34 Chapter 2 expected to be low since reproducible ionization and fragmentations can be achieved as a result of the clean sample extract. Table 2. Performance of various internal standards during sample workup and analysis. Sample preparation LC-MS/MS Analysis Internal Standard Sample Purification Digestion Clean-up and Enrichment Injection Ionization Fragmentation SIL Protein ++ ++ ++ ++ ++ ++ SIL Peptide − − ++ ++ ++ ++ Dimethyl Label − − ++ ++ ++ ++ Flanking SIL (Extended) Peptide − − ++ ++ ++ ++ Analogue Protein ++ + + ++ + + Notes: ++: optimum correction; +: moderate correction, −: no correction. Abbreviation: SIL: stable isotopically labeled. LC-MS/MS optimization Chromatographic separation and MS/MS optimization are critical steps in method development and if done properly can lead to higher assay sensitivity. LC separation and MS/MS optimization are firstly performed on a digested mAb standard and after sample purification and digestion conditions are optimized, LC separation is re-evaluated with a mAb spiked sample of the biological matrix of interest. When sample purification methods are used that result in clean extracts, such as targeted sample purification, short LC runtimes (~5 min) can successfully be achieved [34, 48, 56, 62, 72]. Nevertheless, columns with higher plate numbers and longer gradient times might be required to separate isobaric interferences and matrix effects when generic sample purification methods are used. High resolution instruments such as the Orbitrap, time of flight (TOF) or ion trap (QTap) can also be used and may provide the necessary selectivity. However, matrix effects can only be eliminated through sample cleanup and sufficient LC separation. Detection limits can be lowered by selecting the optimal signature peptide but also by monitoring the most abundant precursor and product-ions. Signature peptides with chain lengths of around 20 amino acids produce precursors consisting of single, double, triple and quadruple charged states. Therefore, a precursor mass-scan needs to be performed to determine the most abundant charged state for quantification (Figure 4B). Consecutively, the most intense product-ion can be found by performing a mass-scan after collision-induced dissociation (CID) of the most abundant precursor peak (Figure 4C). In contrast to triple quadrupole MS, HRMS instrumentations have a higher full mass scan rate of >12 Hz and can easily obtain precursor and product-ion scans of the desired signature peptide with high accuracy and sensitivity using low amount of sample [92]. This combination of most abundant precursor and subsequent production is termed selected reaction monitoring (SRM) and can further be optimized for
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