Comparison of glycopeptide identification performances of collisional fragmentation techniques used in site specific glycosylation investigation

Glycosylation is one of the most important post translational modifications of proteins and is involved in all kinds of biological and physiological processes. In order to have information about the identity of the glycoprotein, information about the peptide sequence, glycosylation site, and glycan structure must be generated. Tandem Mass Spectrometry (MS/MS) is widely used for the analysis of glycopeptides. Collision Induced Dissociation (CID), one of the fragmentation methods, works in harmony with the Quadrupole-Time-of-Flight Mass Spectrometry (QTOF-MS) instrument. Higher-Energy C-trap Dissociation (HCD) method works with the Quadrupole Orbitrap (Q-Exactive Plus) instrument. Also, the very similar technique of HCD and CID, generate primarily b- and y-ions from peptide precursor ions, as well as B-and Y-ions from glycans units. In recent years, to increase the efficiency of fragment ions Stepped Collision Induced Dissociation (sCID) and Stepped Higher‐energy C-trap Dissociation (sHCD) techniques have emerged. With these techniques, different energies are applied to obtain simultaneous information about both glycan and peptide fragments. Within the scope of the thesis, glycopeptide identification performances of QTOF s(CID) (Stepped Collision Induced Dissociation) and Q-Exactive Plus s(HCD) (Stepped Higher‐ energy C-trap Dissociation) systems were compared. These three different variants of haptoglobin (Hpt) protein called Hpt 1-1, Hpt 2-2 and Hpt-M were used in the thesis study. Each variant was analyzed in both QTOF s(CID) and Q-Exactive Plus s(HCD) systems in four replicates. The QTOF s(CID) system defined both more glycosylation sites and more glycopeptide numbers than the Q-Exactive Plus s(HCD) system. The Q Exactive Plus s(HCD) system has a higher total average score than the QTOF s(CID) system.