35 Tutorial on LC-MS/MS methods quantifying mAbs collision gas pressure and collision energy. This can easily be done through infusion of a synthesized peptide directly into the MS or alternatively, can be optimized by performing multiple injections of a digested protein, each time with a different collision setting. Unfortunately, different MS instrumentation can generate different precursor charge ratios and product ion profiles. Therefore, SRM settings are not interchangeable between different instruments. This is demonstrated in Figure 5 where GLEWVAEIR fragments were scanned by 2 different MS instruments. Here, CID with a triple quadrupole we see that fragment 773.80 was higher than fragment 488.55, while this was the opposite with higher energy collision dissociation (HCD) with the orbitrap. Nevertheless, three most intense product ions per signature peptide should be monitored and only after complete validation, the best performing signature peptide with its concomitant product ion can be selected as quantifier. Other signature peptides that have successfully passed validation can be selected as qualifiers and can be used for quality assurance. Here, sample results from the quantifier peptide can be compared with those of the qualifier peptide during routine analysis. Large variations (>15%) between results could be caused by isobaric interferences which can be present in one signature peptide but not necessarily the other and would require further investigation. However, for each signature peptide a SIL internal standard equivalent is required for quantification this would lead to increased costs when SIL peptides are used. Figure 5. Product ion scan of GLEWVAEIR peptide with Quantum Access Max triple quadrupole (A) and with Q Exactive Orbitrap (B). 2
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