Quantification by mass spectrometry

The use of the two instruments in combination allows the optimum utilization of the separating ability of the gas chromatograph with the unequivocal identification possibilities of the mass spectrometer. Most compounds that are amenable to this technique, particularly the abused drugs, can be identified on the basis of the mass spectrum alone, at microgram and submicrogram levels. Quantification by mass spectrometry is possible in the range 10 12 to 10 1 g. 

A stable isotope dilution assay using mass spectrometry to measure insulin, proinsulin, and C-peptide has been developed. The difference in mass among the three analytes allows specific measurement of each protein. Comparison of patient samples revealed that most, but not all, results were higher by immunoassay than mass spectrometry. Thus immunoassays may overestimate insulin, particularly at low concentrations. The high protein concentration in the serum requires extraction of proteins (e.g., by immunoaffinity) and purification by high-performance liquid chromatography (HPLC) before quantification by mass spectrometry. This method is not suitable for routine laboratory analysis. 

Guan, X.L., Riezman, I., Wenk, M.R. and Riezman, H. (2010) Yeast lipid analysis and quantification by mass spectrometry. Methods Enzymol. 470, 369-391. 

Oba, Y., et al. (2004). Identification of the luciferin-luciferase system and quantification of coelenterazine by mass spectrometry in the deep-see luminous ostracod Conchoecia pseudodiscophora. ChemBioChem 5 1495-1499. 

However, IHC as a practical method continues to evolve with increasing demands for standardization, and for true quantification of protein analytes by weight, in the context of their cellular microenvironment. Further studies combining proteomics by mass spectrometry and IHC are likely to lead to the refinement of both methods in the analysis of FFPE tissues. The end result may be the creation of a broader field that defines and quantifies protein expression at a cellular level, incorporating the advantages of the wide spectrum of proteins demonstrable by mass spectrometry and the precise localization offered by IHC. 

M. L. Gross Quantification of protein—ligand interactions by mass spectrometry, titration, and H/D exchange PLIMSTEX./. Am. Chem. Soc. 2003, 125, 5252-5253. 

Presently, FAB-MS spectra are routinely used to characterize synthetic tyrosine O-sulfate peptides.152,57,63-671 Since partial hydrolysis of the sulfate ester occurs in the gas phase, quantification of the tyrosine O-sulfate residue by mass spectrometry is not possible, but combined with one-peak assignment in HPLC, FAB-MS represents a powerful analytical tool. On the other hand, partial hydrolysis in the gas phase excludes the presence of sul-fonated species which should be perfectly stable. In early studies the presence of such species were excluded by quantitative recovery of tyrosine upon acid hydrolysis or upon hydrolysis with arylsulfatase.1361 Recently, even MALDI-TOF-MS spectra of CCK-peptides1441 and of conotoxins a-PnIA and a-PnlB 138 were reported which show that in the positive-ion mode the [M + H-S03]+ ions represent the base peaks, while in the negative-ion mode, [M-H]-ions consistently correspond to the base peaks. In the CCK peptides intramolecular salt bridging of the sulfate hemi-ester with proximal positive charges of arginine or lysine side chains was found to reduce the extent of hydrolysis in the gas phase significantly.144,1491... 

SFC provides complementary quantitative data to the structural information afforded by mass spectrometry. Thermally label materials such as isocyanates can be easily analyzed with minimal sample preparation. Supercritical carbon dioxide is nontoxic and can be obtained in high purity as measured by FID. The easy coupling of SFE with SFC makes the selective isolation and quantification of targeted analytes possible. Furthermore, we are in an age of increased environmental awareness. Solvent disposal is discouraged and has become very expensive. The waste disposal costs associated with supercritical carbon dioxide are negligible when compared to the solvent disposal costs generated by traditional Soxhlet methods. 

FIGURE 1 Example of a gel-free-oriented proteomics nano-LC/MS-MS workflow in which bacterial culture proteins digested to tryptic peptides are separated via LC and peptides subsequently analyzed by mass spectrometry. In the process, the spectrometer rapidly cycles every few seconds and examines a size window in which peptide-derived MSI ions are analyzed to define MS/MS (MS2) spectra. The MS/MS (MS2) spectrum generated for each peptide then enters a bioinformatic pipeline for sequence identification, statistical validation, and quantification. 

W.A. Tao et al., Rapid enantiomeric quantification of an antiviral nucleoside agent (d, l-FMAU, 2 -fluoro-5-methyl-, d, L-arabinofuranosyluracil) by mass spectrometry. J. Med. Chem. 44, 3541-3544 (2001)... 

At present identification and quantification of selenoamino acids in seafood relies solely on matching retention time of peaks with available standards. To unambiguously assign peaks, the use of HPLC coupled to a mass spectrometer is required, as for the identification of As species [158, 159]. However, at present the low concentrations of Se species, the low detection power, and the lack of knowledge about Se species does not make this possible [131, 135, 136, 163], Preparative scale isolation and purification of Se proteins as done for bacterial proteins is required to allow Se proteins and selenoamino acids to be characterized by mass spectrometry [164], Preparative gel and two-dimensional electrophoresis offers promise to obtain pure Se protein fractions. Selenium-containing... 

Lipids are made up of many classes of very different molecules that all show solubility properties in organic solvents. Mass spectrometry plays a key role in the biochemistry of lipids. Indeed, mass spectrometry allows not only the detection and determination of the structure of these molecules but also their quantification. For practical reasons, only the fatty acids, acylglycerols and bile acids are discussed here, although other types of lipids such as phospholipids, [253-256] steroids, [257-259] prostaglandins, [260] ceramides, [261,262] sphingolipids [263,264] and leukotrienes [265,266] have been analysed successfully by mass spectrometry. Moreover, the described methods will be limited to those that are based only on mass spectrometry, even if the majority of these methods generally are coupled directly or indirectly with separation techniques such as GC or HPLC. A book on the mass spectrometry of lipids was published in 1993. [267]... 

The amount of data obtained by mass spectrometry is enormous, particularly when many lipid classes or several lipid species are investigated simultaneously. As such, manual management of the different data sets is impractical. For this reason, various attempts have been initiated to create software that is capable of evaluating and handling the generated data sets in a qualitative, quantitative, and comparative manner. One example is the software called Lipid Profiler, which has been used to achieve automated identification, deconvolution, and absolute quantification... 

Other techniques are based on sample sputtering followed by mass spectrometry of the vaporized products, including secondary ion mass spectrometry (SIMS) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). Beam sizes are in the few to tens of pm range. Elemental sensitivities for SIMS are highly variable depending on ion yield, and quantification can be difficult because of matrix effects in the ion production process. SIMS and LA-ICP-MS have very high sensitivities for some elements and low sensitivity for others. These and other microanalytical techniques used in earth science research are described in Potts et al. (1995). 

Quantification of Triacylglycerols by Mass Spectrometry and Its Use in Olive Oil Analysis... 

Allele-specific quantification can be achieved using a number of different approaches of which primer extension with detection by mass spectrometry will be described here (see Note 13). A number of steps are involved which potentially introduce variance into the experiment for this reason duplication should be included at each step (see Note 14 [1132]). 

---