N-glycan profiling of glycoproteins is essential for discovering novel biomarkers and understanding glycan functions in cellular events. Additionally, N-glycan analysis of protein biopharmaceuticals is very important for human use. In this current article, a high-throughput strategy for identifying and quantifying N-glycan structures was presented using the HILIC-FLD-MS/MS technique.
Glycosylation is a vital modification found in proteins. N-glycan profiling of glycoproteins is required to detect novel biomarker candidates and determine glycan alterations in diseases. Most commercially available biopharmaceutical proteins are glycoproteins. The efficacy of these drugs is affected by glycosylation patterns. Therefore, an in-depth characterization method for the N-glycans is necessary. Here, we present a comprehensive approach for qualitative and quantitative analysis of N-glycans using hydrophilic interaction liquid chromatography equipped with fluorescence detection and tandem mass spectrometry (HILIC-FLD-MS/MS). N-glycans were released from glycoproteins with a facile method and labeled by a procainamide fluorophore tag in the strategy. Subsequently, the procainamide labeled N-glycans were analyzed by a HILIC-FLD-MS/MS technique. In this approach, N-glycan structures were confirmed by the tandem mass spectrometric analysis, whereas fluorescence detection was used for the quantitative analysis. An application for data analysis of the detected N-glycan peaks is described in the study. This protocol can be applied to any glycoprotein extracted from various species.
Glycosylation is a vital post-translational modification observed in proteins1. Multiple enzymatical processes regulate glycosylation modification in cellular organisms. Glycans are attached to the proteins by these enzymatical processes, and the proteins subjected to this modification are called glycoproteins1. Two glycosylation types are commonly observed in proteins. O-glycosylation is the attachment of O-glycans to the side chain of serine or threonine amino acid residues. N-glycosylation is the attachment of N-glycans to the side chain of asparagine amino acid residue in a protein.
The structure, stability, and folding of the proteins are affected by glycan attachments2. The glycosylation process dramatically influences the functions of the proteins, and glycoproteins regulate many cellular functions in organisms3,4. For example, heavily glycosylated proteins protect their glycoproteins from proteolytic degradation5. Another example is glycans of thyroid gland proteins that regulate Tg transport and hormone synthesis6,7. To explain their roles in cellular events, an in-depth characterization of glycoproteins is required8.
N-glycan profiles of the glycoproteins change in disease situations9,10,11,12. Profiling N-glycans derived from crucial glycoproteins or body fluids is required to discover novel biomarkers and understand the enzymatic activity changes in disease cases. On the other hand, most protein biopharmaceuticals are glycoproteins, and their glycan profiles influence drug efficacy13. Therefore, an acceptable method of N-glycan profiling must be performed in developing proper protein biopharmaceuticals for human use14.
Glycomics is an emerging discipline used to identify and quantify glycan structures of glycosylated molecules15,16. Many methods have been utilized for profiling the glycans of glycosylated species, including NMR17 and MS18. Hydrophilic Interaction Liquid Chromatography-with Fluorescence Detection (HPLC-HILIC-FLD) is the gold standard method for profiling N-glycans derived from glycoproteins19. When this strategy is combined with mass spectrometric detection, identifying N-glycan structures could be easier and more reliable. Most fluorescence tags used in N-glycan analysis with mass spectrometry have low ionization efficiencies. In contrast, procainamide increases the ionization efficiencies of N-glycans, which is used to obtain efficient tandem mass spectra of N-glycan structures20,21. Specific fragments can be obtained from this strategy by tandem mass spectrometry for the structural identification of N-glycans such as core fucosylated22 (proc-HexNAc1Fuc1) and bisecting types23 (proc-Hex1HexNAc3, proc-Hex1HexNAc3Fuc1).
This study demonstrates a facile protocol for the N-glycan profiling of glycoproteins with HILIC-FLD-MS/MS. The presented method includes four steps: (1) releasing of N-glycans from glycoproteins (2) labeling of N-glycans by a procainamide tag (3) purification of the procainamide labeled N-glycans, and (4) data analysis.
N-glycan profiling of glycoproteins includes challenging steps. Although there are many different methodologies for this purpose, a suitable approach should be selected for both identification and quantification of N-glycan structures14. HILIC-FLD is the gold standard approach for the quantification of N-glycans. However, identification of all N-glycan types by FLD detection is not achieved. Therefore, tandem MS analysis is needed for confirming N-glyca…
The authors have nothing to disclose.
This work was partly supported by the Ministry of Development-Republic of Turkey with project number: 2016 K121230. Bekir Salih gratefully acknowledges the Turkish Academy of Science (TUBA) for the partial financial support.
Acetic acid | Carlo Erba Reagents | 401413 | Glacial RS For LC/MS |
Acetonitrile | Merck | 1000292500 | LC-MS LiChrosolv |
Agilent 1200 Series HPLC with 1260 Series FLD dedector | Agilent Technologies | ||
Ammoniumm Formate | Carlo Erba Reagents | 419741 | For LC/MS |
Bruker TIMS-TOF (Q-TOF) Mass Spectrometry | Bruker Daltonics | ||
Cellulose | Sigma Aldrich | 310697 | microcrystalline, powder, 20 μm |
Deionized Water | Carlo Erba Reagents | 412111 | For LC/MS |
Dimethyl sulfoxide | Sigma Aldrich | 41639 | BioUltra, for molecular biology, ≥99.5% (GC) |
Empty polypropylene SPE Tube with PE frits | Sigma Aldrich | 54220 | 20 μm porosity,volume 1 mL |
Extraction Manifold, 20-position | Waters | WAT200607 | Complete with rack for 13 x 100 mm tubes |
Human Plasma | Sigma Aldrich | P9523 | lyophilized |
IGEPAL CA-630 | Sigma Aldrich | I8896 | for molecular biology |
IgG | Sigma Aldrich | I4506 | lyophilized powder |
Phosphate buffered saline | Sigma Aldrich | P4417 | Tablet |
PNGase F enzyme | Promega | V483A | |
Procainamide hydrochloride | abcam | ab120955 | |
Sodium cyanoborohydride | Sigma Aldrich | 156159 | reagent grade, 95% |
Sodium dodecyl sulfate | Sigma Aldrich | 71725 | |
trastuzumab | Roche Diagnostics | ||
Trifluoroacetic acid | Sigma Aldrich | 302031 | for HPLC, ≥99.9% |
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