38 Chapter 2 Digestion conditions After sample purification, the therapeutic mAb needs to be denatured (unfolded) to allow, in the subsequent step, the digestive enzyme easier access to the cleavage sites. The protein tertiary structure is maintained by hydrophobic, ionic, hydrogen and disulfide bonds. Thus, abrogation of these interactions and bonds can achieve faster and efficient digestion. Disulfide bonds can be reduced with 5mM DTT or tris (2-carboxyethyl)phosphine (TCEP). TCEP is a stronger and more stable reducing agent compared to DDT. However, DTT is most frequently used due to its neutral pH being more compatible with downstream trypsin digestion. Usually, a reduction is carried out under heating conditions (around 60 °C) to speed up the reaction process and to aid in protein denaturation. Urea concentrations >6M can also unfold the protein structures, but sample dilution or dialysis is then required to lower the urea concentration <1M prior to trypsin digestion. Furthermore, undesirable physiochemical reactions can occur when using urea at elevated temperatures. Recent publications focus mainly on unfolding the protein via heating >70 °C with or without MS compatible surfactants such as RapiGest™ [73, 76, 78]. The use of sodium dodecyl sulfate (SDS) surfactant has also been reported [50, 85]. However, when SDS is left in the buffer solution prior to trypsin digestion, the proteolytic enzyme would denature and subsequent MS analysis would suffer from ionization suppression. As proteolytic enzyme, trypsin is mostly preferred for bottom up proteomics, because it cleaves the peptide bonds following arginine (R) and lysine (K), two basic amino acids that are easily ionized during electrospray ionization. Trypsin is active in a buffered solution with low ionic strength <0.1M with pH 7-9 . Digestion efficiency is dependent on factors such as trypsin to protein ratio, temperature, time, protein accessibility and the presence of trypsin inhibitors such as alpha-1 antitrypsin . Digestion efficiency can further be improved by incorporating methylated trypsin which can retains its activity during digestion and can thus be used in lower amounts  or by addition 1mM calcium ions to the solution which aids in trypsin stability . Treatment with 6-(1-tosylamido-2-phenyl) ethyl chloromethyl ketone (TPCK) is also required to disable chymotrypsin activity in native trypsin , since untreated chymotrypsin would cleave the protein at different locations (tyrosine, tryptophan and phenylalanine) affecting signature peptide recoveries. Immobilized trypsin is increasingly being implemented, since it offers fast and complete digestion and can be used in combination with high temperatures [68, 71, 78, 101, 102]. Immobilization secures trypsin on a silica bead or agarose resin and retains its active conformation during high temperature sample denaturation which is necessary for fast protein digestion. Moreover, efficient digestion is promoted by immobilizing excess mounts of trypsin thus favourable enzyme to protein ratio is achieved. Combinations of enzymes can also be incorporated to improve digestion efficiency such as the inclusion of Enzyme LysC, which tolerates high urea concentrations, in combination with trypsin . Since these combination of enzymes, immobilized trypsin and methylated trypsin are significantly more expensive than TPCK trypsin it is recommended to test these enzymes side by side to determine their added value.