An automated method for obtaining standardized collisionally induced dissociation (CID) spectra using two novel ion activation techniques on a quadrupole ion trap mass spectrometer is described. This strategy simultaneously produces optimal CID spectra "on-the-fly" and maximizes the amount of structurally specific fragment ions obtained in a single experiment, thus eliminating the need for individual tuning of specific mass-to-charge ratios and permitting fragmentation conditions to be more consistently reproduced. Copyright # 1999 John Wiley & Sons, Ltd. Received 17 December 1998; Revised 8 February 1999; Accepted 11 February 1999 Techniques for gas chromatography/mass spectrometry (GC/MS) have traditionally employed high resolution capillary GC separation combined with electron impact (EI) to produce highly specific 'fingerprint-type' spectra. GC/EI-MS provides standardized fragmentation spectra which are rich in structural information but often lack molecular weight information. Chemical ionization (CI) mass spectra typically show abundant molecular ions, thus yielding molecular weight information in addition to structural information. However, both techniques have limitations in that large, thermally labile molecules are often unsuitable for GC analysis, and GC/MS assays often require extensive and time-consuming sample preparation and derivatization, especially for compounds of high polarity or low volatility.In contrast, liquid chromatography (LC) is well suited for the analysis of complex mixtures without the need for sample derivatization. With the development of atmospheric pressure ionization (API) techniques, 1-5 liquid chromatography/mass spectrometry (LC/MS) has become the preferred method of analysis. While LC/MS permits rapid method development, it is not uncommon to see a predominance of the molecular or pseudo-molecular ion with the complete absence of structurally relevant product ions in the mass spectra. As a result, frequently only molecular weight information is obtained.To obtain structural information, liquid chromatography utilized in conjunction with tandem mass spectrometry (LC/ MS/MS) has become a powerful analytical tool.6-9 MS/MS techniques specifically select the desired ion via its molecular mass, and dissociate it to produce a fragmentation pattern. MS/MS fragmentation data are useful because they are derived only from the original selected species and not from other components in the mixture, as is the case with source CID methods. Recently, the development of automated synthetic strategies has placed increased demands upon the analytical methods used to screen and evaluate new compounds. Advances in the automation of data acquisition using intelligent automated data dependent LC/MS/MS and LC/MS n methods have improved analytical productivity. [10][11][12][13][14][15][16][17][18] But in order to keep pace with rising demands, new analytical methods that generate as much information as possible in a single analysis are required.Current MS/MS techniques implemented on ion traps ...
A highly sensitive platform coupling capillary ion chromatography (Cap IC) with Q Exactive mass spectrometer has been developed for metabolic profiling of head and neck squamous cell carcinoma (HNSCC) cells. The Cap IC allowed an excellent separation of anionic polar metabolites, and the sensitivities increased by up to 100-fold compared to reversed-phase liquid chromatography and hydrophilic interaction chromatography performed at either high- or capillary-flow rates. The detection limits for a panel of standard metabolites were between 0.04 to 0.5 nmol/L (0.2 to 3.4 fmol) at a signal-to-noise ratio of 3. This platform was applied to an untargeted metabolomic analysis of head and neck cancer cells and stem-like cancer cells. Differential metabolomics analysis identified significant changes in energy metabolism pathways (e.g., glycolysis and tricarboxylic acid cycle). These experiments demonstrate Cap IC/MS as a powerful metabolomics tool by providing enhanced separation and sensitivity of polar metabolites combined with high resolution and accurate mass measurement (HR/AM) capabilities to differentiate isobaric metabolites.
In this study, we have demonstrated a targeted metabolomics method for analysis of cancer cells, based on high-performance ion chromatography (IC) separation, Q Exactive HF MS for high-resolution and accurate-mass (HR/AM) measurement and the use of stable isotope-labeled internal standards for absolute quantitation. Our method offers great technical advantages for metabolite analysis, including exquisite sensitivity, high speed and reproducibility, and wide dynamic range. The high-performance IC provided fast separation of cellular metabolites within 20 min and excellent resolving power for polar molecules including many isobaric metabolites. The IC/Q Exactive HF MS achieved wide dynamic ranges of 5 orders of magnitude for six targeted metabolites, pyruvate, succinic acid, malic acid, citric acid, fumaric acid, and alpha-ketoglutaric acid, with R(2) ≈ 0.99. Using this platform, metabolites can be simultaneously quantified from low fmol/μL to nmol/μL levels in cellular samples. The high flow rate IC at 380 μL/min has shown excellent reproducibility for a large set of samples (150 injections), with minimal variations of retention time (SD < ± 0.03 min). In addition, the IC-MS-based approach acquires targeted and global metabolomic data in a same analytical run, and the use of stable isotope-labeled standards facilitates accurate quantitation of targeted metabolites in large-scale metabolomics analysis. This metabolomics approach has been successfully applied to analysis of targeted metabolites in head and neck cancer cells as well as cancer stem-like cells (CSCs), and the findings indicate that the metabolic phenotypes may be distinct between high and low invasive head and neck cancer cells and between CSCs and non-SCCs.
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