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.

An isotope tags for relative and absolute quantitation (iTRAQ)-based reversed-phase liquid chromatography (RPLC)-tandem mass spectrometry (MS/MS) method was developed for differential protein expression profiling in complex cellular extracts. The estrogen positive MCF-7 cell line, cultured in the presence of 17␤-estradiol (E2) and tamoxifen (Tam), was used as a model system. MS analysis was performed with a linear trap quadrupole (LTQ) instrument operated by using pulsed Q dissociation (PQD) detection. Optimization experiments were conducted to maximize the iTRAQ labeling efficiency and the number of quantified proteins. MS data filtering criteria were chosen to result in a false positive identification rate of Ͻ4%. The reproducibility of protein identifications was ϳ60%-67% between duplicate, and ϳ50% among triplicate LC-MS/MS runs, respectively. The run-to-run reproducibility, in terms of relative standard deviations (RSD) of global mean iTRAQ ratios, was better than 10%. The quantitation accuracy improved with the number of peptides used for protein identification. From a total of 530 identified proteins (P Ͻ 0.001) in the E2/Tam treated MCF-7 cells, a list of 255 proteins (quantified by at least two peptides) was generated for differential expression analysis. A method was developed for the selection, normalization, and statistical evaluation of such datasets. An approximate ϳ2-fold change in protein expression levels was necessary for a protein to be selected as a biomarker candidate. According to this data processing strategy, ϳ16 proteins involved in biological processes such as apoptosis, RNA processing/metabolism, DNA replication/transcription/repair, cell proliferation and metastasis, were found to be up-or down-regulated. R ecently, two-dimensional liquid chromatography (2DLC) with tandem MS detection has emerged as an attractive technology for quantitative proteomic profiling of complex cellular extracts [1][2][3][4]. Stable isotope labeling and label-free quantitation strategies have been explored [4 -13]. Isotope labeling approaches rely on the covalent attachment of stable isotope tags to specific amino acid residues of proteins or peptides during metabolic, enzymatic, or chemical processes. Label-free quantitation methods rely on measuring peak areas, intensities, or spectral counts, and benefit from not having to chemically alter the sample. Throughput, however, is lower, and quantitation errors are higher, as the samples are processed independently. Among chemical labeling techniques, isotope tags for relative and absolute quantitation (iTRAQ) has received much attention [14 -30]. In this approach, peptides are labeled with isobaric tags at the N-terminus and the lysine side chains. MS/MS fragmentation produces signature ions that can be used to obtain quantitative information. Perhaps the most attractive advantage of this approach is that it can be used for the simultaneous quantification (relative or absolute) of up to four/eight different samples. Simplicity, of course, is an added benefit.A...

No abstract

Amino acid analysis is a powerful tool in life sciences. Current analytical methods used for the detection and quantitation of low abundance amino acids in complex samples face intrinsic challenges such as insufficient sensitivity, selectivity, and throughput. This chapter describes a protocol that makes use of AccQ∙Tag chemical derivatization combined with the exceptional chromatographic resolution of ultra performance liquid chromatography (UPLC), and the sensitivity and selectivity of tandem mass spectrometry (MS/MS). The method has been fully implemented and validated using different tandem quadrupole detectors, and thoroughly tested for a variety of samples such as Plasmodium falciparum, human red blood cells, and Arabidopsis thaliana extracts. Compared to currently available methods for amino acid analysis, the AccQ∙Tag UPLC-MS/MS method presented here provides enhanced sensitivity and reproducibility, and offers excellent performance within a short analysis time and a broad dynamic range of analyte concentration. The focus of this chapter is the application of this improved protocol for the compositional amino acid analysis in A. thaliana leaf extracts using the Xevo TQ for mass spectrometric detection.

In spite of the large arsenal of methodologies developed for amino acid assessment in complex matrices, their implementation in metabolomics studies involving wide-ranging mutant screening is hampered by their lack of high-throughput, sensitivity, reproducibility, and/or wide dynamic range. In response to the challenge of developing amino acid analysis methods that satisfy the criteria required for metabolomic studies, improved reverse-phase high-performance liquid chromatography-mass spectrometry (RPHPLC-MS) methods have been recently reported for large-scale screening of metabolic phenotypes. However, these methods focus on the direct analysis of underivatized amino acids and, therefore, problems associated with insufficient retention and resolution are observed due to the hydrophilic nature of amino acids. It is well known that derivatization methods render amino acids more amenable for reverse phase chromatographic analysis by introducing highly-hydrophobic tags in their carboxylic acid or amino functional group. Therefore, an analytical platform that combines the 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) pre-column derivatization method with ultra performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) is presented in this article. For numerous reasons typical amino acid derivatization methods would be inadequate for large scale metabolic projects. However, AQC derivatization is a simple, rapid and reproducible way of obtaining stable amino acid adducts amenable for UPLC-ESI-MS/MS and the applicability of the method for high-throughput metabolomic analysis in Arabidopsis thaliana is demonstrated in this study. Overall, the major advantages offered by this amino acid analysis method include high-throughput, enhanced sensitivity and selectivity; characteristics that showcase its utility for the rapid screening of the preselected plant metabolites without compromising the quality of the metabolic data. The presented method enabled thirty-eight metabolites (proteinogenic amino acids and related compounds) to be analyzed within 10 min with detection limits down to 1.02 × 10−11 M (i.e., atomole level on column), which represents an improved sensitivity of 1 to 5 orders of magnitude compared to existing methods. Our UPLC-ESI-MS/MS method is one of the seven analytical platforms used by the Arabidopsis Metabolomics Consortium. The amino acid dataset obtained by analysis of Arabidopsis T-DNA mutant stocks with our platform is captured and open to the public in the web portal PlantMetabolomics.org. The analytical platform herein described could find important applications in other studies where the rapid, high-throughput and sensitive assessment of low abundance amino acids in complex biosamples is necessary.