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Peptide/protein separation with cationic polymer brush nanosponges for MALDI-MS analysis.

ABSTRACT: A cationic polymer nanobrush was synthesized, attached to a MALDI target, and used for the fractionation of peptides and proteins based on their pI, prior to analysis by MALDI-MS. The cationic polymer nanobrush was synthesized on a gold substrate by AIBN photoinitiated polymerization, using a 70:30 ratio of 2-aminoethyl methacrylate hydrochloride (AEMA):N-isopropylacrylamide (NIPAAM). This brush showed selectivity for adsorption of acidic peptides and proteins and allowed fractionation of simple two-component mixtures to be completed in less than 10 min. The brush-adsorbed biomolecules were recovered by treating the nanobrush with ammonium hydroxide, which effectively collapsed the brush, thereby releasing the trapped compounds for MALDI MS analysis. These results demonstrate that nanobrush can serve as a convenient platform for rapid fractionation of biomolecules prior to analysis by MALDI-MS.

SUBMITTER: Mitrovic B

PROVIDER: S-EPMC3632665 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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Peptide/protein separation with cationic polymer brush nanosponges for MALDI-MS analysis.

Mitrovic Bojan B Eastwood Stephanie S Wong VenNey V Dyer Daniel D Kinsel Gary G Scott Colleen C

Langmuir : the ACS journal of surfaces and colloids 20121231 2

A cationic polymer nanobrush was synthesized, attached to a MALDI target, and used for the fractionation of peptides and proteins based on their pI, prior to analysis by MALDI-MS. The cationic polymer nanobrush was synthesized on a gold substrate by AIBN photoinitiated polymerization, using a 70:30 ratio of 2-aminoethyl methacrylate hydrochloride (AEMA):N-isopropylacrylamide (NIPAAM). This brush showed selectivity for adsorption of acidic peptides and proteins and allowed fractionation of simple ...[more]

PMID: 23244629

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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

Peptide/protein separation with cationic polymer brush nanosponges for MALDI-MS analysis.

Publications

Peptide/protein separation with cationic polymer brush nanosponges for MALDI-MS analysis.

Similar Datasets

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