High-Throughput Synthesis and Analysis of Intact Glycoproteins Using SAMDI-MS

José Marc Techner; Weston Kightlinger; Liang Lin; Jasmine Hershewe; Ashvita Ramesh; Matthew P. Delisa; Michael C. Jewett; Milan Mrksich

doi:10.1021/acs.analchem.9b04334

High-Throughput Synthesis and Analysis of Intact Glycoproteins Using SAMDI-MS

José Marc Techner, Weston Kightlinger, Liang Lin, Jasmine Hershewe, Ashvita Ramesh, Matthew P. Delisa, Michael C. Jewett^*, Milan Mrksich

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

20 Scopus citations

Abstract

High-throughput quantification of the post-translational modification of many individual protein samples is challenging with current label-based methods. This paper demonstrates an efficient method that addresses this gap by combining Escherichia coli-based cell-free protein synthesis (CFPS) and self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry (SAMDI-MS) to analyze intact proteins. This high-throughput approach begins with polyhistidine-tagged protein substrates expressed from linear DNA templates by CFPS. Here, we synthesized an 87-member library of the E. coli Immunity Protein 7 (Im7) containing an acceptor sequence optimized for glycosylation by the Actinobacillus pleuropneumoniae N-glycosyltransferase (NGT) at every possible position along the protein backbone. These protein substrates were individually treated with NGT and then selectively immobilized to self-assembled monolayers presenting nickel-nitrilotriacetic acid (Ni-NTA) complexes before final analysis by SAMDI-MS to quantify the conversion of substrate to glycoprotein. This method offers new opportunities for rapid synthesis and quantitative evaluation of intact glycoproteins.

Original language	English (US)
Pages (from-to)	1963-1971
Number of pages	9
Journal	Analytical Chemistry
Volume	92
Issue number	2
DOIs	https://doi.org/10.1021/acs.analchem.9b04334
State	Published - Jan 21 2020

Funding

The authors acknowledge J. Stark, A. H. Thames, K. Duncker, and K. Warfel for helpful critiques and the sharing of reagents. We thank X. Zheng and M. Li for helpful discussions. We acknowledge support from the Defense Threat Reduction Agency (HDTRA1-15-10052/P00001), the International Institute for Nanotechnology (Ryan Graduate Fellowship), the Ruth L. Kirschtein National Research Service Award F30 CA196185, National Institutes of Environmental Health Sciences (T32 ES007059), and the National Science Foundation (MCB-1413563 and the Graduate Research Fellowship program under grant No. DGE-1324585). M.C.J. also acknowledges the Packard Foundation and the Dreyfus Teacher–Scholar Program. This work made use of the IMSERC core facility at Northwestern University, the Soft and Hybrid Nanotechnology Experimental (SHyNE) Resource (NSF ECCS-1542205).

ASJC Scopus subject areas

Analytical Chemistry

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10.1021/acs.analchem.9b04334

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title = "High-Throughput Synthesis and Analysis of Intact Glycoproteins Using SAMDI-MS",

abstract = "High-throughput quantification of the post-translational modification of many individual protein samples is challenging with current label-based methods. This paper demonstrates an efficient method that addresses this gap by combining Escherichia coli-based cell-free protein synthesis (CFPS) and self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry (SAMDI-MS) to analyze intact proteins. This high-throughput approach begins with polyhistidine-tagged protein substrates expressed from linear DNA templates by CFPS. Here, we synthesized an 87-member library of the E. coli Immunity Protein 7 (Im7) containing an acceptor sequence optimized for glycosylation by the Actinobacillus pleuropneumoniae N-glycosyltransferase (NGT) at every possible position along the protein backbone. These protein substrates were individually treated with NGT and then selectively immobilized to self-assembled monolayers presenting nickel-nitrilotriacetic acid (Ni-NTA) complexes before final analysis by SAMDI-MS to quantify the conversion of substrate to glycoprotein. This method offers new opportunities for rapid synthesis and quantitative evaluation of intact glycoproteins.",

author = "Techner, {Jos{\'e} Marc} and Weston Kightlinger and Liang Lin and Jasmine Hershewe and Ashvita Ramesh and Delisa, {Matthew P.} and Jewett, {Michael C.} and Milan Mrksich",

note = "Funding Information: The authors acknowledge J. Stark, A. H. Thames, K. Duncker, and K. Warfel for helpful critiques and the sharing of reagents. We thank X. Zheng and M. Li for helpful discussions. We acknowledge support from the Defense Threat Reduction Agency (HDTRA1-15-10052/P00001), the International Institute for Nanotechnology (Ryan Graduate Fellowship), the Ruth L. Kirschtein National Research Service Award F30 CA196185, National Institutes of Environmental Health Sciences (T32 ES007059), and the National Science Foundation (MCB-1413563 and the Graduate Research Fellowship program under grant No. DGE-1324585). M.C.J. also acknowledges the Packard Foundation and the Dreyfus Teacher–Scholar Program. This work made use of the IMSERC core facility at Northwestern University, the Soft and Hybrid Nanotechnology Experimental (SHyNE) Resource (NSF ECCS-1542205). Publisher Copyright: {\textcopyright} 2019 American Chemical Society.",

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doi = "10.1021/acs.analchem.9b04334",

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AU - Techner, José Marc

AU - Kightlinger, Weston

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AU - Hershewe, Jasmine

AU - Ramesh, Ashvita

AU - Delisa, Matthew P.

AU - Jewett, Michael C.

AU - Mrksich, Milan

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PY - 2020/1/21

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N2 - High-throughput quantification of the post-translational modification of many individual protein samples is challenging with current label-based methods. This paper demonstrates an efficient method that addresses this gap by combining Escherichia coli-based cell-free protein synthesis (CFPS) and self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry (SAMDI-MS) to analyze intact proteins. This high-throughput approach begins with polyhistidine-tagged protein substrates expressed from linear DNA templates by CFPS. Here, we synthesized an 87-member library of the E. coli Immunity Protein 7 (Im7) containing an acceptor sequence optimized for glycosylation by the Actinobacillus pleuropneumoniae N-glycosyltransferase (NGT) at every possible position along the protein backbone. These protein substrates were individually treated with NGT and then selectively immobilized to self-assembled monolayers presenting nickel-nitrilotriacetic acid (Ni-NTA) complexes before final analysis by SAMDI-MS to quantify the conversion of substrate to glycoprotein. This method offers new opportunities for rapid synthesis and quantitative evaluation of intact glycoproteins.

AB - High-throughput quantification of the post-translational modification of many individual protein samples is challenging with current label-based methods. This paper demonstrates an efficient method that addresses this gap by combining Escherichia coli-based cell-free protein synthesis (CFPS) and self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry (SAMDI-MS) to analyze intact proteins. This high-throughput approach begins with polyhistidine-tagged protein substrates expressed from linear DNA templates by CFPS. Here, we synthesized an 87-member library of the E. coli Immunity Protein 7 (Im7) containing an acceptor sequence optimized for glycosylation by the Actinobacillus pleuropneumoniae N-glycosyltransferase (NGT) at every possible position along the protein backbone. These protein substrates were individually treated with NGT and then selectively immobilized to self-assembled monolayers presenting nickel-nitrilotriacetic acid (Ni-NTA) complexes before final analysis by SAMDI-MS to quantify the conversion of substrate to glycoprotein. This method offers new opportunities for rapid synthesis and quantitative evaluation of intact glycoproteins.

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JO - Analytical Chemistry

JF - Analytical Chemistry

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ER -

High-Throughput Synthesis and Analysis of Intact Glycoproteins Using SAMDI-MS

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