Advances in protein post-translational modifications (PTMS) using liquid chromatography-mass spectrometry.

摘要

Protein post-translational modifications (PTMs) play significant roles in affecting the physical, chemical, and biological properties of proteins. PTMs can regulate protein functions by modulating protein activity, turnover, cellular location, and protein-protein interaction. In addition, the efficacy and safety of therapeutic protein drugs can be dramatically affected by PTMs. Structural elucidation of complex PTMs provides invaluable insights for the potential roles of PTMs. However, comprehensive characterization of PTMs presents a significant analytical challenge, often requiring the use of multiple orthogonal methods to ensure high confidence in the resulting analytical data. Liquid chromatography coupled with mass spectrometry (LC-MS) is one of the most powerful platforms for determination of PTMs due to its high sensitivity, resolution, and accuracy. LC-MS tools usually can achieve the goal of identification of PTMs, including modified proteins, specific modification sites, and structures of the modifications. This thesis focuses on the methods for the determination of several important PTMs, including deamidation of asparagine, isomerization of aspartic acid, disulfide linkages, and glycosylation using LC-MS approaches.;In chapter 2, a method with differentiation and enrichment of isoaspartic acid (isoAsp) by Asp-N digestion combined with detection and quantification of isoAsp by LC-MS/MS with electron transfer dissociation (ETD) is presented.;In Chapter 3, a proteome-level analysis of isoAsp in urine proteins from the wild type and protein L-isoaspartyl O-methyltransferase (PIMT, a highly conserved isoAsp repair enzyme) deficient mice is reported.;In Chapter 4, a successful and robust methodology for complete characterization of disulfide linkages, including cystine knots and nested disulfides in recombinant human arylsulfatase A (rhASA), is developed using multi-enzyme digestion and LC-MS methods with ETD and sequential collision induced dissociation (CID-MS 3) analysis.;In Chapter 5, the method development for comprehensive characterization of oligosaccharide structures with glycan composition, sequence, linkage, and positional information is described. The newly developed method employs fluoride-mediated negative ionization LC-MS/MS using a microfluidic chip packed with porous graphitized carbon (PGC) for N-glycan separation and an accurate mass quadrupole time of flight (Q-TOF) tandem mass spectrometer for characterization. An application to the characterization of N-glycans released from polyclonal human and murine IgG is described.