Phosphonium labeling for increasing metabolomic coverage of neutral lipids using electrospray ionization mass spectrometry

Abstract

Mass spectrometry has become an indispensable tool for the global study of metabolites (metabolomics), primarily using electrospray ionization mass spectrometry (ESI-MS). However, many important classes of molecules such as neutral lipids do not ionize well by ESI and go undetected. Chemical derivatization of metabolites can enhance ionization for increased sensitivity and metabolomic coverage. Here we describe the use of tris(2,4,6,-trimethoxyphenyl)phosphonium acetic acid (TMPP-AA) to improve liquid chromatography (LC)/ESI-MS detection of hydroxylated metabolites (i.e. lipids) from serum extracts. Cholesterol which is not normally detected from serum using ESI is observed with attomole sensitivity. This approach was applied to identify four endogenous lipids (hexadecanoyl-sn-glycerol, dihydrotachysterol, octadecanol, and alpha-tocopherol) from human serum. Overall, this approach extends the types of metabolites which can be detected using standard ESI-MS instrumentation and demonstrates the potential for targeted metabolomics analysis.

Figure 1

Reaction scheme for the synthesis of the TMPP-AA reagent (top) and derivatization of steroids and fatty alcohols with TMPP-AA (bottom).

Figure 2

ESI-TOF MS spectra of TMPP ester derivatives of model compounds: cholesterol, cortisone, and norethindrone.

Figure 3

Fragmentation pattern and sensitivity of TMPP-cholesterol: (A) Characteristic ESI-MS/MS of TMPP derivatives and fragmentation scheme *TMPP-cholesterol. The product ion at m/z 181 is due to ${(\text{MeO})}_3{\text{PhCH}}_2^{+}$ (B) Extracted ion chromatogram of TMPP-cholesterol as a function of concentration. Inset: Expanded view of the overlay extracted ion chromatograms and mass spectra of TMPP-cholesterol at 400 attomole (S/N ratio = 3).

Figure 4

Comparison of the number of features detected in labeled and unlabeled human serum dichloromethane extracts: Comparison of the LC/MS features detected for the labeled (black circles) reveals an increase in features vs. the unlabeled (red triangles) serum extract. For reference, a blue line is shown to indicate the m/z of the TMPP-AA reagent peak.

Figure 5

Calibration curve for the determination of alpha-tocopherol in human serum. Error bars represent one standard deviation based on four individual measurements.