Detecting O-GlcNAc using in vitro sulfation.

O-linked ß-N-acetylglucosamine (O-GlcNAc) glycosylation, the covalent attachment of N-acetylglucosamine to serine and threonine residues of proteins, is a post-translational modification that shares many features with protein phosphorylation. O-GlcNAc is essential for cell survival and plays important role in many biological processes (e.g. transcription, translation, cell division) and human diseases (e.g. diabetes, Alzheimer's disease, cancer). However, detection of O-GlcNAc is challenging. Here, a method for O-GlcNAc detection using in vitro sulfation with two N-acetylglucosamine (GlcNAc)-specific sulfotransferases, carbohydrate sulfotransferase 2 and carbohydrate sulfotransferase 4, and the radioisotope (35)S is described. Sulfation on free GlcNAc is first demonstrated, and then on O-GlcNAc residues of peptides as well as nuclear and cytoplasmic proteins. It is also demonstrated that the sulfation on O-GlcNAc is sensitive to OGT and O-ß-N-acetylglucosaminidase treatment. The labeled samples are separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by autoradiography. Overall, the method is sensitive, specific and convenient.

Direct detection of S-palmitoylation by mass spectrometry.

Direct detection and quantification of protein/peptide palmitoylation by mass spectrometry (MS) is a challenging task because of the tendency of palmitoyl loss during sample preparation and tandem MS analysis. In addition, the large difference in hydrophobicity between the palmitoyl peptides and their unmodified counterparts could prevent their simultaneous analysis in a single liquid chromatography-MS experiment. Here, the stability of palmitoylation in several model palmitoyl peptides under different incubation and fragmentation conditions was investigated. It was found that the usual trypsin digestion protocol using dithiothreitol as the reducing agent in ammonium bicarbonate buffer could result in significant palmitoyl losses. Instead, it is recommended that sample preparation be performed in neutral tris buffer with tris(2-carboxyethyl)phosphine as the reducing agent, conditions under which palmitoylation was largely preserved. For tandem MS analysis, collision-induced dissociation often led to facile palmitoyl loss, and electron capture dissociation frequently produced secondary side-chain losses remote from the backbone cleavage site, thus discouraging their use for accurate palmitoylation site determination. In contrast, the palmitoyl group was mostly preserved during electron transfer dissociation, which produced extensive inter-residue cleavage coverage, making it the ideal fragmentation method for palmitoyl peptide analysis. Finally, derivatization of the unmodified peptides with a perfluoroalkyl tag, N-[(3-perfluorooctyl)propyl] iodoacetamide, significantly increased their hydrophobicity, allowing them to be simultaneously analyzed with palmitoyl peptides for relative quantification of palmitoylation.

Interlaboratory study on differential analysis of protein glycosylation by mass spectrometry: the ABRF glycoprotein research multi-institutional study 2012.

One of the principal goals of glycoprotein research is to correlate glycan structure and function. Such correlation is necessary in order for one to understand the mechanisms whereby glycoprotein structure elaborates the functions of myriad proteins. The accurate comparison of glycoforms and quantification of glycosites are essential steps in this direction. Mass spectrometry has emerged as a powerful analytical technique in the field of glycoprotein characterization. Its sensitivity, high dynamic range, and mass accuracy provide both quantitative and sequence/structural information. As part of the 2012 ABRF Glycoprotein Research Group study, we explored the use of mass spectrometry and ancillary methodologies to characterize the glycoforms of two sources of human prostate specific antigen (PSA). PSA is used as a tumor marker for prostate cancer, with increasing blood levels used to distinguish between normal and cancer states. The glycans on PSA are believed to be biantennary N-linked, and it has been observed that prostate cancer tissues and cell lines contain more antennae than their benign counterparts. Thus, the ability to quantify differences in glycosylation associated with cancer has the potential to positively impact the use of PSA as a biomarker. We studied standard peptide-based proteomics/glycomics methodologies, including LC-MS/MS for peptide/glycopeptide sequencing and label-free approaches for differential quantification. We performed an interlaboratory study to determine the ability of different laboratories to correctly characterize the differences between glycoforms from two different sources using mass spectrometry methods. We used clustering analysis and ancillary statistical data treatment on the data sets submitted by participating laboratories to obtain a consensus of the glycoforms and abundances. The results demonstrate the relative strengths and weaknesses of top-down glycoproteomics, bottom-up glycoproteomics, and glycomics methods.

Highly sulfated nonreducing end-derived heparan sulfate domains bind fibroblast growth factor-2 with high affinity and are enriched in biologically active fractions.

Human fibroblast growth factor-2 (FGF2) regulates cellular processes including proliferation, adhesion, motility, and angiogenesis. FGF2 exerts its biological function by binding and dimerizing its receptor (FGFR), which activates signal transduction cascades. Effective binding of FGF2 to its receptor requires the presence of heparan sulfate (HS), a linear polysaccharide with N-sulfated domains (NS) localized at the cell surface and extracellular matrix. HS acts as a platform facilitating the formation of a functional FGF-FGFR-HS ternary complex. Crystal structures of the signaling ternary complex revealed two conflicting architectures. In the asymmetrical model, two FGFs and two FGFRs bind a single HS chain. In contrast, the symmetrical model postulates that one FGF and one FGFR bind to the free end of the HS chain and dimerization require these ends to join, bringing the two half-complexes together. In this study, we screened a hexasaccharide HS library for compositions that are able to bind FGF2. The library was composed primarily of NS domains internal to the HS chain with minor presence of non-reducing end (NRE) NS. The binders were categorized into low versus high affinity binders. The low affinity fraction contained primarily hexasaccharides with low degree of sulfation that were internal to the HS chains. In contrast, the high affinity bound fraction was enriched in NRE oligosaccharides that were considerably more sulfated and had the ability to promote FGFR-mediated cell proliferation. The results suggest a role of the NRE of HS in FGF2 signaling and favor the formation of the symmetrical architecture on short NS domains.

Tandem mass spectrometry for structural characterization of proline-rich proteins: application to salivary PRP-3.

Proline-rich proteins (PRPs), including collagens, complement 1q, and salivary PRPs, are unusually difficult to sequence by mass spectrometry, due to the high efficiency of cleavage at the amide bond on the N-terminal of proline residues and the consequently low relative abundance of fragment arising from cleavages at other amide bonds. To fully characterize these proteins by mass spectrometry, specialized approaches to fragmentation are needed for the peptides with high proline content. Our work reported herein focused on the analysis of the set of peptides derived by tryptic cleavage of the salivary protein PRP-3. Two methods of fragmentation were compared: Collision-induced dissociation (CID) and electron capture dissociation (ECD). The data obtained demonstrated that ECD spectra of peptides containing more than 30% proline residues are simpler and easier to interpret than are CID spectra of those peptides. Factors that limit the two methods of fragmentation include the complexity of information contained in the CID spectra and the low efficiency of ECD processes. A complementary approach using both decomposition methods provides more complete and interpretable sequence information and yielded >93% sequence coverage for the 11-kDa PRP-3 isolated from human saliva.