PhD Project

To utilize the electron-based fragmentation and photodissociation methods available on the Bruker solariX 12T FT-ICR MS to differentiate isomers of amino acids in peptides and proteins.

Current work:

1. Differentiation and quantitation of the isomeric products of deamidation in peptides using ECD MS/MS.

The spontaneous, non-enzymatic deamidation of asparagine (Asn) (and to a lesser extent glutamine, Gln) is a common PTM, playing a significant role in diseases related to ageing such as Alzheimer’s and Parkinson’s disease.

The ability to distinguish between the isomerization products of Asn deamidation and the quantitation of the ratio of these isomers will help us to gain a deeper understanding of the effect that they have on protein structure, which is known to alter the activity and function of the proteins. For example, in monoclonal antibodies, the isomerisation of Asp to isoAsp reduces the antigen binding affinity and thereby reduces the overall antibody activity.

Chromatography and tandem MS methods such as collisionally activated dissociation can be used to separate Asp from isoAsp but it can be challenging due to the slight structural differences between the isomers. Hence using ultra-high resolution FT-ICR MS and employing electron based fragmentation methods may be more beneficial as we can generate diagnostic fragment ions for Asp and isoAsp and quantify the ratio of these isomers using electron capture dissociation (ECD).

Cleavage of the C_α - C_β bonds in the peptide backbone by ECD MS/MS generates diagnostic fragment ions for isoAsp residues, c_l-n + 57 and z_n—57.

 

 Figure 1. a) deamidation of Asn and isomerization of Asp to isoAsp via hydrolysis of the succinimide intermediate and b) deamidation of Gln.

Figure 2. MS and ECD MS/MS spectra of a bovine serum albumin (BSA) trypsin digested deamidated peptide.

2. Distinguishing between methylated histidine isomers generated as a post-translational modification of actin

This is a collaborative project with the division of Biomedical Sciences (cell development and biology programme) at Warwick Medical School. 3-methylhistidine (3-MH) is formed by methylation of histidine as a post-translational modification of the muscle and motor proteins, actin and myosin. 3-MH is of particular interest as it is liberated during degradation of these proteins and therefore can be used a biomarker for muscle degeneration when found in urine or plasma.