Quantitative LC-MS/MS analysis of 5-hydroxymethyl-2'-deoxyuridine to monitor the biological activity of J-binding protein

Jeroen Roosendaal; Tatjana Heidebrecht; Hilde Rosing; Anastassis Perrakis; Jos H Beijnen

doi:10.1016/j.ab.2020.113930

Quantitative LC-MS/MS analysis of 5-hydroxymethyl-2'-deoxyuridine to monitor the biological activity of J-binding protein

Jeroen Roosendaal, Tatjana Heidebrecht, Hilde Rosing, Anastassis Perrakis, Jos H Beijnen

Department of Pharmacy & Pharmacology, Netherlands Cancer Institute - Antoni van Leeuwenhoek, Amsterdam, the Netherlands; Division of Pharmacoepidemiology and Clinical Pharmacology, Science Faculty, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, the Netherlands.
Department of Biochemistry, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, the Netherlands; Oncode Institute, the Netherlands.
Department of Pharmacy & Pharmacology, Netherlands Cancer Institute - Antoni van Leeuwenhoek, Amsterdam, the Netherlands.

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

Abstract

Base J replaces 1% of thymine in most kinetoplastid flagellates, and is implicated in transcription regulation. Base J is synthesized in two steps: first, a thymine base in DNA is converted to 5-hydroxymethyluracil by J-binding proteins (JBP1, JBP2); secondly, a glucosyl transferase glycosylates the 5-hydroxymethyluracil to form base J. Here, we present a highly sensitive and selective LC-MS/MS method to quantify the in vitro JBP1 activity on synthetic oligonucleotide substrates. The method demonstrated successful to support biochemical studies of JBPs and can be used as a template for additional JBP activity studies or for inhibitor screening in the future.

Original language	English
Article number	113930
Number of pages	4
Journal	Analytical Biochemistry
Volume	610
DOIs	https://doi.org/10.1016/j.ab.2020.113930
Publication status	Published - 1 Dec 2020

Keywords

JBP1/2
LC-MS/MS
5-hydroxymethyl-2′ -deoxyuridine
Base J

Access to Document

10.1016/j.ab.2020.113930

1-s2.0-S0003269720304620-mainFinal published version, 1.26 MBLicence: Taverne

Cite this

@article{943d7054b92e4475ae904ae0946a6e98,

title = "Quantitative LC-MS/MS analysis of 5-hydroxymethyl-2'-deoxyuridine to monitor the biological activity of J-binding protein",

abstract = "Base J replaces 1% of thymine in most kinetoplastid flagellates, and is implicated in transcription regulation. Base J is synthesized in two steps: first, a thymine base in DNA is converted to 5-hydroxymethyluracil by J-binding proteins (JBP1, JBP2); secondly, a glucosyl transferase glycosylates the 5-hydroxymethyluracil to form base J. Here, we present a highly sensitive and selective LC-MS/MS method to quantify the in vitro JBP1 activity on synthetic oligonucleotide substrates. The method demonstrated successful to support biochemical studies of JBPs and can be used as a template for additional JBP activity studies or for inhibitor screening in the future.",

keywords = "JBP1/2, LC-MS/MS, 5-hydroxymethyl-2′ -deoxyuridine, Base J",

author = "Jeroen Roosendaal and Tatjana Heidebrecht and Hilde Rosing and Anastassis Perrakis and Beijnen, {Jos H}",

year = "2020",

month = dec,

day = "1",

doi = "10.1016/j.ab.2020.113930",

language = "English",

volume = "610",

journal = "Analytical Biochemistry",

issn = "0003-2697",

publisher = "Academic Press Inc.",

}

TY - JOUR

T1 - Quantitative LC-MS/MS analysis of 5-hydroxymethyl-2'-deoxyuridine to monitor the biological activity of J-binding protein

AU - Roosendaal, Jeroen

AU - Heidebrecht, Tatjana

AU - Rosing, Hilde

AU - Perrakis, Anastassis

AU - Beijnen, Jos H

PY - 2020/12/1

Y1 - 2020/12/1

N2 - Base J replaces 1% of thymine in most kinetoplastid flagellates, and is implicated in transcription regulation. Base J is synthesized in two steps: first, a thymine base in DNA is converted to 5-hydroxymethyluracil by J-binding proteins (JBP1, JBP2); secondly, a glucosyl transferase glycosylates the 5-hydroxymethyluracil to form base J. Here, we present a highly sensitive and selective LC-MS/MS method to quantify the in vitro JBP1 activity on synthetic oligonucleotide substrates. The method demonstrated successful to support biochemical studies of JBPs and can be used as a template for additional JBP activity studies or for inhibitor screening in the future.

AB - Base J replaces 1% of thymine in most kinetoplastid flagellates, and is implicated in transcription regulation. Base J is synthesized in two steps: first, a thymine base in DNA is converted to 5-hydroxymethyluracil by J-binding proteins (JBP1, JBP2); secondly, a glucosyl transferase glycosylates the 5-hydroxymethyluracil to form base J. Here, we present a highly sensitive and selective LC-MS/MS method to quantify the in vitro JBP1 activity on synthetic oligonucleotide substrates. The method demonstrated successful to support biochemical studies of JBPs and can be used as a template for additional JBP activity studies or for inhibitor screening in the future.

KW - JBP1/2

KW - LC-MS/MS

KW - 5-hydroxymethyl-2′ -deoxyuridine

KW - Base J

U2 - 10.1016/j.ab.2020.113930

DO - 10.1016/j.ab.2020.113930

M3 - Article

C2 - 32866463

SN - 0003-2697

VL - 610

JO - Analytical Biochemistry

JF - Analytical Biochemistry

M1 - 113930

ER -

Quantitative LC-MS/MS analysis of 5-hydroxymethyl-2'-deoxyuridine to monitor the biological activity of J-binding protein

Abstract

Keywords

Access to Document

Fingerprint

Cite this