There are infact two reasons:

The first is indeed that the glycosylation is hetrogenous and the mass can change dramatically by several thousand daltons because of the extent of protein glycosylation variou s so much - MALDI mass spec is very good at resolving glycoforms of several glyoproteins.

The second is the physico-chemistry of SDS-PAGE. The role of SDS is to denature a protein into as tight a ball or spheroid possible so they are all approximately the same shape and importantly swamp the surface so they all have the same charge to mass ratio. Thus when the electrical current is applied they all move at the same speed (i.e same mass to charge ratio) and are resolved according to their overall size which should be proportional to mass as they all have the same shape (e.g. are either big balls or little balls of proteins). The trouble is that the sugar side chains are huge and don't interct with SDS in the same way as amino-acids in a peptide chain. As a result glycoproteins are not perfectly rolled up into tight balls, and the terminal sugars are sometimes charged and thus alter the perfect matching of charge to match ratio fundemental to SDS-PAGE electrophoresis.

The result of these combined effects are often a huge smear on SDS-PAGE.

There are infact two reasons:

The first is indeed that the glycosylation is hetrogenous and the mass can change dramatically by several thousand daltons because of the extent of protein glycosylation variou s so much - MALDI mass spec is very good at resolving glycoforms of several glyoproteins.

The second is the physico-chemistry of SDS-PAGE. The role of SDS is to denature a protein into as tight a ball or spheroid possible so they are all approximately the same shape and importantly swamp the surface so they all have the same charge to mass ratio. Thus when the electrical current is applied they all move at the same speed (i.e same mass to charge ratio) and are resolved according to their overall size which should be proportional to mass as they all have the same shape (e.g. are either big balls or little balls of proteins). The trouble is that the sugar side chains are huge and don't interct with SDS in the same way as amino-acids in a peptide chain. As a result glycoproteins are not perfectly rolled up into tight balls, and the terminal sugars are sometimes charged and thus alter the perfect matching of charge to match ratio fundemental to SDS-PAGE electrophoresis.

The result of these combined effects are often a huge smear on SDS-PAGE.

I completely agree with Ray's answer. Still, there is a possibility that the separation is affected by the density of the gel. Too dense gels will cause greater smear than the less concentrated ones. The separation conditions should be optimized in each case.

Yes, it is due to heterogeneity in glycosylation. By the way, not all glycoproteins produce a smear on SDS-PAGE. If glycosylation is relatively consistent (as in the case of RNAse B, to cite one example) the protein will produce a single band on SDS-PAGE, albeit at an altered mr -as explained by Ray above.

To add my two cents to those of Ray and Alejandro:  The smear is a result of negatively charged sugars (sialic acid, aka neuraminic acid) that are added when glycoproteins are destined for secretion, and/or phosphates in the form of mannose-6-phosphate on lysosomal glycoproteins/enzymes that escaped or secreted in an overexpression system.  These charged groups resist or interfare with the SDS effects mentioned by Ray, and thus cause a retarded or delayed migration toward the anode (+ terminal) thus appear to be "bigger".  The plasma glycoprotein transferrin is a good example for that, and treatment of transferrin with neuraminidase will reduce or eliminate the smear and the remaining oligosaccharide mass will still follow the size scale in SDS-PAGE as in MS.

Ray,

would you know a reference that explains why proteins that have been unfolded by SDS would assume a spherical shape? I find this quite interesting and a bit counterintuitive.

Dear Achim - this is an interesting comment and you will find most "classic" text books show a diagrammatic protein (a convoluted 2D circle squiggle) being denatured into a linear peptide. Well this is a two dimensional explanation model not the reality of three dimensional space. Like why planets are circular and we don’t have, in fact, a flat earth. Thermodynamically the most stable minimal energy state in 3D space is a circle or spheroid (think soap bubbles and water droplets). Now in reality proteins are held in non- spherical shapes (even straight lines) with all kinds of protrusion because of the various polar and non-polar and charge interactions. Indeed, as the thermodynamics of the charges and polarities of the interacting amino acids alter with environmental pH and salinity, so cysteine bonds stabilise the protrusion etc once a protein is shaped (and why reducing with beta-mercaptoethanol of DTT gives better SDS PAGE results). As explained, SDS brings all these down to a common charge and polar state; virtually throughout the peptide chain. Thus, we reason what is the most thermodynamic shape in 3D space if there are no other significant influencing forces? A spheroid - might not be perfect and a bit Rod like, but stil spherical.

PS RNA and even DNA won’t drift around as linear entities. In your DNA extraction the purified DNA supercoils and its biological shapes (sequence exposure etc) is due to interactions with other structures holding the required shape; RNA bends back on itself to find complementary base pairs which are thermodynamically favourable.

Another thing to consider is the effect of glycosylation on protein staining. Coomassie blue in particular, is affected by glycosylation and may reduce the sensitivity to the point that you see a 'negative' stain around the glycoprotein band in a gel. Glycosidases can help with heterogeneity (and staining). Sigma have a good primer for this at http://tinyurl.com/no46mqy (and I have no affiliations with Sigma).

After decades out of lab science, I now write biographies.

Speculation that I published years ago:

The synthesis of Protein, DNA and RNA are directed by a template, but sugar polymers are not, allowing for greater flexibility of synthesis--perhaps their synthesis reflects certain, local  specific conditions at the time of polymerization (pH, temp, availabilty of activated sugars, etc). Conditions change and heterogeneity results. Does the smear pattern change if cells in culture are grown under different conditions?--(or fish cultured at different temps). Perhaps patterns of heterogeneity respond-a response to  environmental conditions--somewhat Lamarckian.

Pretreatment with neuraminidase (and/or other glycosidases) should clean up a lot of the smear.

Removing the glycosylation improves the behaviour of many glycoproteins on SDS-PAGE but it is worth considering what biological role the carbohydrate might play. As Leonard notes, the addition of CHO follows different kinds of rules to the Central Dogma and there remains much unclear about why some glycosylation sites are 'active' and others are ignored. The biology of such glycosylation remains unclear in many (perhaps most) cases so it is important not to consider the attached CHO as a nuisance but to realise it may be important for function.

I agree with craig, but I would like to emphasize my unproven faith that a phenomenon like heterogeneity of CHOs is not  random and meaningless, and It might be worth digging in to, despite the disorder, interference with rational thought, and lack of aesthetics. In almost all cases we do not know what CHOs are really doing and beyond that we do not know what the "use" of heterogeneity is--but in fact it many help answer the first uncertainty.

Leonard's answer raises some very interesting questions. It seems likely that at least some of the glycosylation is biological meaningful in some way. That comprises two main areas: which residues are glycosylated (and there are quite a number of options) and which are not, and exactly what kind of glycosylation is added at a given site.

There seems to be reasonable evidence that certain sites are selected on the basis of 'context' but I'm not sure that the roles that govern this context are well understood. However, exactly what form the glycosylation takes is less well understood. Some additions are very specific (the glycosylation of antifreeze glycopeptides in both Antarctic and Arctic fish for example) but others seem to be less defined (some mucins for example).

Presumably there is some context to the addition of carbohydrate and presumably at least some of these additions have some biological significance (although in the case of mucins it might be more how much is added rather than exactly what).

All in all, an area that could benefit from some more work, perhaps not least in tools for effectively analysing the carbohydrate content of glycoproteins.

Try tricine PAGE to get rid of smear. It works best for the glycoproteins.

Bushra Bilal could you recommend a protocol or even a kit for Tricine PAGE

Here is an interesting paper to discuss Glyosylated gag in MLV. It clearly points out the residues that are involved in glyosylation, and the amount of shift/smear is directly proportional to the number of amino acids involved in binding CHOs.

After an absence from the lab of a quarter of a century, I can still be aroused to think about the intimate nature of biochemical processes. Is it possible that the smears we see, perhaps due to heterogeneity of the carbohydrate structure of glycoproteins, is a true reflection of the range of physical and chemical structures of a gycoprotein? Within the cell, the various sites on the protein and polypeptide components of a glycoprotein available to natural substances (Ions, drugs, dyes, other macromolecules etc) reflects a complementary set of heterogeneous binding sites revealed on glycoproteins in a gel? on the various molecules within the cell. Whatever the action of a glycoprotein, there are a range of molecules carrying out the process, but they may vary (perhaps measurable in the various physic chemical constants). Whatever the environment within the cell at any given moment some forms are better than others, allowing a cell to operate more effectively at the moment.  This putative ability to operate more effectively may be a kind of "preadaptation." Heterogeneity is probably the result of polymerization without the guidance of a template, and might be a mechanism to help the organism to adapt to a new set of conditions.

Hi Haitham 

here is the protocol for Tricine PAGE that I am using in my lab.

Another rambling thought: carbohydrate molecules attached to a polypeptide broaden the range of folded forms possible for the polymer (however slight the differences), and/or expand the degree,  nature, and range of interactions of the polymer with other molecules--polymers, drugs, metabolites, other cellular components, gel and buffer molecules etc. But what are the forces at work? If Tricine  eliminates variation, how and why is it different from a conventional buffering agent? Is the variation and its elimination purely an experimental artifact of no biological significance ? ..to be continued.

 The smear may be revealing a critically important property, a capability, that a glycoprotein molecule possesses in an aqueous environment, which the experimentalist dismises as a nuisance. [In fact, there is every reason to believe it is an experimental artefact, and very possibly is of no biological relevance.] Perhaps, in an electromagnetic environment in a gel (with its own structure), many many variations of a molecular species can form, each slightly different from its neighbor to form a molecular series--a smear, as the environment changes. However whatever activity the glycoprotein can carry out, the activity each form is capable of, varies from one to the next form. A selection of the most effective forms in a specific environment follows resulting in a living cell even as the environment changes.

Is this the contribution that accounts for the essentiality of protein-bound carbohydrates?

It is a very complicated process, but isn't the operation of a cell impossibly complicated?