Poly(ethylene glycol)-Based Stable Isotope Labeling Reagents for the Quantitative Analysis of Low Molecular Weight Metabolites by LC−MS

Abstract

Stable isotope labeling (SIL) in combination with liquid chromatography-mass spectrometry is one of the most widely used quantitative analytical methods due to its sensitivity and ability to deal with extremely complex biological samples. However, SIL methods for metabolite analysis are still often limited in terms of multiplexing, the chromatographic properties of the derivatized analytes, or their ionization efficiency. Here we describe a new family of reagents for the SIL of primary amine-containing compounds based on pentafluorophenyl-activated esters of 13C-containing poly(ethylene glycol) chains (PEG) that addresses these shortcomings. A sequential buildup of the PEG chain allowed the introduction of various numbers of 13C atoms opening extended multiplexing possibilities. The PEG derivatives of rather hydrophilic molecules such as amino acids and glutathione were successfully retained on a standard C18 reversed-phase column, and their identification was facilitated based on m/z values and retention times using extracted ion chromatograms. The mass increase due to PEG derivatization moved low molecular weight metabolite signals out of the often noisy, low m/z region of the mass spectra, which resulted in enhanced overall sensitivity and selectivity. Furthermore, elution at increased retention times resulted in efficient electrospray ionization due to the higher acetonitrile content in the mobile phase. The method was successfully applied to the quantification of intracellular amino acids and glutathione in a cellular model of human lung epithelium exposed to cigarette smoke-induced oxidative stress. It was shown that the concentration of most amino acids increased upon exposure of A549 cells to gas-phase cigarette smoke with respect to air control and cigarette smoke extract and that free thiol-containing species (e.g., glutathione) decreased although disulfide bond formation was not increased. These labeling reagents should also prove useful for the labeling of peptides and other compounds containing primary amine functionalities.

Development of versatile isotopic labeling reagents for profiling the amine submetabolome by liquid chromatography–mass spectrometry

“…To date, several CIL reagents have been developed to quantify organic amines, carboxylic acids, carbonyl compounds and some other types of metabolites with varying degrees of performance [1,4,5,[7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24]. These research activities are mainly driven by the need of dealing with different classes of metabolites; a universally applicable derivatization reagent for LC-MS is not available at the present.…”

Electron Transfer and Collision Induced Dissociation of Non-Derivatized and Derivatized Desmosine and Isodesmosine

“…For their use, the products were dissolved in acetonitrile (HPLC supra gradient grade, Biosolve), divided into 1 μmol portions, vacuum dried and stored at −80°C until use. Pentafluorophenyl-activated PEG (mediumPEG, 4 ethylene oxide units) esters containing zero or five 13 C atoms (unlabeledmediumPEG, 13 C 5 -mediumPEG) were synthesized as described previously [29], dissolved in acetonitrile, divided into 1 μmol portions, vacuum dried and stored at −80°C until use.…”

“…We recently showed that PEG derivatization may also be useful in enhancing the ionization efficiency of small molecules such as amino acids, as well as to increase their hydrophobicity allowing analysis by reversed-phase LC-MS [29]. In addition, PEG ether functionalities may be protonated which could affect the ETD and CID pattern of derivatized DES [19,35,36].…”

“…Since derivatization of DES/IDS is often used to increase retention on C18 stationary phases as well as to enhance ESI ionization efficiency, we studied ETD and CID of derivatized and nonderivatized DES/IDS. DES/IDS were derivatized with polyethyleneglycol (PEG) based on our finding that derivatization of amino acids with PEG increases ionization efficiency by a factor 10-500 ( [29] and unpublished results). In addition, DES/IDS were derivatized with propionic anhydride, as this approach has been employed for the analysis of DES/IDS in urine [30].…”

Qualitative Metabolome Analysis of Human Cerebrospinal Fluid by13C-/12C-Isotope Dansylation Labeling Combined with Liquid Chromatography Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

“…Unlike other chemical derivatization schemes where the main purpose was to introduce a mass tag to the metabolites for quantitative metabolite or metabolome analysis [21][22][23][24][25][26][27][28], dansylation can not only effectively introduce an isotope tag for accurate metabolite quantification, but also improve the chromatographic retention of the metabolites in reversed-phase LC and enhance the efficiency of electrospray ionization (ESI). In our previous work, sensitive detection of metabolites from human urine samples was demonstrated.…”