Patent Application
20110143386
The present disclosure pertains to the fields of biochemistry and analytical chemistry. More particularly, the present disclosure pertains to a method for analysis of oligosaccharides.
O-glycosylation is a common post-translational modification of proteins. O-linked oligosaccharides play a significant role in development, immunity, infectious diseases and cancer. The functions of O-linked oligosaccharides vary from cell-cell recognition to protein-protein interaction. Many studies of O-linked oligosaccharides have been performed using antibody analysis and nuclear magnetic resonance (NMR). The high sensitivity and scalability of matrix assisted laser desorption ionization time of flight (MALDI-TOF) mass spectroscopy make it an attractive option for the analysis of O-glycans; however, has been limited by sample preparation protocols.
The existing techniques for the analysis of O-glycans rely on microgram amounts of starting material, and they are not feasible in all situations given sample limitations. Therefore, it would be advantageous to develop a technique utilizing sub-microgram levels of glycoproteins which is both simple and reproducible.
Another caveat of the existing chemical cleavage protocols is the associated ‘peeling reactions’ that occur. This further complicates the analysis of simple dimeric and trimeric oligosaccharides, as they could represent a simple structure on the glycoprotein or be the result of a peeling reaction on a larger sugar. The ability to limit or eliminate these reactions will greatly reduce the ambiguity associated with analyzing O-linked oligosaccharides.
Another problem associated with existing chemical cleavage protocols is the tendency for large glycoproteins to mask or bury small O-linked oligosaccharides. We encountered this problem while analyzing human IgA, in which a dimeric oligosaccharide, HexNAc1Hex1, was not observed following a standard chemical cleavage protocol. After digesting the glycoprotein with trypsin, followed by the chemical cleavage protocol, we were able to cleave the dimeric oligosaccharide. Thus, the previously existing techniques for oligosaccharide analysis are found to have disadvantages as described above. We disclose herein a new glycomics technique for analysis of O-linked oligosaccharides with improved sensitivity, reproducibility, and an ability to accommodate smaller sample sizes.
A method for analyzing oligosaccharides comprises one or more of the following features or combinations thereof:
According to one illustrative embodiment of the invention, a new glycomics technique is utilized for O-glycan analysis. The technique is a combination of non-specific proteolysis using PRONASE, in combination with solid-phase permethylation, which results in free, permethylated and non-reduced O-linked oligosaccharides.
Glycoproteins are digested with PRONASE at a high ratio of enzyme to protein with a 48 hour reaction time. Samples are then dried and subjected to solid-phase permethylation followed by MALDI analysis. This technique provides a sensitive and reproducible method for glycomic analysis of O-linked oligosaccharides.
In another illustrative embodiment of the invention, the method can be applied for analysis of N-glycans.
While the invention is susceptible to various modifications and alternative forms, specific embodiments will herein be described in detail. It should be understood, however, that there is no intent to limit the invention to the particular forms described, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention.
In accordance with one embodiment of the invention, a method of analyzing O-linked oligosaccharides is provided. The method comprises the steps of digesting a glycoprotein with a proteolytic enzyme, performing solid-phase permethylation of the oligosaccharide, and analyzing the permethylated and non-reduced O-linked oligosaccharides using MALDI-TOF mass spectrometry.
Chemicals and Materials. Sodium hydroxide, 20-40 mesh beads, 97%, iodomethane (including isotopic versions), 2,5-dihydroxybenzoic acid (DHB) and acetonitrile were acquired from Aldrich (Milwaukee, Wis.). Chloroform and dimethylsulfoxide (DMSO) were obtained from EM Science (Gibbstown, N.J.). Borane-ammonia complex, proteomics-grade trypsin, bovine serum fetuin, human IgA and 28% aqueous ammonium hydroxide were acquired from Sigma Co. (St. Louis, Mo.). PRONASE was obtained from Roche Applied Science (Mannheim, Germany).
The bile-salt-stimulated lipase from human milk was obtained from the Department of Clinical Chemistry, University Hospital, Linkoping, Sweden.
Digestion with PRONASE. The glycoproteins were dissolved in water to a final concentration of 2 mg/ml and PRONASE was added to a final concentration of 0.2 mg/ml. Reaction mixture was then incubated at 55° C. for 48 hours unless otherwise described.
Digestion with Trypsin. The glycoproteins were dissolved in water to a final concentration of 2 mg/ml and trypsin was added to a final ratio of 20:1 protein to enzyme. Reaction mixture was then incubated at 37° C. overnight.
β-Elimination of O-linked Oligosaccharides. Glycoproteins digested using trypsin were subjected to a modified β-elimination protocol. The peptides were first dried using a speed-vac and then had a small volume of 5 mg/ml ammonia borane complex in 28% ammonium hydroxide added to each sample to final concentration of 1 μl per 1 μg of protein. Samples were then incubated for 24 hours at 60° C. After allowing the samples to cool, 1M HCl was added to destroy any residual ammonia borane complex. Finally, the samples were dried and washed three times using methanol prior to solid-phase permethylation.
Permethylation. Solid-phase permethylation was performed using the spin-column technique developed in the lab. Samples were first dissolved in 90 μl of DMSO, 2.7 μl of water and 35 μl of iodomethane. Samples were then passed over a spin-column packed with sodium hydroxide mesh beads a total of eight times. The columns were then washed once with acetonitrile. Chloroform (400 μl) was then added to the samples followed by three 1 ml extractions using 500 mM NaCl. The chloroform layer was saved and dried using a speed-vac and the extracted material was resolubilized using 4 μl of a 50:50 water/methanol mix.
MALDI-TOF Analysis. The permethylated samples were spotted using equal volumes of the sample and a DHB matrix containing 10 mg/ml DHB and 1 mM sodium actetate. Mass spectra were then acquired using an Applied Biosystems 4800 Proteomic Analyzer (Applied Biosystems Inc., Framingham, Mass.). Mass spectra were acquired in positive ion mode.
Permethylation of Intact Proteins. We created a new method for the analysis of O-linked oligosaccharides which is an improvement over existing methodologies. High molecular weight proteins have the ability to bury small O-linked oligosaccharides making chemical cleavage procedures inefficient. In the absence of an enzymatic means of cleaving O-linked oligosaccharides these chemical procedures need to be more efficacious to ensure quality data is being collected. The existing chemical cleavage procedures are flawed by another undesirable result, the ability to initiate ‘peeling reactions’. These reactions take place when oligosaccharides undergo cleavages to bonds other than the protein-oligosaccharide link, and the products of these reactions further obfuscate data interpretation. Solid-phase permethylation confers added stability to both N- and O-linked oligosaccharides while improving sensitivity of glycans for MS analysis. The permethylation reaction conditions occur in a highly basic milieu which may have the ability to hydrolyze the linkage between the protein and oligosaccharide. In order to test the ability of the permethylation conditions to release O-linked oligosaccharides, we first began with permethylating intact proteins.
A reaction mechanism has been proposed for the cleavage of O-linked oligosaccharides from the serine or threonine residue (
Digestion of Glycoproteins. We improved the ability of the permethylation reaction to cleave O-linked oligosaccharides from glycoproteins. To overcome problems associated with steric interference we used a non-specific protease, PRONASE. Previous work using PRONASE on glycoproteins focused on N-linked oligosaccharides from either standard proteins or bacteria. The study also relied on an alternative method of permethylation which occurs under anhydrous conditions. For the digestion, we utilized ratios of PRONASE to glycoproteins in the 1:1 to 1:10 PRONASE/protein range. For the digestion, we incubated the samples at 55° C. for 24 to 48 hours. After digestion the samples were dried and subjected to solid-phase permethylation using the spin column technique. As seen in
The O-glycans with the lowest relative intensities did have a much higher RSD then glycans with greater relative intensities, a statistical issue that is difficult to resolve. We have demonstrated that this technique is both robust and reliable when compared to previously published methods. To investigate the role of the PRONASE digestion, we performed a time course experiment using calf serum fetuin. Samples were incubated for either 24 or 48 hours in the presence of PRONASE and then dried to completion. After solid phase permethylation using the C-GlycoMAP method, we were able to directly compare the results from the two time points. The mass sepectrum, seen in
C-GlycoMAP Comparison of O-Glycan Cleavage Techniques. We performed a direct comparison of the β-elimination technique and the PRONASE/permethylation technique of O-glycan cleavage using C-GlycoMAP. To facilitate this comparison we utilized equal masses of both calf serum fetuin and human IgA and subjected them to three different protocols. The first sets of proteins subjected to the PRONASE/permethylation protocol were permethylated using the CH3I methyl iodide. The second sets of proteins were subjected to the previously described method of β-elimination using ammonia-borane complex and the resulting sugars were then permethylated using CHD2I methyl iodide. The final sets of proteins were subjected to a modified method of the β-elimination technique in which the proteins were first digested using trypsin and then subjected to the ammonia-borane complex. These final sets of O-linked oligosaccharides were then permethylated using CD3I methyl iodide. These samples were then pooled and analyzed together using MALDI. As seen in
Profiling O-glycans on a Complex Sample. In order to demonstrate the potential of this technique we analyzed the O-linked oligosaccharides on a complex protein, bile-salt-stimulated lipase (BSSL) from human milk. This enzyme is a 100 kDa protein that is known for having a region in its C-terminus which contains many serine and threonine residues resulting in a complex web of O-glycosylation. As seen in
The current methods that exist for the analysis of O-linked oligosaccharides have left much room for improvement. In the absence of a consistent enzymatic release of O-linked oligosaccharides there is a need to develop techniques which mimic these results. We have developed a method of O-linked oligosaccharide cleavage that utilizes non-specific proteolysis in combination with solid-phase permethylation which results in free non-reduced O-glycans. This allows further processing of the samples to include separation using liquid chromatography or direct infusion for analysis using mass spectrometry. The result is an inexpensive, consistent and more sensitive technique which allows minimal processing of the samples. Furthermore, the technique does not increase the amount of time a sample is processed. This technique also eliminates the use of several potentially hazardous chemicals when compared to existing β-elimination techniques. This method provides the ability to perform analysis of O-linked oligosaccharides on samples previously thought to be impossible given sample size limitations or buffer related complications.
While the invention has been illustrated and described in detail in the foregoing description, such an illustration and description is to be considered as exemplary and not restrictive in character, it being understood that only the illustrative embodiments have been described and that all changes and modifications that come within the spirit of the invention are desired to be protected. Those of ordinary skill in the art may readily devise their own implementations that incorporate one or more of the features described herein, and thus fall within the spirit and scope of the present invention.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US09/53385 | 8/11/2009 | WO | 00 | 2/10/2011 |
| Number | Date | Country | |
|---|---|---|---|
| 61090081 | Aug 2008 | US |
