Multiply charged peptide ions

Figure 6.4. Fragmentation spectrum of a tryptic peptide obtained from bovine serum albumin. Peptide sequence LGEYGFQNALIVR, monoisotopic [M + H]+ = 1479.796, monoisotopic [M+2H]2+ =740.402. Upper panel full scan MS spectrum. Lower panel MS/MS spectrum of a doubly-charged ion at 740.7 m/z with a ladder of y ions, the distances between which correspond to amino acid residues (upper row of letters). A shorter series of b ions is also seen (lower row of letters). See Fig. 6.5 for description of nomenclature. Note the often observed phenomenon where multiply-charged ions lose the charge during fragmentation process and, therefore, have higher m/z values than the original parent ion.

Usually the number of charges on an ion will not be known, but it can be calculated using a formula based on two different ions appearing in the spectrum. Actually, the molecular mass of a sample can be calculated automatically, or semiautomatically, by the processing software associated with the mass spectrometer. Experimentally, the automatic calculation of molecular mass is very helpful because a complex peptide or protein mixture will display an m/z spectrum with several overlapping series of multiply charged ions. 

In tandem MS mode, because the product ions are recorded with the same TOF mass analyzers as in full scan mode, the same high resolution and mass accuracy is obtained. Isolation of the precursor ion can be performed either at unit mass resolution or at 2-3 m/z units for multiply charged ions. Accurate mass measurements of the elemental composition of product ions greatly facilitate spectra interpretation and the main applications are peptide analysis and metabolite identification using electrospray iomzation [68]. In TOF mass analyzers accurate mass determination can be affected by various parameters such as (i) ion intensities, (ii) room temperature or (iii) detector dead time. Interestingly, the mass spectrum can be recalibrated post-acquisition using the mass of a known ion (lock mass). The lock mass can be a cluster ion in full scan mode or the residual precursor ion in the product ion mode. For LC-MS analysis a dual spray (LockSpray) source has been described, which allows the continuous introduction of a reference analyte into the mass spectrometer for improved accurate mass measurements [69]. The versatile precursor ion scan, another specific feature of the triple quadrupole, is maintained in the QqTOF instrument. However, in pre-... 

ESI tandem MS stands for electro spray ionization mass spectrometry performed in multistage. This technique is conducted based on the production of multiply charged ions from proteins and peptides. In this technique, ionization procedure is carried out within the instrument. Three types of mass analyzers are used individually or in combination. 

A new ionization method called desorption electrospray ionization (DESI) was described by Cooks and his co-workers in 2004 [86]. This direct probe exposure method based on ESI can be used on samples under ambient conditions with no preparation. The principle is illustrated in Figure 1.36. An ionized stream of solvent that is produced by an ESI source is sprayed on the surface of the analysed sample. The exact mechanism is not yet established, but it seems that the charged droplets and ions of solvent desorb and extract some sample material and bounce to the inlet capillary of an atmospheric pressure interface of a mass spectrometer. The fact is that samples of peptides or proteins produce multiply charged ions, strongly suggesting dissolution of the analyte in the charged droplet. Furthermore, the solution that is sprayed can be selected to optimize the signal or selectively to ionize particular compounds. 

Figure 5.11 displays the analysis of a mixture of peptides from ovalbumin obtained by CE/MS coupled to ESI. The spectrum shown is a mean of the spectra acquired during the elution of the indicated broad peak. It corresponds to a mixture of doubly or triply charged ions of several glycopeptides [27]. The displayed structures were actually deduced from MS/MS fragmentation spectra of these multiply charged ions. 

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