Multiple Reaction Monitoring Mass Spectrometry for the Discovery and Quantification of O‑GlcNAc-Modified Proteins

O-linked <i>N</i>-acetylglucosamine (O-GlcNAc) is a post-translational modification regulating proteins involved in a variety of cellular processes and diseases. Unfortunately, O-GlcNAc remains challenging to detect and quantify by shotgun mass spectrometry (MS) where it is time-consuming and tedious. Here, we investigate the potential of Multiple Reaction Monitoring Mass Spectrometry (MRM-MS), a targeted MS method, to detect and quantify native O-GlcNAc modified peptides without extensive labeling and enrichment. We report the ability of MRM-MS to detect a standard O-GlcNAcylated peptide and show that the method is robust to quantify the amount of O-GlcNAcylated peptide with a method detection limit of 3 fmol. In addition, when diluted by 100-fold in a trypsin-digested whole cell lysate, the O-GlcNAcylated peptide remains detectable. Next, we apply this strategy to study glycogen synthase kinase-3 beta (GSK-3β), a kinase able to compete with O-GlcNAc transferase and modify identical site on proteins. We demonstrate that GSK-3β is itself modified by O-GlcNAc in human embryonic stem cells (hESC). Indeed, by only using gel electrophoresis to grossly enrich GSK-3β from whole cell lysate, we discover by MRM-MS a novel O-GlcNAcylated GSK-3β peptide, bearing 3 potential O-GlcNAcylation sites. We confirm our finding by quantifying the increase of O-GlcNAcylation, following hESC treatment with an O-GlcNAc hydrolase inhibitor. This novel O-GlcNAcylation could potentially be involved in an autoinhibition mechanism. To the best of our knowledge, this is the first report utilizing MRM-MS to detect native O-GlcNAc modified peptides. This could potentially facilitate rapid discovery and quantification of new O-GlcNAcylated peptides/proteins

Representative size exclusion chromatogram and distribution of complete IgG monomer, aggregate, and incomplete IgG fragments produced in stable transfection pools at different LC:HC ratios.

<p>Components within protein A purified supernatant collected at the end of culture were separated by size exclusion chromatography followed by the identification and quantification of species by light scattering and UV detection, respectively. Analysis was done for duplicate stable transfection pools. (A) Representative chromatogram obtained by UV detector for the pool of LC:HC = 0.24. Agg: Aggregates; IgG: complete IgG monomer; Frag: Incomplete IgG fragments. (B) Quantitative comparison of complete IgG monomer, aggregates, and incomplete IgG fragments for different LC:HC ratios. Each value in figure B represents the average of four measurements from two independent stable transfection pools.</p

Effect of LC:HC ratios on mAb productivity in stable transfection pools.

<p>CHO DG44 stable transfection pools at different LC:HC ratios generated were cultured in shake flask batch cultures. Titers of monoclonal antibody at the end of culture were determined using a nephelometric method or ELISA. Each value represents the average and standard deviation of measurements from two independent stable transfection pools.</p

Comparison of non-AUG translation efficiency in cap-dependent and cap-independent translations.

a<p>Cap-dependent translation efficiencies of non-AUG condons were determined in COS-1 cells in transient transfections <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082100#pone.0082100-Mehdi1" target="_blank">[35]</a>.</p

Western blot analysis of supernatant in stable transfection pools at different LC:HC ratios.

<p>CHO DG44 stable transfection pools at different LC:HC ratios were cultured in shake flask batch cultures. Crude supernatant collected at the end of culture was analyzed under both non-reducing and reducing conditions by western blot. Positive and negative controls are the same as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082100#pone-0082100-g004" target="_blank">Figure 4</a>.</p

Relative strength of EMCV IRES variants in different mammalian cell lines in transient transfections.

<p>The strengths of IRES variants in different cell lines were obtained by transfection of dual-luciferase vectors (refer <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082100#pone-0082100-g001" target="_blank">figure 1A</a>) containing different IRES variants including IRESv3, IRESv10, or IRESv18 on the Fluc gene. At 48 h post-transfection, the luciferase activities of Rluc and Fluc gene were quantified by Dual-Glo Luciferase Assay Systems. Results represent the strength of each IRES variant calculated as the ratios of luciferase activities of Fluc to Rluc normalized to the control, the wild type EMCV IRES (IRESwt). Each value represents the average and standard deviation of sixteen measurements from four independent transfections for CHO K1 cells and eight measurements from two independent transfections for other cell lines.</p

Schematic representation of vectors.

<p>(A) Structure of dual-luciferase bicistronic vectors for determination of IRES variants' strengths. (B) Structure of monoclonal antibody expressing tricistronic vectors with specific IRES variants applied on the light chain (LC) or heavy chain (HC) genes. (C) Amino acid sequences of signal peptide, N- and C-terminal end of LC and HC. CMV, human cytomegalovirus IE gene promoter; IRESwt, wild type encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES); IRESvn, a specific EMCV IRES variant; SpA, SV40 polyadenylation signal; Rluc, renilla luciferase coding region; Fluc, firefly luciferase coding region; SP_L, light chain signal peptide; LC, light chain coding region; SP_H, heavy chain signal peptide; HC, heavy chain coding region; DHFR, dihydrofolate reductase coding region; HP, DNA sequence that can form a hairpin structure and contains an upstream AUG that is out of frame with the coding sequence.</p

Control of LC and HC expression using different IRES variants in stable transfections.

<p>CHO DG44 stable transfection pools were generated by transfection of tricistronic vectors with different IRES variants applied on the LC and HC cDNA (refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082100#pone-0082100-g001" target="_blank">Figure 1B</a>). LC:HC ratios listed in the table were calculated as the concentration of intracellular LC polypeptides determined by ELISA divided by the HC polypeptides. Each value represents the average and standard deviation of four measurements from two independent stable transfection pools. The intracellular abundance of LC and HC polypeptides were also analyzed using western blot under reducing conditions. Cell lysates containing equal amounts of proteins were loaded into each lane. A commercial human affinity purified myeloma Ig1 (Sigma-Aldrich) and supernatants from cells transfected with either a vector expressing only HC or a vector expressing only LC were used as positive control, and supernatant from non-transfected cells as negative control (N).</p

Relative strength of IRES variants in expressing a gene.

<p>Relative strength of IRES variants in expressing a gene.</p

Relative strength of EMCV IRES variants in CHO K1 cells in transient transfections.

<p>Equal amounts of dual-luciferase bicistronic vectors (refer <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082100#pone-0082100-g001" target="_blank">Figure 1A</a>) encoding renilla luciferase (Rluc) and firefly luciferase (Fluc) genes were transfected into CHO K1 cells. At 48 h post-transfection, cell pellets were collected for analysis of Fluc and Rluc luciferase activities by using Dual-Glo Luciferase Assay system. Transfection of each vector was done in duplicates and repeated a second time using independently prepared plasmids and cultures. (A) and (B) Measured Fluc and Rluc activities. (C) Ratios of luciferase activities of Fluc to Rluc gene for each IRES variant normalized to the wild-type IRES (WT). Each individual bar represents the average and standard deviation of eight measurements from two transfections in one experiment. Black bars represent the results from experiment 1 and gray bars from experiment 2.</p