Screening combinatorial libraries of de Novo proteins by hydrogen- deuterium exchange and electrospray mass spectrometry

Daniel M. Rosenbaum, Sushmita Roy, Michael H. Hecht

Research output: Contribution to journalArticle

33 Citations (Scopus)

Abstract

Combinatorial methods have emerged as valuable tools for the discovery of proteins, nucleic acids, and small molecules with novel structures and properties. While combinatorial methods can generate de novo proteins with native-like properties, finding such proteins in libraries containing an abundance of non-native structures has proved difficult and tedious. To overcome these difficulties, we developed a rapid screen for native-like properties. The screen uses electrospray mass spectrometry (ESMS) to monitor the hydrogen-deuterium (H-D) exchange kinetics in semicrude samples of de novo proteins expressed in Escherichia coli. To demonstrate the utility of the approach, we screened two libraries of de novo sequences and identified proteins whose amide protons were protected from exchange with solvent. The results of the screen correlate well with orthogonal methods for detecting native-like structures. As protection of amide protons from exchange is a hallmark of well-folded proteins, this screen can be used to identify native- like proteins from combinatorial libraries containing both native-like and molten globule-like structures. Moreover, since the screen can be applied to semicrude samples and does not require extensive protein purification, it can be used for medium throughput screening of large combinatorial libraries.

Original languageEnglish (US)
Pages (from-to)9509-9513
Number of pages5
JournalJournal of the American Chemical Society
Volume121
Issue number41
DOIs
StatePublished - Oct 20 1999

Fingerprint

Deuterium
Mass spectrometry
Hydrogen
Mass Spectrometry
Screening
Proteins
Amides
Libraries
Protons
Ion exchange
Nucleic acids
Escherichia coli
Nucleic Acids
Purification
Molten materials
Throughput
Molecules
Kinetics

ASJC Scopus subject areas

  • Chemistry(all)

Cite this

Screening combinatorial libraries of de Novo proteins by hydrogen- deuterium exchange and electrospray mass spectrometry. / Rosenbaum, Daniel M.; Roy, Sushmita; Hecht, Michael H.

In: Journal of the American Chemical Society, Vol. 121, No. 41, 20.10.1999, p. 9509-9513.

Research output: Contribution to journalArticle

@article{624babe5eb6e4c8aa9ba01c08e8bbb3b,
title = "Screening combinatorial libraries of de Novo proteins by hydrogen- deuterium exchange and electrospray mass spectrometry",
abstract = "Combinatorial methods have emerged as valuable tools for the discovery of proteins, nucleic acids, and small molecules with novel structures and properties. While combinatorial methods can generate de novo proteins with native-like properties, finding such proteins in libraries containing an abundance of non-native structures has proved difficult and tedious. To overcome these difficulties, we developed a rapid screen for native-like properties. The screen uses electrospray mass spectrometry (ESMS) to monitor the hydrogen-deuterium (H-D) exchange kinetics in semicrude samples of de novo proteins expressed in Escherichia coli. To demonstrate the utility of the approach, we screened two libraries of de novo sequences and identified proteins whose amide protons were protected from exchange with solvent. The results of the screen correlate well with orthogonal methods for detecting native-like structures. As protection of amide protons from exchange is a hallmark of well-folded proteins, this screen can be used to identify native- like proteins from combinatorial libraries containing both native-like and molten globule-like structures. Moreover, since the screen can be applied to semicrude samples and does not require extensive protein purification, it can be used for medium throughput screening of large combinatorial libraries.",
author = "Rosenbaum, {Daniel M.} and Sushmita Roy and Hecht, {Michael H.}",
year = "1999",
month = "10",
day = "20",
doi = "10.1021/ja991843x",
language = "English (US)",
volume = "121",
pages = "9509--9513",
journal = "Journal of the American Chemical Society",
issn = "0002-7863",
publisher = "American Chemical Society",
number = "41",

}

TY - JOUR

T1 - Screening combinatorial libraries of de Novo proteins by hydrogen- deuterium exchange and electrospray mass spectrometry

AU - Rosenbaum, Daniel M.

AU - Roy, Sushmita

AU - Hecht, Michael H.

PY - 1999/10/20

Y1 - 1999/10/20

N2 - Combinatorial methods have emerged as valuable tools for the discovery of proteins, nucleic acids, and small molecules with novel structures and properties. While combinatorial methods can generate de novo proteins with native-like properties, finding such proteins in libraries containing an abundance of non-native structures has proved difficult and tedious. To overcome these difficulties, we developed a rapid screen for native-like properties. The screen uses electrospray mass spectrometry (ESMS) to monitor the hydrogen-deuterium (H-D) exchange kinetics in semicrude samples of de novo proteins expressed in Escherichia coli. To demonstrate the utility of the approach, we screened two libraries of de novo sequences and identified proteins whose amide protons were protected from exchange with solvent. The results of the screen correlate well with orthogonal methods for detecting native-like structures. As protection of amide protons from exchange is a hallmark of well-folded proteins, this screen can be used to identify native- like proteins from combinatorial libraries containing both native-like and molten globule-like structures. Moreover, since the screen can be applied to semicrude samples and does not require extensive protein purification, it can be used for medium throughput screening of large combinatorial libraries.

AB - Combinatorial methods have emerged as valuable tools for the discovery of proteins, nucleic acids, and small molecules with novel structures and properties. While combinatorial methods can generate de novo proteins with native-like properties, finding such proteins in libraries containing an abundance of non-native structures has proved difficult and tedious. To overcome these difficulties, we developed a rapid screen for native-like properties. The screen uses electrospray mass spectrometry (ESMS) to monitor the hydrogen-deuterium (H-D) exchange kinetics in semicrude samples of de novo proteins expressed in Escherichia coli. To demonstrate the utility of the approach, we screened two libraries of de novo sequences and identified proteins whose amide protons were protected from exchange with solvent. The results of the screen correlate well with orthogonal methods for detecting native-like structures. As protection of amide protons from exchange is a hallmark of well-folded proteins, this screen can be used to identify native- like proteins from combinatorial libraries containing both native-like and molten globule-like structures. Moreover, since the screen can be applied to semicrude samples and does not require extensive protein purification, it can be used for medium throughput screening of large combinatorial libraries.

UR - http://www.scopus.com/inward/record.url?scp=0032724728&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0032724728&partnerID=8YFLogxK

U2 - 10.1021/ja991843x

DO - 10.1021/ja991843x

M3 - Article

AN - SCOPUS:0032724728

VL - 121

SP - 9509

EP - 9513

JO - Journal of the American Chemical Society

JF - Journal of the American Chemical Society

SN - 0002-7863

IS - 41

ER -