Qualitative and quantitative analysis of proteolytically digested glycoproteins by mass spectrometry

Glycoproteins are a very large and biologically relevant class of proteins that comprise more than 50 % of proteins in the human body. The glycosylation present on proteins, specifically N-linked glycosylation has been shown to be important for a variety of processes including protein folding, protein stability, and cell-cell interactions. Many glycoproteins are currently being considered as therapeutic drug targets. Glycosylation on proteins has also been shown to be altered with the onset of diseases, such as cancer, which has opened up the field of glycoproteomics, which aims to detect glycosylation changes for earlier detection of disease states. Mass spectrometry is a versatile technique that is frequently utilized for the analysis of glycoproteins, and it is particularly useful in the detection of glycosylation present on proteins. Most glycoproteins are prepared for mass spectrometric analysis by performing a protease digestion, followed by either a separation by HPLC or some other technique for enrichment of glycopeptides. In this work, the protease digestion procedure was optimized for maximized protein sequence coverage and detection of N-linked glycopeptides and other post-translational modifications. This method was applied to a recombinant glycoprotein that had never before been fully characterized by mass spectrometry and is a potential protein therapeutic as well as known to play a role in different types of cancer. Furthermore, a mass spectrometric relative quantitation method was developed by creating glycosylation profiles from glycopeptides detected at individual glycosylation sites on different glycoproteins. This method allowed for distinguishing between changes in protein concentration from changes in glycosylation. Lastly, glycoprotein structure and stability was probed by circular dichroism spectroscopy before and after glycan removal on glycoproteins containing high mannose type glycans with the enzyme peptide-N-glycosidase F. Protease digestion and mass spectrometry was performed to ensure that the deglycosylation reaction went to completion

A general protease digestion procedure for optimal protein sequence coverage and PTM analysis of recombinant glycoproteins: Application to the characterization of hLOXL2 glycosylation

Using recombinant DNA technology for expression of protein therapeutics is a maturing field of pharmaceutical research and development. As recombinant proteins are increasingly utilized as biotherapeutics, improved methodologies ensuring the characterization of post-translational modifications (PTMs) are needed. Typically, proteins prepared for PTM analysis are proteolytically digested and analyzed by mass spectrometry. To assure full coverage of the PTMs on a given protein, one must obtain complete sequence coverage of the protein, which is often quite challenging. The objective of the research described here is to design a protocol that maximizes protein sequence coverage and enables detection of post-translational modifications, specifically N-linked glycosylation. To achieve this objective, a highly efficient proteolytic digest protocol using trypsin was designed by comparing the relative merits of denaturing agents (urea and Rapigest™ SF), reducing agents (dithiothreitol, DTT, and tris(2-carboxyethyl)phophine, TCEP), and various concentrations of alkylating agent (iodoacetamide, IAM). After analysis of human apo-transferrin using various protease digestion protocols, ideal conditions were determined to contain 6 M urea for denaturation, 5 mM TCEP for reduction, 10 mM IAM for alkylation, and 10 mM DTT, to quench excess IAM before the addition of trypsin. This method was successfully applied to a novel recombinant protein, human lysyl oxidase-like 2 (hLOXL2). Furthermore, the glycosylation PTMs were readily detected at two glycosylation sites in the protein. These digestion conditions were specifically designed for PTM analysis of recombinant proteins and biotherapeutics, and the work described herein fills an unmet need in the growing field of biopharmaceutical analysis

GlycoPep Grader: A web-based utility for assigning the composition of N-linked glycopeptides

GlycoPep Grader (GPG) is a freely-available software tool designed to accelerate the process of accurately determining glycopeptide composition from tandem mass spectrometric data. GPG relies on the identification of unique dissociation patterns shown for high mannose, hybrid, and complex N-linked glycoprotein types, including patterns specific to those structures containing fucose or sialic acid residues. The novel GPG scoring algorithm scores potential candidate compositions of the same nominal mass against MS/MS data through evaluation of the Y1 ion and other peptide-containing product ions, across multiple charge states, when applicable. In addition to evaluating the peptide portions of a given glycopeptide, the GPG algorithm predicts and scores product ions that result from unique neutral losses of terminal glycans. GPG has been applied to a variety of glycoproteins, including RNase B, asialofetuin and transferrin, and the HIV envelope glycoprotein, CON-S gp140 CFI. The GPG software is implemented predominantly in PostgreSQL, with PHP as the presentation tier, and is publically accessible online. Thus far, the algorithm has identified the correct compositional assignment from multiple candidate N-glycopeptides in all tests performed

GlycoPep Grader: A Web-Based Utility for Assigning the Composition of <i>N</i>-Linked Glycopeptides

GlycoPep grader (GPG) is a freely available software tool designed to accelerate the process of accurately determining glycopeptide composition from tandem mass spectrometric data. GPG relies on the identification of unique dissociation patterns shown for high mannose, hybrid, and complex <i>N</i>-linked glycoprotein types, including patterns specific to those structures containing fucose or sialic acid residues. The novel GPG scoring algorithm scores potential candidate compositions of the same nominal mass against MS/MS data through evaluation of the Y₁ ion and other peptide-containing product ions, across multiple charge states, when applicable. In addition to evaluating the peptide portion of a given glycopeptide, the GPG algorithm predicts and scores product ions that result from unique neutral losses of terminal glycans. GPG has been applied to a variety of glycoproteins, including RNase B, asialofetuin, and transferrin, and the HIV envelope glycoprotein, CON-S gp140ΔCFI. The GPG software is implemented predominantly in PostgreSQL, with PHP as the presentation tier, and is publicly accessible online. Thus far, the algorithm has identified the correct compositional assignment from multiple candidate <i>N</i>-glycopeptides in all tests performed

Monitoring changes in membrane phosphatidylinositol 4,5-bisphosphate in living cells using a domain from the transcription factor tubby

Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) is a key component in signal transduction, being a precursor to other signalling molecules and itself associated with roles in signal transduction and cell biology. Tubby is a membrane bound transcription factor whose dysfunction results in obesity in mice. It contains a domain that selectively binds PtdIns(4,5)P2. We have investigated the use of a fluorescently tagged version of this domain to monitor changes in PtdIns(4,5)P2 concentration in living cells and compared it to the pleckstrin homology domain of PLCδ1. Our results show that selected mutants of this domain report receptor-mediated changes in cellular PtdIns(4,5)P2. In contrast to the pleckstrin homology domain of PLCδ1 it does not have a significant affinity for inositol 1,4,5-trisphosphate (IP3). Using a selected mutant, we examine the regulation of ATP-sensitive K+ channels via a Gq/11-coupled receptor. These experiments reveal a correlation between reporter translocation and the onset of current inhibition whilst the recovery of current after agonist removal is delayed when compared to the reporter. Furthermore our studies reveal the importance of Ca2+ in determining the overall activity of phospholipase C in living cells. This probe may be valuable in examining changes in PtdIns(4,5)P2 distinct from those of IP3 in intact cells in a variety of physiological settings