Characterizing The Poly(A) Tail In mRNA Using Ion-Pairing Reversed-Phase LC/MS (IPRP-LC/MS) And Deconvolution Tools

Poly(A) tail length can influence mRNA stability, translation efficiency, and overall performance, making accurate characterization an important part of mRNA quality assessment. RNase 4 digestion paired with ion-pairing reversed-phase LC/MS offers a targeted way to analyze the poly(A) region of Firefly Luciferase mRNA and measure tail-length distribution with mass-based confidence. In this work, deconvoluted spectra confirmed poly(A) tail lengths ranging from 119 to 128 adenosines. Method development with an RNA standard also addressed common LC/MS considerations, including sodium adduction, retention time precision, and extracted ion chromatogram reproducibility. Hexylamine reduced sodium adduction compared with DIPEA, while replicate testing showed retention time RSDs below 0.2% and EIC peak height RSDs below 2%.

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