9.1 Conclusions
The advanced performance of FTICR MS, in particular high resolution, high mass accuracy, and flexible tandem mass spectrometry capability, has been demonstrated by a number of applications in the thesis, including the isotopic fine structure measurement of Aβ peptides (Chapters 2 and 3), the compositional and structural investigation of a polymeric mixture (Chapter 4), the characterization of the fragmentation of chlorophyll-a and pheophytin-a
(Chapter 6 and 7), and a little bit of top-down proteomics (Chapter 8).
In Chapter 2 and 3, to estimate the 17O labelling ratio in several Aβ peptides, their isotopic fine structures were obtained. High resolving power is vital for the measurement because the mass difference between 13C- and 17O-substited species is about 0.86 mDa. Three peptides, Aβ
16-22 (MW: 894.498124 Da), Aβ11-25 (MW: 1757.885869 Da), and Aβ37-42 (MW: 515.30855 Da), were analyzed with ultra-high resolving power, 5.0 M, 6.0 M, and 3.5 M, respectively. Without adding any standard control, the uncertainty of the 17O labelling ratio measurement in each peptide could be calculated according to the difference between the theoretical and experimental ratio of 13C in the same peptide, which is ~2.0% for small peptides, Aβ16-22 and Aβ37-42, and ~5.2% for Aβ11-25. The results will be helpful in interpretating the NMR signal to estimate the distance between atoms. In addition to 17O labelling, all of the three peptides are also labelled with 15N or/and 13C, so it is crucial to assign each isotopic peak correctly using high mass accuracy. Dual nano- electrospray experiments were introduced to internally calibrate the spectra
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by a standard (HPmix or PEG400 depending on the mass range), and less than 0.1 ppm errors were obtained. MS/MS methods are particularly valuable to determine the isotopic labelling position. Isotope labelled counterparts have similar chemical and physical properties, which are very difficult to be separated using traditional techniques, however, could be qualitatively and quantitatively distinguished by FTICR MS without any extra separation/chemistry.
In chapter 4, the composition of TPGS samples, a PEGylated drug excipient, was studied, because differing compositions of TPGS between batches could result in variable performance of the formulated product. In addition to TPGS, Di-TPGS and free PEG were also detected. The coexistent of different polymeric components and different adducts such as H+, NH4+, and Na+, arises the complexity of the spectra, so that a high resolving power (450,000 at m/z 500) and high mass accuracy (˂ 1 ppm) are important to assign peaks with high confidence. For TPGS samples, the ionization solution condition was shown to be a very crucial factor influencing the peaks observed in the MS spectrum, indicating that the solvent selection can be particularly important for the compositional study of some polymers by MS. On the other hand, the fragmentation pattern of TPGS was investigated using different MS/MS methods and with different adducts. Diagnostic fragments from CAD and ECD MS/MS can be used for the identification of the TPGS structure in the future, where ECD and CAD show different preferences in bond cleavages. In addition to the methodology study, based on the MS and MS/MS results of TPGS samples from different suppliers, it was found that the composition of each TPGS sample and the structure of TPGS in each
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sample were the same; however, the relative intensity of free PEG and TPGS shows differences between samples from two suppliers. FTICR MS demonstrated the promising role in studying complex mixtures, and FTICR MS/MS is a valuable structural characterization tool for polymers complementary to traditional spectroscopy techniques.
Cationization is a hot-topic in MS/MS field, as introducing different cations usually gives different fragments, and there are also cases that fragmentation patterns were fully changed by simply using different adducts.285 To understand the role of adducts in MS/MS processes is always of great interest. In Chapter 4, silver adducted TPGS showed different ECD behaviour in contrast to the precursors with Li+ or Na+ ions, so the influence of several adduct ions on the fragmentation processes of CAD and ECD was further studied in Chapter 5. Li+, Na+, K+, Ag+, and H+ were chosen for this study, and the competitive influence of each ion was investigated by fragmenting TPGS attached with two different cations, [M+X1+X2]2+ (X1 and X2 refer to Li+, Na+, K+, Ag+, H+). The observation in CAD experiments agrees with previous research carried on other molecules that the dissociation of ionic adducts from the precursor is most likely depending on the binding strength, and, to some extent, ions binding stronger increase fragmentation. While during ECD, the charge carriers are lost in an order which appears to correlate with the relative reduction potential of the ions, which suggests that the reduction potential of the charge carrier is an important factor influencing the fragmentation pattern. The ion with a high reduction potential seems to be more effective in capturing electrons; however, it may also be lost easily before leading to any fragmentation. The trends observed from TPGS are of
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benefit for selecting adducts in MS/MS investigations and may facilitate the understanding of the ECD results when cationization is involved.
Porphyrins include a large category of compounds that play important roles in life, where chlorophylls are the ones significant in plants. In contrast to the biosynthesis of chlorophylls, the biodegradation process is less well understood. One of the key steps on the way to unveil the enigma is to identify the metabolites in degradation; therefore, an efficient structural characterization strategy accessible to species of low concentration would be significant. In Chapter 6 and 7, chlorophyll-a and the corresponding Mg- depleted species, pheophytin-a, were exposed to three different MS/MS methods, CAD, EID, and IRMPD, respectively to investigate the fragmentation efficiency of the two analogues. Again, the importance of high mass accuracy and high resolution were demonstrated in peak assignment. For both molecules, most abundant and extensive fragments were observed from IRMPD, which technique shows great potential in studying the structures of porphyrin like molecules. On the other hand, the EID spectrum of chlorophyll-a resembles its IRMPD spectrum, but EID results of pheophytin-a
show differences. In comparison, the macrocycle of pheophytin-a having no central Mg2+ ion is more fragile compared to chlorophyll-a because fragments from cleavages of the pyrrole groups were only observed from pheophytin-a. Moreover, the unique macrocyclic structure shows tolerence to radicles, and odd-electron species were detected from all three MS/MS methods.
In an on-going project, a segment of protein p65 having a mass of ca 35 kDa was explored using the top-down approach. FTICR MS displayed capabilities in dealing with big molecules; along with MS/MS, the molecular
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weight and some information of the sequence were obtained. Even though it is not feasible to locate the phosphorylation site of phosphorylated p65 so far since only a few sequential fragments were assigned from CAD and ECD, MS/MS definitely has the potential to be used in studying PTMs. A modification of ~144.04 Da, suggesting a formula of C6H8O4 (MW: 144.04223 Da), in the N-terminal from aa 12-14 was detected by MS/MS. Sample preparation was found particularly crucial to the success of the MS detection of big proteins. For FTICR MS, the ion capacity and detection sensitivity of an ICR cell is limited, which means good signal could only be approached with right amount of ions in the cell. As proteins tend to present with different charge states and attach with different adducts, all of these could dilute the signal of target ions disturbing the detection. Moreover, the present of modifications and wide isotopic distribution will further exacerbate the signal. 9.2 Future work
Using isotopic fine structure to measure the isotope ratio of an element in a molecule has distinct advantages and shows as a promising complementary strategy. In our experiments, the uncertainty of the measurement is approximately 0.5% for molecules smaller than 300 Da, and the error is bigger for large molecules. Because the ion population in the cell was minimized in practice, a small change of ion number during detection may have big influence on the isotope ratio measurement. In theory, the precision could be improved by signal average, but the peak shift between scans can kill the resolution or lead to split peaks. To avoid frequency shift and obtain a better S/N, one solution has been used in Marshall’s group is to align spectra before frequency-domain coaddition,121 hence, the experimental S/N and