As members of the glycosaminoglycan (GAG) family, heparin and heparan sulfate (HS) are responsible for mediation of a wide range of essential biological actions, most of which are mediated by specific patterns of modifications of regions of these polysaccharides. To fully understand the regulation of HS modification and the biological function of HS through its interactions with protein ligands, it is essential to know the specific HS sequences present. However, the sequencing of mixtures of HS oligosaccharides presents major challenges due to the lability of the sulfate modifications, as well as difficulties in separating isomeric HS chains. Here, we apply a sequential chemical derivatization strategy involving permethylation, desulfation, and trideuteroperacetylation to label original sulfation sites with stable and hydrophobic trideuteroacetyl groups. The derivatization chemistry differentiates between all possible heparin/HS sequences solely by glycosidic bond cleavages, without the need to generate cross-ring cleavages. This derivatization strategy combined with LC-MS/MS analysis has been used to separate and sequence five synthetic HS-like oligosaccharides of sizes up to dodecasaccharide, as well as a highly sulfated Arixtra-like heptamer. This strategy offers a unique capability for the sequencing of microgram quantities of HS oligosaccharide mixtures by LC-MS/MS.

Protein oxidation is typically associated with oxidative stress and aging and affects protein function in normal and pathological processes. Additionally, deliberate oxidative labeling is used to probe protein structure and protein–ligand interactions in hydroxyl radical protein footprinting (HRPF). Oxidation often occurs at multiple sites, leading to mixtures of oxidation isomers that differ only by the site of modification. We utilized sets of synthetic, isomeric “oxidized” peptides to test and compare the ability of electron-transfer dissociation (ETD) and collision-induced dissociation (CID), as well as nano-ultra high performance liquid chromatography (nanoUPLC) separation, to quantitate oxidation isomers with one oxidation at multiple adjacent sites in mixtures of peptides. Tandem mass spectrometry by ETD generates fragment ion ratios that accurately report on relative oxidative modification extent on specific sites, regardless of the charge state of the precursor ion. Conversely, CID was found to generate quantitative MS/MS product ions only at the higher precursor charge state. Oxidized isomers having multiple sites of oxidation in each of two peptide sequences in HRPF product of protein Robo-1 Ig1-2, a protein involved in nervous system axon guidance, were also identified and the oxidation extent at each residue was quantified by ETD without prior liquid chromatography (LC) separation. ETD has proven to be a reliable technique for simultaneous identification and relative quantification of a variety of functionally different oxidation isomers, and is a valuable tool for the study of oxidative stress, as well as for improving spatial resolution for HRPF studies.

Unlike small molecule drugs, therapeutic protein pharmaceuticals must not only have the correct amino acid sequence and modifications, but also the correct conformation to ensure safety and efficacy. Here, we describe a method for comparison of therapeutic protein conformations by hydroxyl radical protein footprinting using liquid chromatography-mass spectrometry (LC-MS) as an analytical platform. Hydroxyl radical protein footprinting allows for rapid analysis of the conformation of therapeutic proteins based on the apparent rate of oxidation of various amino acids by hydroxyl radicals generated in situ. Conformations of Neupogen®, a patented granulocyte colony-stimulating factor (GCSF), were compared to several expired samples of recombinant GCSF, as well as heat-treated Neupogen®. Conformations of different samples of the therapeutic proteins interferon α-2A and erythropoietin were also compared. Differences in the hydroxyl radical footprint were measured between Neupogen® and the expired or mishandled GCSF samples, and confirmed by circular dichroism spectroscopy. Samples that had identical circular dichroism spectra were also found to be indistinguishable by hydroxyl radical footprinting. The method is applicable to a wide variety of therapeutic proteins and formulations through the use of separations techniques to clean up the protein samples after radical oxidation. The reaction products are stable, allowing for flexibility in sample handling, as well as archiving and reanalysis of samples. Initial screening can be performed on small amounts of therapeutic protein with minimal training in LC-MS, but samples with structural differences from the reference can be more carefully analyzed by LC-MS/MS to attain higher spatial resolution, which can aid in engineering and troubleshooting.

Improved methods for structural analyses of glycosaminoglycans (GAGs) are required to understand their functional roles in various biological processes. Major challenges in structural characterization of complex GAG oligosaccharides using liquid chromatography-mass spectrometry (LC-MS) include the accurate determination of the patterns of sulfation due to gas-phase losses of the sulfate groups upon collisional activation and inefficient on-line separation of positional sulfation isomers prior to MS/MS analyses. Here, a sequential chemical derivatization procedure including permethylation, desulfation, and acetylation was demonstrated to enable both on-line LC separation of isomeric mixtures of chondroitin sulfate (CS) oligosaccharides and accurate determination of sites of sulfation by MSn. The derivatized oligosaccharides have sulfate groups replaced with acetyl groups, which are sufficiently stable to survive MSn fragmentation and reflect the original sulfation patterns. A standard reversed-phase LC-MS system with a capillary C18 column was used for separation, and MSn experiments using collision-induced dissociation (CID) were performed. Our results indicate that the combination of this derivatization strategy and MSn methodology enables accurate identification of the sulfation isomers of CS hexasaccharides with either saturated or unsaturated nonreducing ends. Moreover, derivatized CS hexasaccharide isomer mixtures become separable by LC-MS method due to different positions of acetyl modifications.

Hydroxyl radical footprinting is a valuable technique for studying protein structure, but care must be taken to ensure that the protein does not unfold during the labeling process due to oxidative damage. Footprinting methods based on submicrosecond laser photolysis of peroxide that complete the labeling process faster than the protein can unfold have been recently described; however, the mere presence of large amounts of hydrogen peroxide can also cause uncontrolled oxidation and minor conformational changes. We have developed a novel method for submicrosecond hydroxyl radical protein footprinting using a pulsed electron beam from a 2 MeV Van de Graaff electron accelerator to generate a high concentration of hydroxyl radicals by radiolysis of water. The amount of oxidation can be controlled by buffer composition, pulsewidth, dose, and dissolved nitrous oxide gas in the sample. Our results with ubiquitin and β-lactoglobulin A demonstrate that one submicrosecond electron beam pulse produces extensive protein surface modifications. Highly reactive residues that are buried within the protein structure are not oxidized, indicating that the protein retains its folded structure during the labeling process. Time-resolved spectroscopy indicates that the major part of protein oxidation is complete in a time scale shorter than that of large scale protein motions.

Hydroxyl radical footprinting is a technique for studying protein structure and binding that entails oxidizing a protein system of interest with diffusing hydroxyl radicals, and then measuring the amount of oxidation of each amino acid. One important issue in hydroxyl radical footprinting is limiting amino acid oxidation by secondary oxidants to prevent uncontrolled oxidation, which can cause amino acids to appear more solvent accessible than they really are. Previous work suggested that hydrogen peroxide was the major secondary oxidant of concern in hydroxyl radical footprinting experiments; however, even after elimination of all hydrogen peroxide, some secondary oxidation was still detected. Evidence is presented for the formation of peptidyl hydroperoxides as the most abundant product upon oxidation of aliphatic amino acids. Both reverse phase liquid chromatography and catalase treatment were shown to be ineffective at eliminating peptidyl hydroperoxides. The ability of these peptidyl hydroperoxides to directly oxidize methionine is demonstrated, suggesting the value of methionine amide as an in situ protectant. Hydroxyl radical footprinting protocols require the use of an organic sulfide or similar peroxide scavenger in addition to removal of hydrogen peroxide to successfully eradicate all secondary oxidizing species and prevent uncontrolled oxidation of sulfur-containing residues.The formation of peptidyl hydroperoxides on aliphatic residues in hydroxyl radical footprinting experiments causes uncontrolled secondary oxidation of sulfur-containing residues.Figure optionsDownload full-size imageDownload high-quality image (57 K)Download as PowerPoint slide