Targeted and Untargeted Amine Metabolite Quantitation in Single Cells with Isobaric Multiplexing

Abstract

We developed a single cell amine analysis approach utilizing isobarically multiplexed samples of 6 individual cells along with analyte abundant carrier. This methodology was applied for absolute quantitation of amino acids and untargeted relative quantitation of amines in a total of 108 individual cells using nanoflow LC with high-resolution mass spectrometry. Together with individually determined cell sizes, this provides accessible quantification of intracellular amino acid concentrations within individual cells. The targeted method was partially validated for 10 amino acids with limits of detection in low attomoles, linear calibration range covering analyte amounts typically from 30 amol to 120 fmol, and correlation coefficients (R) above 0.99. This was applied with cell sizes recorded during dispensing to determine millimolar intracellular amino acid concentrations. The untargeted approach yielded 249 features that were detected in at least 25 % of the single cells, providing modest cell type separation on principal component analysis. Using Greedy forward selection with regularized least squares, a sub-selection of 100 features explaining most of the difference was determined. These features were annotated using MS2 from analyte standards and accurate mass with library search. The approach provides accessible, sensitive, and high-throughput method with the potential to be expanded also to other forms of ultrasensitive analysis.

Keywords: Amino acids; Mass spectrometry (MS).; Metabolomics; Nano liquid chromatography (nLC); Single cell.

Figure 1

Isobaric labeling concept for single cell samples. Multiplexed sample consists of six individual single cells prepared and labeled individually on a 384‐well plate along with analyte abundant carrier, blank, and IS‐blank prepared and labeled in bulk. From the ten TMT10plex reagents, nine (126, 127 N, 128 N, 128 C, 129 N, 129 C, 130 N, 130 C, and 131) were used, whereas one (127 C) was not, due to isotope impurity effect of analyte abundant carrier. These multiplexed samples were measured using nLC‐MS2, where each isobarically labeled analyte from the multiplexed sample produces a superimposed MS1 peak that can be decomposed to sample reagent (126–131) specific reporter ions with HCD fragmentation and quantitatively measured with high resolution tandem MS.

Figure 2

MS2 EICs of integrated reporter ion (m/z 126.1277) peaks of tPRM acquisition of TMT10plex isotopomer labeled 120 fmol analyte amounts from multiplexed calibration sample.

Figure 3

A) EICs of TMT10plex reporter ions of cystine (TMT10plex‐cystine precursor, m/z 350.1824) from multiplexed calibration sample with amounts of 0.03–120 fmol. B) The constructed calibration line of integrated reporter ion chromatographic peaks after isobaric impurity correction and internal standard normalization. 0.03 fmol sample was removed as being below LOQ and 0.3 fmol that was an outlier with most analytes (Grubb's outlier test on residuals). These were subsequently excluded from the calibration.

Figure 4

LC–MS2 integrated EICs of TMT10plex labeled amino acids in a single TYK‐nu.CP‐r cell (ID65) and the used 12 fmol internal standard (EICs inverted)

Figure 5

A) Absolute amounts of ten amino acids in single cells along with the analyte lower limit of quantitation (LOQ, blue dashed line) and upper limit of quantitation (red dashed line) as in calibration (Table 1 and Figure S1). Calculated analyte amounts outside the scope of calibration are excluded (marked red), and the cell ID85 (TYK‐nu.CP‐r cell) is highlighted. B) Cell volume distribution of measured cells by cell type, with the cell ID85 highlighted, along with its CellenONE image recorded during cell sorting. C). Cellular concentrations (mol/L) of amino acids in individual HEK‐293, TYK‐nu, and TYK‐nu.CP‐r cells based on absolute sample amount and cell size. Significance at p <0.05 (*) and p <0.01 (**) determined by Kruskal‐Wallis with Dunn's post‐hoc test.

Figure 6

Metabolite comparison of confidently identified (Level 2, matched to analyte standard MS2) analytes from Greedy RLS sub selection of untargeted metabolomics.