Metabolic Profiling of Human Blood by High Resolution Ion Mobility Mass Spectrometry (IM-MS)

Abstract

A high resolution ion mobility time-of-flight mass spectrometer with electrospray ionization source (ESI-IM-MS) was evaluated as an analytical method for rapid analysis of complex biological samples such as human blood metabolome was investigated. The hybrid instrument (IM-MS) provided an average ion mobility resolving power of ~90 and a mass resolution of ~1500 (at m/z 100). A few µL of whole blood was extracted with methanol, centrifuged and infused into the IM-MS via an electrospray ionization source. Upon IM-MS profiling of the human blood metabolome approximately 1,100 metabolite ions were detected and 300 isomeric metabolites separated in short analyses time (30 minutes). Estimated concentration of the metabolites ranged from the low micromolar to the low nanomolar level. Various classes of metabolites (amino acids, organic acids, fatty acids, carbohydrates, purines and pyrimidines etc) were found to form characteristic mobility-mass correlation curves (MMCC) that aided in metabolite identification. Peaks corresponding to various sterol derivatives, estrogen derivatives, phosphocholines, prostaglandins, and cholesterol derivatives detected in the blood extract were found to occupy characteristic two dimensional IM-MS space. Low abundance metabolite peaks that can be lost in MS random noise were resolved from noise peaks by differentiation in mobility space. In addition, the peak capacity of MS increased six fold by coupling IMS prior to MS analysis.

Figure 1

Schematic of the electrospray ionization atmospheric pressure ion mobility time of-flight mass spectrometer used for the analysis of human blood metabolome. This instrument is comprised of nine primary units: (1) electrospray ionization source; (2) heated atmospheric pressure desolvation region; (3) Bradbury-Nielsen ion gate; (4) counter-flow atmospheric pressure drift region; (5) differentially pumped interface; (6) ion guide with ion lenses; (7) extraction region; (8) reflectron time-of-flight mass analyzer, (9) bipolar MCP detector and (10) time to digital converter for data acquisition.

Figure 2

2D IMMS spectrum of an equimolar (500 n Molar) metabolite mixture solution containing serine, nicotinamide, adenine, lysine, glutamine, ribose, phenylalanine, arginine, glucose, lactose, and maltose in 50:50 MeOH and H₂O with 1% acetic acid and 30 minute data acquisition time is shown. All metabolites in the mixture were detected as one or more than one of the following adducts: {M (H₂O)n + H}⁺, {M (MeOH)n + H}⁺, {M (H₂O)n + Na}⁺ (Table 1).

Figure 3

Demonstration of reproducibility of reduced mobility value (K_o) of ribose as (M + Na)⁺ ion measured for four different measurements at identical experimental conditions except for atmospheric pressure. Notice the reproducibility of K_o between measurements taken three months apart (between a and b/c/d). Figure also shows the effect of charge competition, ion suppression and preferential ionization of metabolites at different concentrations (1 mM to 500 nM).

Figure 4

Figure 4a: Two dimensional spectrum of metabolic features measured in methanol extract of human blood. Figure 4b: A zoomed in section of the IM-MS spectrum in the m/z range of 300–400 Da illustrating the peaks detected in the region with 5 ion counts or more along with separation of isomers and isobars.

Figure 5

One dimensional mass spectrum (1) compared to one dimensional mass spectrum with mobility differentiation (2). Three main features observed: a) noise peak accounted as real peak (bold line), b) contribution of random noise to peak intensity (dashed line), and c) real peak lost in noise (dotted line).

Figure 7

A: MMCC for amino acids detected in the blood extract. Peaks identified as amino acids in blood extract based on mass and reduced mobility data matched with that measured for standard solutions of amino acids. B: MMCC for various classes of metabolites detected in the blood sample. Only protonated ions of metabolites constitute the MMCC (except for the sugars as sodium adducts).

Figure 8

a) Enlarged section of Figure 4a in the m/z range of 190–225 Da showing the separation of isomers/isobars by IMS. Four mobility separated peaks identified as potassium adducts of hexose sugars at m/z value of 219 Da along with four mobility separated peaks at m/z 195 Da and two mobility separated peaks each at m/z values of 191, 197, 209, 217, and 203 Da is illustrated. b) plot of drift time Vs. m/z values of ions showing separation of over 200 isomeric/isobaric metabolite ions in blood extract detected by IMMS with ≥ 10 ion counts.

Figure 9

Two dimensional space (shaded area in the X-Y quadrant) occupied by metabolites that were detected in human blood extract by ESI- IM-MS (Peak capacity) is illustrated. The theoretical MMCC (dotted line) is the centre line of the shaded area along which a constant drift time deviation should be observed. MMCC for the experimental data is depicted as bold line. The length of the 2D space is defined by the m/z range and width is defined by the maximum and minimum drift time deviation observed at a particular m/z value shown as double sided in the figure.