Quantitative Characterization of Glycopeptide Isomers

Texas Tech University, Lubbock TX

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Abstract

Because of the pressing needs to comprehensively understand the biological attributes of glycosylation in many critical biological functions such as the immune response, cell development, cellular differentiation/adhesion and host-pathogen interactions, glycoproteomics continues to be a highly dynamic research area. Aberrant glycosylation for decades has been recognized as the attribute of many mammalian diseases, including osteoarthritis, cystic fibrosis, and cancer. Moreover, isomeric alterations of glycoproteins have been observed in diseases such as Alzheimer’s Disease and cancers. Recently, glycans and their isomers have been reported to be vital to the SARS-CoV-2 viral infection, making them a crucial target for the drug development of COVID-19. Therefore, reliable, and efficient characterization of glycopeptides and their isomers is necessary to better understand the attributes of glycosylation in biological and biomedical processes. We are proposing here four specific aims: Aim 1. To enhance the separation and identification of glycopeptide isomers using mesoporous graphitized carbon (MGC)-LC-MS and hydrophobicity index of peptides (HIP); Aim 2. To enhance the quantification of glycopeptide isomers using a 15N metabolic-TMT multiplexing approach and a parallel reaction monitoring (PRM) method; Aim 3. To enhance the glycopeptide isomeric characterization using novel derivatization methods; and Aim 4. To enhance automated isomeric glycopeptide data processing by the development of improved software. The outcome of these aims will provide reliable and efficient glycoproteomic platforms and algorithms for a better isomeric characterization of glycopeptides which can be employed to address biomedical issue, thus contributing to the glycoscience community. The innovations of this proposal originate from the uniqueness of the proposed analytical methods and software. The isomeric separation of glycopeptides using MGC-LC-MS/MS is a highly innovative method, developed in our lab, permitting efficient separation of glycopeptide isomers on a 1 cm short column. The retention time normalization of glycans intra and inter-laboratories has been introduced and demonstrated to be necessary previously through a glucose unit index (GUI), but not in glycoproteomic analysis. For the first time, we will investigate the retention time normalization of glycopeptides and glycopeptide isomers using a set of peptides which have known hydrophobicity factors on different instruments and different laboratories. The combination of 15N stable isotope labeling of glycopeptides and TMT will double the multiplexing capacity of TMT to 36-plex when studying in vitro cell line glycoproteomics. Although PRM has been utilized for glycoproteomic profiling, the analysis of isomeric glycoproteomics is lacking. Thus, this will be the first comprehensive investigation of glycopeptide isomers using PRM. Moreover, the derivatization methods we proposed are of great innovation. It will be the first time to achieve 2-aminobenzamide (2-AB) labeling on sialic acids of glycopeptides, and the first time to achieve efficient isomeric separation of sialoglycopeptides on a 15 cm C18 column via a two-step oxidation-reductive amination reaction. In addition, we have achieved a derivatization of sialylated glycopeptides (DOSG) method that will introduce mass difference to distinguish α2,3 and α2,6 linked sialoglycopeptides. Consequently, the combination oxidation- reductive amination and DOSG methods leads to an innovative enrichment method for sialoglycopeptides. The derivatization of carboxyl groups of sialic acids on glycans and amino acids on peptide backbone neutralizes additional charges and the addition of quaternary ammonium functionalized molecules provides controllable positive charges which make the enrichment using strong cation exchange (SCX) possible. This is a novel method for the efficient enrichment of sialoglycopeptides where sialic acid linkage isomers can be distinguished at the same time. The deliverables of this proposal are reliable, adaptable, and affordable strategies and improved software to enhance the isomeric glycopeptide studies by any laboratory interested in defining comprehensive protein glycosylation using LC-MS/MS. The proposed technologies are expected to enable a better understanding of the biological attributes of glycoprotein isomers in the development and progression of diseases.