Why is site-specific N- and O-glycopeptide analysis needed?
The in-depth investigation of glycosylation, a complex post-translational modification process that confers carbohydrate moieties to proteins, requires a thorough analysis of N-glycopeptides and O-glycopeptides on a site-specific basis. The involvement of glycosylation in critical biological processes such as protein folding, stability, function, and recognition, as well as its implication in multiple ailments like cancer, autoimmune disorders, and genetic disorders, stresses the importance of this research area.
The site-specific N- and O-glycopeptide analysis entails the identification and characterization of glycosylation sites and their attached glycans' structures and compositions on proteins. This data holds valuable insights into the functional significance of glycosylation and its potential role in disease. The analysis is crucial due to the heterogeneous and intricate nature of the glycosylation process, which allows for the emergence of multiple feasible glycan structures and attachment sites.
In the realm of mass spectrometry-based approaches, site-specific N- and O-glycopeptide analysis is typically carried out to identify and quantify specific glycopeptides. This methodology involves the enzymatic cleavage of the protein of interest, followed by the enrichment of glycopeptides, fragmentation of glycan and peptide moieties, and lastly, analysis of the resulting fragments using mass spectrometry.
1 SDS-PAGE
1) Prepare 5–10 μg of glycoprotein dissolved in water, mix it with sample buffer (ratio 3:1 (v/v)) and incubate the sample for 10 min at 70 °C for denaturation.
2) Load the sample onto the SDS- PAGE and separate proteins for 50 min at 200 V.
3) Wash the gel three times for 5 min with water on a shaker.
4) Stain the gel for 1 h on a shaker.
5) Destain the gel in water over night on a shaker.
2 In-Gel Reduction and Alkylation of Cysteine Bonds Followed by Pronase Treatment
1) Cut the gel bands of interest into small pieces of around 1 mm3.
2) Transfer the gel pieces into a 1.5 ml reaction vial.
3) Wash the gel pieces with 100 μl AmBiC buffer for 5 min and then remove the liquid.
4) Shrink the gel pieces with 100 μl ACN for 2 min and discard the liquid, followed by another 100 μl of fresh ACN for 10 min and remove the liquid.
5) Swell the gel pieces with 50 μl reduction buffer and incubate it for 30 min at 60 °C to reduce cysteines and then remove the liquid.
6) Shrink the gel pieces with 50 μl ACN for 2 min and discard the liquid, followed by another 50 μl of fresh ACN for 5 min and remove the liquid.
7) Swell the gel pieces again with 50 μl alkylation buffer and incubate for 20 min in dark to alkylate cysteines and then remove the liquid.
8) Wash the gel pieces with 100 μl AmBiC buffer for 2 min and discard the liquid, followed by another 100 μl of fresh AmBiC buffer for 10 min and remove the liquid.
9) Shrink the gel pieces with 100 μl ACN for 5 min and remove the liquid.
10) Repeat the steps 8 and 9 to destain the gel bands ( see Note 8 ).
11) Dry down the gel pieces in a centrifugal vacuum concentrator for approximately 5 min.
12) Add 30 μl digestion buffer to the gel pieces and let it swell on ice for 45 min. Check after 45 min if the gel pieces are fully covered with liquid and add another 5–10 μl AmBiC buffer if necessary and let it incubate at 37 °C for 20 h.
13) Collect the liquid in a separate reaction vial.
14) Extract (glyco)peptides by adding 30 μl of AmBiC buffer for 1 h at maximum speed on a shaker.
15) Remove the liquid and combine it with the first one.
16) Keep the sample at −20 °C or dry down the liquid in a centrifugal vacuum concentrator if longer storage is needed.
3 C18-PGC- LC Setup
1) Connect the C18 columns and connecting tubing.
2) Connect the PGC precolumn on both ends to 30 μm ID × 10 cm connecting tubing via 1/16″ stainless steel ZDV unions and the attach it to the valve.
3) Connect the PGC analytical column to the valve via a 1/16″ stainless steel ZDV union and 30 μm ID × 10 cm connecting tubing.
4) Connect the outlet of the PGC analytical column to the fused silica tubing by a conductive micro union.
5) Attach grounding cables to the stainless steel ZDV unions of the PGC precolumn and analytical column as well as to the conductive micro union.
4 C18-PGC-LC-ESIQTOF- MS/MS Analysis of Glycopeptides
1) Calibrate the mass spectrometer according to the manufacturer's instructions.
2) Apply all QTOF-MS/MS settings.
3) Equilibrate both precolumn and analytical column with 1 % solvent B.
4) Set the column oven temperature to 36 °C.
5) Dilute the sample in water to a protein concentration corresponding to approximately 20–50 ng/μl.
6) Inject 1 μl of sample (or any other volume if needed).
7) Set both valves to position 1_2 to load the sample for 6 min at 6 μl/min and then switch to 1_6.
8) Set the nano valve to guide the C18 elution to the MS.
9) Elute first analytes from the C18 columns with a gradient from 1 % to 55 % solvent B in 30 min.
10) Switch the nano valve at 35 min to guide the elution of the PGC columns to the MS.
11) Elute analytes from the PGC column with a gradient from 1 % to 60 % solvent B in 48 min.
5 Data Analysis
1) Screen fragmentation spectra for oxonium ions 204.0867 [N-acetylhexosamine (HexNAc) + H] +, 366.1394 [HexNAc + Hexose (Hex) + H] +, 292.1027 [N-acetyl neuraminic acid (NeuAc) + H] +, 657.2348 [HexNAc + Hex + NeuAc + H] + to classify spectra of glycopeptides.
2) Identify the intact peptide mass by following the glycanderived B- and Y-ions.
3) Search for potential peptide sequences using findpept with a mass deviation of maximum 10 ppm.
4) Verify the peptide sequence manually by matching b- and y-ions.
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Reference
- Lauc, G., & Wuhrer, M. (2017). High-throughput glycomics and glycoproteomics. Springer New York.
