide predictions.

Publications

Generating high quality libraries for DIA MS with empirically corrected peptide predictions.

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