Development of a Click-Chemistry Reagent Compatible with Mass Cytometry

Jessica Shaklee,Kriti Srivastava,Jop H Van Berlo,Edgar A Arriaga,Heather Brown,Valerie C Pierre

doi:10.1038/s41598-018-25000-y

Jessica Shaklee, Kriti Srivastava + Show 4 more

Open Access

https://doi.org/10.1038/s41598-018-25000-y

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Abstract

The recent development of mass cytometry has allowed simultaneous detection of 40 or more unique parameters from individual single cells. While similar to flow cytometry, which is based on detection of fluorophores, one key distinguishing feature of mass cytometry is the detection of atomic masses of lanthanides by mass spectrometry in a mass cytometer. Its superior mass resolution results in lack of signal overlap, thereby allowing multiparametric detection of molecular features in each single cell greater than that of flow cytometry, which is limited to 20 parameters. Unfortunately, most detection in mass cytometry relies on lanthanide-tagged antibodies, which is ideal to detect proteins, but not other types of molecular features. To further expand the repertoire of molecular features that are detectable by mass cytometry, we developed a lanthanide-chelated, azide-containing probe that allows click-chemistry mediated labeling of target molecules. Following incorporation of the thymidine analog 5-ethynyl-2′-deoxyuridine (EdU) during DNA synthesis in S-phase of the cell cycle, we demonstrate that the probe introduced here, tagged with Terbium-159 (159Tb), reacts via copper-catalyzed azide-alkyne Huisgen cycloaddition (click-chemistry) with Edu. Thus, detection of 159Tb makes it possible to measure DNA synthesis in single cells using mass cytometry. The approach introduced here shows similar sensitivity (true positive rate) to other methods used to measure DNA synthesis in single cells by mass cytometry and is compatible with the parallel antibody-based detection of other parameters in single cells. Due to its universal nature, the use of click-chemistry in mass cytometry expands the types of molecular targets that can be monitored by mass cytometry.

Highlights

Multiparametric detection by mass cytometry has many applications and has made it possible to identify populations of immune cells with higher precision, characterize drug responses in multiple cell types simultaneously, and describe dynamics of cell differentiation[9,10]
Thymidine analog 5-bromo-2′-deoxyuridine (BrdU) does not have inhibitory effects on cellular proliferation and detection of BrdU incorporated during DNA synthesis is achievable by mass cytometry using anti-Brdu antibodies tagged with lanthanide metal ions
To demonstrate feasibility of a generic click-chemistry approach that results in labeling of molecular targets with lanthanides that are commonly used in mass cytometry analyses, we detected by mass cytometry the incorporation of EdU, a thymidine analog, into DNA during DNA replication in currently or previously proliferating cells (Fig. 1 and Methods)

Summary

Introduction

Multiparametric detection by mass cytometry has many applications and has made it possible to identify populations of immune cells with higher precision, characterize drug responses in multiple cell types simultaneously, and describe dynamics of cell differentiation[9,10]. Incorporation of 5-iodo-2′-deoxyuridine (IdU) during DNA synthesis can be monitored by mass cytometry because iodine-127 is isotopically stable and falls within the measurable mass range of mass cytometry[12]. Thymidine analog 5-bromo-2′-deoxyuridine (BrdU) does not have inhibitory effects on cellular proliferation and detection of BrdU incorporated during DNA synthesis is achievable by mass cytometry using anti-Brdu antibodies tagged with lanthanide metal ions. An alternative to BrdU that is detectable without denaturing DNA has been developed for immunocytochemistry and flow cytometry[11,18] This alternative uses incorporation of 5-ethynyl-2′-deoxyuridine (EdU) followed by copper-catalyzed azide-alkyne Huisgen cycloaddition (click-chemistry) with a fluorescent probe to detect EdU11,19,20. A recent publication laid out one possible strategy to detect incorporated EdU with mass cytometry, we propose a more versatile strategy that allows labeling with a lanthanide ion of choice[21]

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