Chem 8157

Milan gave a very interesting and fascinating talk that was directly related to course material we have covered. I especially took interest in his work regarding carbohydrate arrays. He was able to develop a new method of synthesis on a solid phase to drastically reduce the amount of time required in synthesis. His group was able to turn weeks of carbohydrate synthesis into a 1 day synthesis AND assay time. This is remarkable and will drastically improve the throughput when studying substrate specificity of enzymes through the use of carbohydrates.

Today's seminar speaker was Dr. Milan Mrksich from the Howard Hughes Medical Institute in Chicago. His group is developing a technique called SAMDI, which links self-assembled monolayers with MALDI mass spectrometry. A variety of molecules can be immobilized on alkane thiols which self-assemble in an ordered monolayer on a gold surface. Chemical reactions, affinity or binding arrays, or even kinetic studies can then be performed on the immobilized ligands and MALDI can be used as a label-free method of detection to classify products on the array. This technique is highly relevant to bioanalytical chemistry because it can be used to study bio-interactions between enzymes and substrates, inhibition, and binding between proteins and carbohydrates. The advantages of this technique are that it is not necessary to use labels as MALDI should provide information to identify structure, that enzymes are more likely to retain their function, and that a large number of different substrates can be tested for affinity or activity at the same time.

I thought the presentation itself was very well organized and easy to follow - Dr. Mrksich did a very good job of addressing the appropriate audience. One criticism I had is that this method could be called quantitative - while MALDI signal intensity does correspond to the concentration of analyte, I'm not sure that the extent of binding of any given substrate or ligand to the alkane-thiol monolayer can be characterized precisely enough for quantification.

Milan Mrksich's discussion on designing surfaces to probe biological problems was very interesting and seemed very potent to solve many problems. I feel that this work should theoretically be able to monitor many various reactions and processes from kinase activity, phosphorylase activity, farnesylation, etc. Showing that this technology is very efficient in terms of time and yield only enhances its potential. The huge number of probes that could be screened for with this method makes it very attractive. Using a gold slide and alkane thiols with various ligands attached, numerous potential 'surfaces' can be made to react with a cornicopia of compounds. After screening various compounds for a reaction, the products can be observed by mass spectroscopy. When struck with a laser, the monolayers can disattach from the gold slide and the molecular weight of the formed compounds can be measured to the molecular weight of the initial compounds. Since the user should know what they are trying to bind/react, the theoretical molecular weight should also be known and can be searched for in the products. A couple of things I would have liked to seen were L.O.D.s for the mass spec work done on the carbohydrate addition. Some of the labeled minor peaks appeared very similar to the baseline peaks which were unlabeled. Spectroscopic confirmation of the products vs. reactants would also have been good to have.

I have to agree with both Josh and Greg that this morning's seminar speaker Dr. Milan Mrksich provided a very interesting talk about the new developments in his lab. The ability to do synthesis of small molecules in such a short amount of time directly on the surface of the self-assembled monolayers is really quite impressive. The coupling of this fast synthesis to the development of microarrays for a variety of small molecules and then using SAMDI to characterize the surface provides the bioanalytical researcher with a powerful new tool for interrogating biointeractions. I also agree with Chad in the fact that more information obtained via spectroscopy would go a long way to confirming the usefullness of this tool

The seminar presented by Milan was wonderful in both style and content. He seemed to have a talent for addressing an audience and presenting the truly pertinent information. I agree with Chad in that I would like to have seen some spectroscopic data to support his conclusions from mass spectrometry. His method for building probes directly on the slide surface was ingenious and I am curious as to how far he can extend the probes before they either lose reactivity to the enzyme or lose the ability to extend the probe further. Also, to what extent can relatively hydrophobic probes, such as protein sequences, be attached to the surface before they are solvated by the poly(ethylene glycol) background that was used to prevent nonspecific adsorption of enzymes to the slide surface?

I agree with Greg on whether the method is quantitative, since the intensity of MS signal relies on to many factors. Bulk analytes such as proteins and carbohydrate may not be ionized efficiently; and the bio-molecules may decompose as time goes by. These factors are inevitable and hardly quantified. So in my opinion this method is qualitative.

Reply to Eric’s question, I read the Milan’s paper about the detection of protein-protein interactions on biochip. They use 1:20 (the maleimide groups to the tri(ethylene glycol) groups ratio) for the self-assembly monolayer preparation. I think they must have tried different ratio for SAMs preparation. The 1:20 ratio might be the optimized condition for preventing nonspecific binding and getting the protein attachment.

Reference: W. S. Yeo, D. H. Min , R. W. Hsieh, G. L. Greene and M. Mrksich, Angew. Chem. Int. Ed., 2005, 44(34)., 5480-5483.

Dr. Milan Mrksich from Chemistry Department in University Chicago gave a very good presentation today. The main goal is to design BioChip Arrays to determine the interaction between biomolecules and monitor the reactions and processes. The challenges are non-specific interaction, inconsistent activity of immobilized protein and labels restrict assay types. Mrksich group uses self-assembled monolayers to design and synthesis the surface which can immobilize protein, providing specific interaction. The mass spectrometry of the array shows that it is label free. That is to say, it is not necessary to design a specific label to determine a certain interaction, which is good for finding new interaction. This method also well illustrates the interaction between two proteins and can be used to do kinetic studies. I agree with Greg. The future work could be combining this method with spectroscopic techniques and they can go further step for quantitative analysis.

One area of Mrksich's research that I find interesting is the carbohydrate work. I think partly because there are so many unknowns in carbohydrate chemistry and partly because Nicola Pohl's seminar also peaked my interest in the area. Mrksich's bio-chips with carbohydrates are incredibly important because they are a part of PTM for proteins, and as we learned, understanding PTMs are critical for understanding protein function and/or behavior. As their results showed, the biochips with tethered sugars could bind peptides and we could observe, really quickly, which peptides interact with a particular sugar. One limitation of this work is that the peptide chains are not very long. That is, protein-saccharide interactions may be missed because the peptide may not accurately represent the protein.

The seminar presented by professor Milan Mrksich is frontier in terms of surface chemistry study. It is very interesting to find new reaction mechanisms on the surface which can reduce the time needed in synthesis of carbohydrate and potentially other organic compounds. Also it’s a useful tool to either trace the reactions step by step with out labels or study the orientation of proteins on surface. From biotechnology point of view, the screening program on enzyme activity attracts me the most. Experiment with immobilized proteins on different spots on substrate can be tested in present of enzyme and small molecules (acting either as an inhibitor or activator). Inhibitors can easily be identified after incubation because the intact proteins will give known SAMDI signals. This is promising in drug design, metabolism process study and so on. Advantages of this assay are high sensitivity and low DL (one property of MALDI) and easy sample treatment (as it’s much easier to just wash away nonspecific bonding on surface than do purification and separation in solution). However, there are also drawbacks in terms of long time consume (since every spot should be screened separately) and it costs a lot to do so many MALDI trials.

I agree with Melissa! It is pretty rad that Dr. Mrksich works extensively with carbohydrates, as this field seems to have much yet to discover and understand. I find it particularly interesting that he is using many analytical techniques, including biochips to be analyzed by mass spectrometry, yet he is not working selectively with proteins are nucleic acids. I find it pretty innovative that instead of tethering an oligonucleotide or peptide chain, he tethers a sugar. Interesting talk.

Oh, I forgot - what does the acronym SAMDI stand for? I am unsure, and would like to know!

Professor Mrksich gave an interesting talk about label-free bioassay. MALDI is used as the detection method which can not only avoid the label problem but also provide the information of the target molecule. Further more MALDI could be used to collect the binding molecule for further study.

But I think the method has some limitation too. First, the target need to immobilize on the chip. The 3-dimensional structure of the target may change and it may lose some of the interaction sites due to the steric hindrance. Second, before and after binding of a second layer, the surface environment is differenct. So the desorption intensities of the surface molecule are different. So I think it is not so reliable to do a quantitative detecion too.

The professor presenting was Dr. Milan Marksich who hails from the Howard Hughes Medical Institute in Chicago. I was not able to attend the seminar topic because of conflict of scheduling, but from reading his biography I can tell that Dr. Markisch and his group is interested in studying self-assembled monlayers in order to design and synthesize surfaces having well-defined structures and properties. His group is doing this in ordert to study cell adhesion and migration patterns. Dr. Markish is also interested in studying electroactive substrates. In order to do this he wants to develop interfaces that can present ligands for selective interaction with proteins. Although I was not at the seminar, I know, from reading other comments, that Dr. Marksich spoke about carbohydrate arrays as well as the SAMDI method that can be coupled to MALDI in order to link self-assembled monlayers.

[Edited by blog owner]…It is very interesting that Dr. Milan Mirksich also focuses some of his labs energy on Drug Discovery. As a Medicinal Chemist, I was very interested to see that his lab develops small molecule drugs that act as targets against bio-warfare agents. Also, it is interesting that he uses a label free high throughput strategy. Any strategy that can eliminate the labeling step can save both time and money and this is very important in the pharmaceutical industry. He uses the bioanalytical technique MALDI-TOF mass spectrometry to formulate a monolayer substrate. Also, with this method so far Dr. Mrksich has identified lead compounds for anthrax. He is already in the process of mapping out the structure-activity relationships of these compounds and beginning developing preclinical candidates. I would very much be interested in reading more details about this research.

Following up on Li and Gregs’ posts, I was curious how Dr. Mrksich technique could be used for more of a quantitative assay. I think that right know he has a very interesting technique and his work with carbohydrate-protein interaction looks very promising. It will be interesting to see how this technique can be adapted to looking at other biological interactions. But getting back to my earlier comment it seemed that his technique is more of a qualitative assay, because the techniques looks at whether there is binding or not. He did present some inhibition study information, which was I thought was pretty good, but I was wondering if he could get even more quantitative information for his assay. Also I think his technique would be even more powerful if it could be used determine the mechanism of biological interactions especially for things like protein-protein interactions. But overall I was really impressed by the talk.

Following up on Li and Gregs’ posts, I was curious how Dr. Mrksich technique could be used for more of a quantitative assay. I think that right know he has a very interesting technique and his work with carbohydrate-protein interaction looks very promising. It will be interesting to see how this technique can be adapted to looking at other biological interactions. But getting back to my earlier comment it seemed that his technique is more of a qualitative assay, because the techniques looks at whether there is binding or not. He did present some inhibition study information, which was I thought was pretty good, but I was wondering if he could get even more quantitative information for his assay. Also I think his technique would be even more powerful if it could be used determine the mechanism of biological interactions especially for things like protein-protein interactions. But overall I was really impressed by the talk.

Reply to Courtney’s question, SAMDI is the acronym of self-assembled monolayers for MALDI. Dr. Mrkisch's group developed this method for the detection of several types of biomolecular interactions. In their publications, they always use SAMDI to stand for this technique.

In response to Courtney's question about SAMDI, I was perusing on of Professor Mrksich's papers and found that SAMDI-TOF mass spectrometry stands for
self-assembled monolayers for matrix-assisted laser desorption
ionization time-of-flight mass spectrometry.
The paper reference in case your interested is:
Marin, V. L. et. al. Angew. Chem. Int. Ed. 2007, 46, 8796–8798

Due to the conflict of the schedule, I cannot attend Professor Mrksich's seminar. But from the paper of Professor Mrksich, I know one of his research is about SAMDI which applys self assembled monolayer to modify the physical property of the surface and identify proteins, polymers and other molecules.
I agree with Jon's opinion. This technique is more about qualitative. The intensity of the m/z peak is related to the density of the analyte on the monolyare. But different molecules have different ionization efficiency. So we cannot get the amount of the analyte according to the intensity of the peak directly. Even though the peak of unfunctionalized alkanethiolate in the monolayer can be used to calibrate the intensity of the analyte peaks, it seems we can compare the amount of different analytes. It needs to set up an authentic standards and is time consuming. So it decreases the throughput of the SMDI method. I think it is also one of the limitations of this technique.