Sensitive and Rapid Method for Amino Acid Quantitation in Malaria Biological Samples Using AccQ•Tag Ultra Performance Liquid Chromatography-Electrospray Ionization-MS/MS with Multiple Reaction Monitoring

Abstract

An AccQ•Tag Ultra performance liquid chromatography-electrospray ionization-tandem mass spectrometry (AccQ•Tag -UPLC-ESI-MS/MS) method for fast, reproducible and sensitive amino acid quantitation in biological samples, particularly, the malaria parasite Plasmodium falciparum is presented. The Waters Acquity TQD UPLC/MS system equipped with photodiode array (PDA) detector was used for amino acid separation and detection. The method was developed and validated using amino acid standard mixtures containing acidic, neutral, and basic amino acids. For MS analysis, the optimum cone voltage implemented, based on direct infusion analysis of a few selected AccQ•Tag amino acids with multiple reaction monitoring, varied from 29-39 V, whereas the collision energy varied from 15-35 V. Calibration curves were built using both internal and external standardization. Typically, a linear response for all amino acids was observed at concentrations ranges of 3 × 10−3-25 pmol/μL. For some amino acids, concentration limits of detection were as low as 1.65 fmol. The coefficients of variation for retention times were within the ranges of 0.08-1.08%. The coefficients of variation for amino acid quantitation, determined from triplicate UPLC-MS/MS runs, were below 8% on the average. The developed AccQ•Tag-UPLC-ESI-MS/MS method revealed good technical and biological reproducibility when applied to P. falciparum and human red blood cells samples. This study should provide a valuable insight into the performance of UPLC-ESI-MS/MS for amino acid quantitation using AccQ•Tag derivatization.

Temporal-Spatial Interaction between Reactive Oxygen Species and Abscisic Acid Regulates Rapid Systemic Acclimation in Plants

“…To examine the accumulation of stress-associated metabolites in systemic and local leaves of Arabidopsis, we performed gas chromatography-mass spectrometry (Fiehn et al, 2000;Roessner et al, 2000;Shuman et al, 2011) and liquid chromatography-mass spectrometry (Armenta et al, 2010;Salazar et al, 2012) analyses and expressed the level of individual compounds as relative to an internal control (e.g., ribitol; Rizhsky et al, 2004b). For gas chromatography-mass spectrometry analysis, extraction and derivatization were performed as described previously (Fiehn et al, 2000;Roessner et al, 2000;Shuman et al, 2011).…”

“…For gas chromatography-mass spectrometry analysis, extraction and derivatization were performed as described previously (Fiehn et al, 2000;Roessner et al, 2000;Shuman et al, 2011). Amino acids were extracted and quantified by liquid chromatography-mass spectrometry analysis as previously described (Armenta et al, 2010;Salazar et al, 2012). ABA was extracted as previously described (Pan et al, 2010).…”

Metabolomic profiling reveals a finely tuned, starvation-induced metabolic switch in Trypanosoma cruzi epimastigotes

“…Metabolite extraction protocol was adapted from Ref. 39. Briefly, extraction was initiated by adding 400 l (sample up to 5 ϫ 10 7 parasites) or 800 l (sample Ն5 ϫ 10 7 parasites) of extraction mixture containing methanol/water (1:1, v/v) and the necessary amount of internal standards to obtain the final concentration indicated after metabolite extraction…”

Amino acid analysis of sub-picomolar amounts of proteins by precolumn fluorescence derivatization with 6-aminoquinolyl-<i>N</i>-hydroxysuccinimidyl carbamate

“…Furthermore, precolumn derivatization has enabled the sub-picomolar quantitation of amino acids with the development of fluorescent reagents such as OPA, 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F), 9-fluorenylmethyl-chloroformate (FMOCCl), and 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) (See details in Section 2.2.). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) detection of pre-labeled amino acids has been also reported including pre-labeling by aminereactive isobaric tagging reagents that include different mass group of stable isotope (7,8), by p-N,N,Ntrimethylammonioanilyl-N'-hydroxysuccinimidyl carbamate iodide (9), and by AQC (10).…”