Low-nanogram Fourier Transform Isotopic Ratio Mass Spectrometry of Proteins

Abstract

Stable carbon and nitrogen isotope ratios are widely used in the life sciences to investigate diet, trophic interactions, and metabolic fluxes, but conventional isotope ratio mass spectrometry requires milligram-scale samples, limiting its applicability to small or rare biological specimens. Fourier Transform Isotopic Ratio Mass Spectrometry (FT IsoR MS) enables amino acid-resolved isotope analysis in a proteomics-compatible workflow and has previously been demonstrated at the microgram scale. Here, we assess the lower sample limit of FT IsoR MS by integrating it with single-cell proteomics-style sample preparation. Using human HeLa cells cultured in 13C-glucose-enriched and control media, we show that reliable relative {delta}13C measurements can be obtained from as few as 50 cells, corresponding to <10 ng of total protein, with a precision of approximately {+/-}9{per thousand}. The observed amino acid-specific labeling patterns are metabolically coherent and consistent with bulk measurements, while smaller cell numbers ([≤]10 cells) do not yield statistically robust results. These findings establish the practical sensitivity threshold of FT IsoR MS at the low-nanogram level and demonstrate its suitability for isotope-resolved analyses of small cell populations, micro-organoids, and other low-input biological samples, thereby extending stable isotope analysis toward single-cell-scale applications.