Single-stranded DNA as a cleavable linker for bioorthogonal click chemistry-based proteomics

Abstract

In this communication, we report a new class of cleavable linker based on automatically synthesized, single-stranded DNAs. We incorporated a DNA oligo into an azide-functionalized biotin (biotin-DNA-N3) and used the probe to enrich for alkyne-tagged glycoproteins from mammalian cell lysates. Highly efficient and selective release of the captured proteins from streptavidin agarose resins was achieved using DNase treatment under very mild conditions. A total of 36 sialylated glycoproteins were identified from the lysates of HL60 cells, an acute human promyeloid leukemia cell line. These sialylated glycoproteins were involved in many different biological processes ranging from glycan biosynthesis to cell adhesion events.

Figure 1

(a) Biotin-DNA-N₃ was reacted with 488-alkyne in the presence of BTTP/Cu(I), followed by incubation with or without (control) streptavidin agarose resins. After resins were removed, supernatant was transferred into a 96-well microtiter plate to quantify the residual fluorescence using a fluorescence plate reader (excitation 488 nm/emission 520 nm). (b) A silver-stained gel image showing specific release of the captured BSA from streptavidin agarose resins. Jurkat cell lysates containing the alkyne-modified BSA were reacted with biotin-DNA-N₃ or biotin-N₃ in the presence of the BTTP/Cu(I) catalyst. Following incubation with streptavidin agarose resins, BSA was released from resins by benzonase (lane 1) or by boiling resins in SDS buffer (lane 3). After the bezonase treatment, resins were boiled in SDS buffer to release residual proteins for SDS-PAGE analysis (lane 2). The black arrows indicate the dimmeric and monomeric BSA (BSA dimerization is irreversible between pH 4.2 and 7.0 even after treatment with DTT). The red arrows indicate contaminated proteins binding to immobilized streptavidin through nonspecific, hydrophobic interactions or proteins that are endogenous biotinylated.

Figure 2

LC/MS analysis of the cleaved products of biotin-DNA-488 by benzonase (a-c) and PvuII (d-f). (a) the HPLC trace with absorbance at 488 nm. Two major peaks (a1 and a2) and two minor peaks (a3 and a4) were observed as indicted with arrows. (b) the MS spectrum of peak a1. (c) the MS spectrum of peak a2. The MS spectra of both peak a1 and peak a2 indicate that the major cleaved product of biotin-DNA-488 is 488-5′GT-3′ (Chemical Formula: C₅₈H₇₁N₁₃O₂₂P₂; molecular weight 1364.22). (d) the HPLC trace with absorbance at 488 nm. Two major peaks (d1 and d2) were observed as indicted with arrows. (e) the MS spectrum of peak d1. (c) the MS spectra of peak d2. The MS spectra of both peak d1 and peak d2 show that the cleaved product of biotin-DNA-488 is 488-5′GTAACGATCCAG-3′ (Chemical Formula: C₁₅₆H₁₉₄N₅₄O₇₈P₁₂; molecular weight 4445.25).

Figure 3

(a) Metabolic labeling of sialic acid using the unnatural sugar Ac₄ManNAl. Cells were cultured in medium containing Ac₄ManNAl. Ac₄ManNAl was taken up by cells, processed by a series of cellular enzymes, and converted to the alkyne-modified sialic acid and incorporated into cell-surface sialylated glycoproteins. (b) Classification of the 36 sialylated glycoproteins identified in HL60 cells.

Scheme 1. A DNA oligo-based cleavable linker for the CuAAC-based bioorthogonal proteomics

Scheme 2. Synthesis of biotin-DNA-N₃ on the solid phase CPG^a

^aReagents: (a) solid phase synthesis using Ac-dC-CE (β-cyanoethyl) Phosphoramidite, dA-CE Phosphoramidite, dT-CE Phosphoramidite, dG-CE Phosphoramidite; (b) solid phase synthesis using 5′-Bromohexyl Phosphoramidite; (c) NaN₃/NaI; (d) CH₃NH₂/NH₄OH