Mass spectrometry has become an invaluable tool when it comes to identifying proteins. Current mass spectrometry research is focused on not only protein identification but acquiring information about post-translational modifications found on a particular sub-set or group proteins. Recently, there has been an increasing interest in mapping sites of O-glycosylation. In comparison to identifying sites of N-glycosylation, mapping sites of O-glycosylation has proven to be a challenging task, as currently there is not a defined consensus sequence to identify the site of modification. Additionally, O-linked glycans have been shown to modify both serine and threonine residues, which are often adjacent to each other for long stretches within the protein sequence and not every serine or threonine residue is modified. Perhaps one of the more challenging aspects to mapping sites of O-linked glycosylation is due to a loss of information of the attached O-glycan when using collision induced dissociation (CID) type approaches. Thus, different fragmentation methods such as electron transfer dissociation (ETD) have been applied to aid in mapping sites of O-glycosylation. ETD allows retention of the modifying O-glycan upon fragmentation facilitating the site of modification being determined.
However, ETD, or the more recently described HCD-triggered ETD, is not always available. Thus, beta-elimination/Michael addition methods have been developed for site mapping where the labile modification is replaced with a more stable modification for CID experiments. One drawback to be noted of these approaches is that since the modifying moiety is removed, one can not determine the exact species that occupied the residue originally. For small samples sizes where sample cleanup steps are likely to result in poor or no yields we have begun using a simple one step method that uses a volatile buffer (ammonium hydroxide) as both the base and the nucleophile1). |
| Category | Glyco-proteomic mass spectrometry protocols |
| Protocol Name | Methods for mapping sites of O-linked glycosylation |
Authors
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Stalnaker H., Stephanie
Complex Carbohydrate Research Center, University of Georgia
Wells, Lance
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Complex Carbohydrate Research Center, University of Georgia
*To whom correspondence should be addressed.
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Reagents
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55 mM Iodoacetamide (1 mg/mL in 40mM NH4HCO3) |
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Sequence Grade Trypsin (Promega Corp., Fitchburg, WI) |
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C18 MicroSpin Column (The Nest Group, Inc., Southborough, MA) |
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Buffer A: 0.1% Formic Acid |
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Buffer B: 80% Acetonitrile, 0.1% Formic Acid |
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Instruments
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Obritrap (Thermo Fisher Scientific Inc., Waltham, MA) |
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Tryptic digestion of glycoprotein mixture |
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Measure the volume of each sample with pipette. |
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Add sufficient amount of 40 mM NH4HCO3 to adjust the pH value (the pH should be approximately 7–7.5) and bring up sample volume to 200 μL. |
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Add 1 M DTT at a ratio of 100:1 (ex. 200 μL sample - 2 μL 1 M DTT) making final concentration ~10 mM DTT. |
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Incubate sample at 56°C for 1 h. |
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Let it cool to room temperature (5 min). |
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Add 100 mL of 55 mM iodoacetamide (C2H4INO, IA, 10 mg/mL in 40 mM NH4HCO3). |
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Incubate at room temperature in dark for 45 min, vortexing every 15 min. |
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Resuspend 20 μg trypsin in 100 μL of 40 mM NH4HCO3. |
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Add 50 μL of trypsin to each (10 μg trypsin, 1:50 to 1:10 ratio). |
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Incubate overnight at 37°C (14–16 h). |
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Add 150 μL 1 % TFA to stop reaction. |
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Reverse phase clean-up of glycoprotein tryptic digest |
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Add 250 μL of buffer B to column. Spin at 2,000 rpm for 4 min. Discard flow through. |
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Add 250 μL of buffer A to column and spin at 2,000 rpm for 4 min. Discard flow through. (Repeat ×2) (3× total) |
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Add 250 μL of sample, spin at 2,000 × g for 4 min. Place flow through back into column & spin again. Discard flow through. Repeat for any remaining sample volume. |
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Wash column 2× with 250 μL buffer A. Discard flow through. |
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Add 150 μL of buffer B to elute. Spin at 2,000 rpm for 4 min. Retain flow through containing eluted glycopeptides. Repeat & combine flow through. Final volume ~300 μL. |
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β-elimination/Michael addition of glycopeptides with ammonium hydroxide (see Note 1) |
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Add 500 μL of ammonium hydroxide to dried glycopeptide/peptide mixture. |
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Add 500 μL of 18 megaOhm purified water to sample. Repeat 2×. |
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Analyze glycopeptide/peptide mixture by LC-MS/MS using Orbitrap (expected shift of -1 daltons for modified Ser/Thr residues). |
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| Notes | 1. Alternatively, especially if one wishes to enrich their peptides following beta-elimination/conjugate addition or introduce isotopes for comparative proteomics, one can use the BEMAD protocol described in References 2 and 3. |
| Copyrights |
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This work is released underCreative Commons licenses
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| Date of registration：2015-02-18 16:03:55 |
- Rademaker, G. J., Pergantis, S. A., Blok-Tip, L., Langridge, J. I., Kleen, A., and Thomas-Oates, J. E. (1998) Mass spectrometric determination of the sites of O-glycan attachment with low picomolar sensitivity. Anal Biochem 257, 149-160 [PMID : 9514784]
- Wells, L., Vosseller, K., Cole, R. N., Cronshaw, J. M., Matunis, M. J., and Hart, G. W. (2002) Mapping sites of O-GlcNAc modification using affinity tags for serine and threonine post-translational modifications. Mol Cell Proteomics 1, 791-804 [PMID : 12438562]
- Vosseler, K., Hansen, K. C., Chalkley, R. J., Trinidad, J. C., Wells, L., Hart, G. W., and Burlingame, A. L. (2005) Quantitative analysis of both protein expression and serine / threonine post-translational modifications through stable isotope labeling with dithiothreitol. Proteomics 5, 388-398 [PMID : 15648052]
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Wells, Lance,
(2015).
Methods for mapping sites of O-linked glycosylation.
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Wells, Lance,
(2015).
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