Apparent mass resolution

Recent years have also witnessed approaches to increase the apparent mass resolution [85, 209, 210]. Most genotyping assay formats were designed such that the instrumental mass resolution of approximately 1 Da, available for linear MALDI-TOF instruments, does not compromise the mass accuracy. However, more challenging assays and higher degrees of multiplexing would benefit from increased mass resolution and accuracy. 

On the one hand, the use of isotopically depleted nucleotides for MALDI-TOF-MS represents an option to increase the apparent mass resolution. In combination with linear TOFs, however, the instrumental limitation in mass accuracy is still dominating. A gain in mass resolution by a factor of 2 does not merit the costs associated with the production of isotopically depleted nucleotides. Newer instruments such as orthogonal TOFs and Orbitraps, on the other hand, will alone also not overcome all of the current limitations, because the isotopic envelope of the analytes will be limiting when natural nucleotides are used. Hence, the success of more challenging applications in NA analysis is linked to both, instrumental as well as biochemical improvements, that is, the large-scale use of isotopically depleted nucleotides. 

As illustrated in Figure 8.8, the apparent mass resolution gradually decreases as the ion mass increases. The actual value of the upper mass limit where oligomers are resolved is dependent on several factors, including the number of components present in a polymer sample, the mass(es) of the repeat units(s), and the matrix/sample preparation method. For the latter, whenever possible, the formation of two or more cation adducts should be avoided. The addition of a preferred cation suppresses other cationization and can improve the quality of the spectrum by reducing peak overlap. 

Figure 6.6. Metabolite profiles of omeprazole in human plasma (a) base peak ion chromatogram of unprocessed data and (b) base peak ion chromatogram of MDF-processed data exhibiting all metabolite peaks present and some endogenous peaks. High-resolution LC-MS data were obtained for a human plasma sample spiked with omeprazole metabolites generated by microsomal incubation (the equivalent of a 1.0-mL plasma injection). A MDF ivas set at 50 mDa around the apparent mass defect of the omeprazole ion.

ISD fragmentations lead to product ions that are always apparent in the MALDI spectra, whereas the observation of product ions from PSD fragmentation needs certain instrumental conditions. For example, a MALDI source coupled to a linear TOF analyser allows detection of fragment ions produced in the source at their appropriate m/z ratio. On the contrary, fragment ions produced after the source cannot be resolved from their precursor ions and are detected at the same apparent m/z ratio. This induces a broadening of the peaks with a concomitant loss of mass resolution and sensitivity. 

The ion issuing from the metastable fragmentation shows up at the apparent mass rrf, linked to mp and mf through the relation m = ml/mp. The kinetic energy released during the fragmentation brings up a dispersion in velocity, thus an alteration in the resolution. 

It is seen that an excellent separation is obtained and apparently little resolution is lost in the capillary interface. The mass spectrum of the peak marked T2 in the chromatogram is shown in figure 30. It is clear that good quality spectra can be obtained up to ion masses of at least 900. Such a combination of techniques can be invaluable for the structure elucidation of compounds generated in biochemical research. 

Improvement in mass resolution by MALDI of samples loaded on synthetic membranes was particularly apparent in the MALDI of contaminated samples. We systematically examined the ability to remove measured amounts of contaminants from peptide and protein samples by doping previously pure samples with glycerol and salts. Samples doped with 5 % glycerol and 500 mM sodium were prepared for MALDI-MS analysis using the method described above. 

Baveye, P, Boast, C.W., Ogawa, S., Parlange, J.-Y. and Steenhuis, T. (1998). Influence of image resolution and thresholding on the apparent mass fractal characteristics of preferential flow patterns in fleld soils. Water Resour. Res., 34(11), 2783-2796. 

As discussed in the sputtering section and illustrated in Figs. 4.1 and 4.2, ion yields vary widely. When combined with the choice of analytical conditions, it is apparent why there is a wide range of detection limits. Typical detection limits in dynamic SIMS are from the ppm level to the ppb level and, in some cases, sub ppb. Interferences, the use of high mass resolution, or the use of energy offset... 

High-resolution mass spectrometers have been used to obtain electrospray spectra and have the added advantage that they allow the direct determination of the charge state of the ions being observed, e.g. if the apparent separation of the and isotopic contributions is 0.1 Da, the charge state is 10, while if it is 0.05 Da, the charge state is 20, etc. 

The sustained attractiveness of photolabeling is apparent from its prominence in studies of y-secretase, an intramembrane protease that contributes to forming amyloid-p peptides and is a major target in Alzheimer s disease [60-62]. y-Secretase is a complex of at least four different polypeptides, and is difficult to engage with high-resolution structural methods. However, in a case of this kind that involves a known target, immunodetection of proteins can often specify the target of y-secretase inhibitor photoaffinity probes such as 19, and proteomic mass spectrometry is not needed. 

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