Isobaric compounds

The protocol for using isobaric tags differs from that described previously for the ICAT or ECAT type reagents. In the following method, the proteins are denatured and the disulfides reduced and then alkylated to block them permanently. This eliminates disulfide re-association and also prevents the isobaric tags from forming thioester modification with cysteine thiols. Next, the proteins are digested with trypsin and then modified with an isobaric tag. Each sample is labeled with a different isobaric compound so that the samples can be differentiated upon MS/MS analysis. 

Resolution (or resolving power) plays an important role in mass spectrometry for applications requiring the characterization of very similar chemical species. The ability to detect and accurately measure the m/z ratio of a particular ion depends directly on the resolving power of the mass analyzer. For example, if a sample contains two isobaric compounds (i.e., having the same nominal molecular mass but different elemental formulae) the difference in the exact masses of the molecular ions will be much less than 1 m/z unit. Any mass analyzer possessing a nominal resolving power (e.g., RP< 1000) will register only one peak in the mass spectrum of such a binary mixture. Attempts to measure the... 

From the FIA—MS overview spectrum, speculation that there can be more than just one structurally defined molecule type behind an observable signal i.e. the presence of isobaric compounds, cannot be excluded whenever one signal defined by the m/z-ratio is examined in FIA-MS spectra. Consequently, the information obtained by FIA-MS is quite limited whenever we deal with complex mixtures of environmental pollutants rather than the analysis of pure products or formulations with a known range of ingredients. LC separation is inevitable when mixtures of isomeric compounds should be identified with MS-MS. Therefore, in FIA-MS-MS special attention has to be paid to avoid the generation of mixed product ion spectra from isomeric parent compounds. This would block identification by library search and may lead to misinterpretations of product ion spectra because of the fragmentation behaviour observed. 

The importance of a good chromatographic separation prior to the mass spectrometric analysis has to be underlined in face of the large number of possible surfactant-derived isobaric compounds to be encountered during the microbial degradation of LAS containing traces of DATS. For example, Cn-LAS and methylated C8-DATSC yield the same molecular ion [M — H] at m/z 311 identical masses of the [M — H] ions (m/z 355) are calculated for methylated Cn-SPC and dimethylated C8-DATSdC. 

Initial lead discovery Start with prefer 10-100 hits Limited by massive installed base of other methods. Current paradigm requires only singlepoint estimate of activity, because low-potency hits are expected (therefore, power of MS-based systems may overmatch the task). Mass-based recognition of compounds may be thwarted by isobaric compounds or impurities. 

Kalinoski et al. [32] has applied this method to the determination of tri-chothecene mycotoxins in wheat. The methods were based on chemical ionisation MS and collision-induced dissociation tandem MS and enabled the rapid identification of ppm levels of several trichothecene mycotoxins. Supercritical carbon dioxide is shown to allow identification of mycotoxins with minimum sample handling in complex natural matrices such as wheat. Tandem MS techniques are employed for unambiguous identification of compounds of varying polarity, and false positives from isobaric compounds are avoided. Capillary column SCFC-MS of a SCF extract of the same sample was also performed, and detection limits in the ppb range appear feasible. 

Stage, there is no indication from Figure 13.13a whether the ions observed represent one or more isobaric compounds. However, careful analysis of the tandem MS data can identify multiple components or yield valuable structural information. For example, the MS/MS spectrum of m/z 456 (Figure 13.13b) shows five main ions m/z 438, 396, 378, 352, 338, and 254). By then conducting MS/MS/MS experiments (Figure 13.13c-f) it can be deduced that the ion peak at m/z 456 represents two compounds and, further, that derivatization appears to occur at either of two positions on the C-9 esterifying acid [35]. 

Accurate isotope patterns or isotopic signature and unique mass differences between isobaric compounds can be obtained using these two MS formats. 

In general, MSn is also highly effective for differentiation of isobaric compounds. The detection of fragment ions [M-162]+ (Y0+), [M-324]+ (due to consecutive losses of two sugar residues), [M-204]+, [M-308]+... 

Figure 3.17 Differentiation of two isobaric compounds with the same aglycone moiety by deuterium exchange experiment positive-ion ESI mass spectrum of Mv-3-0(6-0-caffeoyl)monoglucoside (above) and of Mv-3,5-0-diglucoside (below) dissolved in D20. (Reprinted from American Journal of Enology and Viticulture 51, Favretto and Flamini, Copyright 2000)...

The advantage of GC/MS is the capacity to separate these isomeric (or isobaric) compounds. Its disadvantage is, among others, to introduce coeluting compounds, leading to uncertainties and to a reduced sensitivity (in the case where high resolution becomes necessary) this no longer occurs when MS/MS is utilized. 

A large number of isobaric compounds, especially those of uric acid, was studied. The spectrum of this compound is distinguished considerably from those of its isobars. Using FI with their instrument, they detected small quantities of caffeine in complex samples (urine). Maquestiau et al. [245] also demonstrated the same compound in tea samples with the same method, but they used Cl conditions to produce protonated molecules. 

However, it will be shown later that single stage MS can be unreliable for the analysis of AN as it cannot discriminate this toxin from the isobaric compound, phenylalanine (Phe), that occurs commonly in environmental water bodies. 

McEwen et al. have utilized another ambient technique called atmospheric pressure solids analysis probe (ASAP) [84,85] and interfaced it with TWIM-MS [86], This source is now available commercially on the Synapt instrument. They have used m/z vi drift time plots to define compound classes in CTude oil analysis and to resolve isobaric compounds. Excellent agreement was demonstrated for drift times obtained from standards run individually and doped into the crude oil mixture. This work demonstrates the utility of mobility experiments to provide a degree of separation and classification in the study of very complex mixtures where chromatographic separation is either not possible or too time consuming. 

The combination provides enhanced sensitivity, selectivity, speed of analysis, an opportunity for automation, feasibility of quantification of coeluting components or isobaric compounds, and use of the stable-isotope labeled analogs as internal standards. 

While MSI can provide images that allow the user to discriminate between drug and metabolite, it is far more difficult to differentiate between isobaric compounds. Due to the complex nature of tissue, many different lipids, peptides, and other biological compounds (not to mention MALDI matrix clusters) can complicate the MSI analysis for drug compounds. Several... 

By connecting an ion mobility spectrometry (IMS) in front of a QTOF-MS, another dimension of separation is realized. Unseparated isobaric compounds, which have the same m/z value, can be separated after ionization by the structure-dependent drift time through the IMS. The combination of IMS with QTOF is also a powerful tool for nontarget analysis in complex samples, due to the fact that the chemical noise is drastically reduced by IMS. 

Figure 1.9 Distinction of isobaric compounds by RP chromatography and UV detection for a reaction controi anaiysis (N-aryiation of an E/Z acryiic ester mixture).

In all cases where molecules cannot be distinguished based on their molecular mass within the experimental error (isobaric compounds), additional and complementary detection principles are imperative. The related detector modules can be added either serially or in parallel by splitting the LC column effluent, depending on the detection principle. 

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