MS/MS Analysis and Automated Tool Development for Protein Post-Translational Modifications

Protein post-translational modifications (PTMs) are important for a variety of reasons. PTMs confer the final protein product and biological functionality onto a nascent protein chain. Two common PTMs are glycosylation and disulfide bond formation. Both glycosylation and disulfide bond formation contribute to a variety of biological processes, including protein folding and stabilization. Mass spectrometry (MS) has shown to be an essential technique to study PTMs, especially when tandem mass spectrometry (MS/MS) experiments are performed. In the characterization of PTMs using MS/MS, different fragmentation techniques are often used. Regardless of the dissociation method that is employed, MS/MS data interpretation is a tedious and lengthy process. To render this analysis more efficient, the use of automated tools is necessary. In this work, collision induced dissociation (CID) MS/MS experiments were carried out in order to create a set of fragmentation rules applicable to any N-linked glycopeptide. These rules were then used to develop an algorithm to power publicly available software that accurately determines glycopeptide composition from MS/MS data. This program greatly reduces the time it takes researchers to manually assign the identity of an N-linked glycopeptide to an acquired CID spectrum. In addition, electron transfer dissociation (ETD) experiments were performed in order to devise a computational approach that works to determine precursor charge state directly from MS/MS data of peptides containing disulfide bonds. Lastly, alternate fragmentation patterns found to be detected in glycopeptides containing labile monosaccharide residues such as sialic acid are discussed. These patterns, along with other trends noticed after extensive analysis of N-linked glycopeptide CID data, were then used to propose future updates to the GPG analysis tool

Software for Automated Interpretation of Mass Spectrometry Data from Glycans and Glycopeptides

The purpose of this review is to provide those interested in glycosylation analysis with the most updated information on the availability of automated tools for MS characterization of N-linked and O-linked glycosylation types. Specifically, this review describes software tools that facilitate elucidation of glycosylation from MS data on the basis of mass alone, as well as software designed to speed the interpretation of glycan and glycopeptide fragmentation from MS/MS data. This review focuses equally on software designed to interpret the composition of released glycans and on tools to characterize N-linked and O-linked glycopeptides. Several websites have been compiled and described that will be helpful to the reader who is interested in further exploring the described tools

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

Binding of Pseudomonas aeruginosa Apo-Bacterioferritin Associated Ferredoxin to Bacterioferritin B Promotes Heme Mediation of Electron Delivery and Mobilization of Core Mineral Iron†

The bfrB gene from Pseudomonas aeruginosa was cloned and expressed in E. coli. The resultant protein (BfrB), which assembles into a 445.3 kDa complex0020from 24 identical subunits, binds 12 molecules of heme axially coordinated by two Met residues. BfrB, isolated with 5–10 iron atoms per protein molecule, was reconstituted with ferrous ions to prepare samples with a core mineral containing 600 ± 40 ferric ions per BfrB molecule and approximately one phosphate molecule per iron atom. In the presence of sodium dithionite or in the presence of P. aeruginosa ferredoxin NADP reductase (FPR) and NADPH the heme in BfrB remains oxidized and the core iron mineral is mobilized sluggishly. In stark contrast, addition of NADPH to a solution containing BfrB, FPR and the apo-form of P. aeruginosa bacterioferritin associated ferredoxin (apo-Bfd) results in rapid reduction of the heme in BfrB and in the efficient mobilization of the core iron mineral. Results from additional experimentation indicate that Bfd must bind to BfrB to promote heme mediation of electrons from the surface to the core to support the efficient mobilization of ferrous ions from BfrB. In this context, the thus far mysterious role of heme in bacterioferritins has been brought to the front by reconstituting BfrB with its physiological partner, apo-Bfd. These findings are discussed in the context of a model for the utilization of stored iron in which the significant upregulation of the bfd gene under low-iron conditions [Ochsner, U.A., Wilderman, P.J., Vasil, A.I., and Vasil, M.L. (2002) Mol. Microbiol. 45, 1277–1287] ensures sufficient concentrations of apo-Bfd to bind BfrB and unlock the iron stored in its core. Although these findings are in contrast to previous speculations suggesting redox mediation of electron transfer by holo-Bfd, the ability of apo-Bfd to promote iron mobilization is an economical strategy used by the cell because it obviates the need to further deplete cellular iron levels to assemble iron sulfur clusters in Bfd before the iron stored in BfrB can be mobilized and utilized

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

Binding of Pseudomonas aeruginosa apobacterioferritin-associated ferredoxin to bacterioferritin B promotes heme mediation of electron delivery and mobilization of core mineral iron

The bfrB gene from Pseudomonas aeruginosa was cloned and expressed in Escherichia coli. The resultant protein (BfrB), which assembles into a 445.3 kDa complex from 24 identical subunits, binds 12 molecules of heme axially coordinated by two Met residues. BfrB, isolated with 5-10 iron atoms per protein molecule, was reconstituted with ferrous ions to prepare samples with a core mineral containing 600 ± 40 ferric ions per BfrB molecule and approximately one phosphate molecule per iron atom. In the presence of sodium dithionite or in the presence of P. aeruginosa ferredoxin NADP reductase (FPR) and NADPH, the heme in BfrB remains oxidized, and the core iron mineral is mobilized sluggishly. In stark contrast, addition of NADPH to a solution containing BfrB, FPR, and the apo form of P. aeruginosa bacterioferritin- associated ferredoxin (apo-Bfd) results in rapid reduction of the heme in BfrB and in the efficient mobilization of the core iron mineral. Results from additional experimentation indicate that Bfd must bind to BfrB to promote heme mediation of electrons from the surface to the core to support the efficient mobilization of ferrous ions from BfrB. In this context, the thus far mysterious role of heme in bacterioferritins has been brought to the front by reconstituting BfrB with its physiological partner, apo-Bfd. These findings are discussed in the context of a model for the utilization of stored iron in which the significant upregulation of the bfd gene under low-iron conditions [Ochsner, U. A., Wilderman, P. J., Vasil, A. I., and Vasil, M. L. (2002) Mol. Microbiol. 45, 1277-1287] ensures sufficient concentrations of apo-Bfd to bind BfrB and unlock the iron stored in its core. Although these findings are in contrast to previous speculations suggesting redox mediation of electron transfer by holo-Bfd, the ability of apo-Bfd to promote iron mobilization is an economical strategy used by the cell because it obviates the need to further deplete cellular iron levels to assemble iron-sulfur clusters in Bfd before the iron stored in BfrB can be mobilized and utilized

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