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- Feb 1, 2011
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Alright i am really tired of all the posts and emails i get from different sponsors that claim "receptor grade", "99.xxx purity" and a whole host of other useless claims. I am here to tell you that most of this is pure garbage. These last few weeks have been very eye opening. Most sponsors (and i mean owners of the companies) are completely clueless on testing methods or even how to read their own tests they post on their websites.
And i am here to tell you that not ONE sponsor is innocent of this. When i did the recent LR3 analysis i had a chance to discuss the testing methods and procedures with many of the sponsors and was shocked that most did not even know what should be the actual procedure for testing their peptide or protein. Even the manufacturer that sells them peptide provides them with whatever BS tests that mean nothing. So i am going to explain some tests and graphs you see and what they mean and what they dont mean.
This is meant to be strictly educational so you guys understand what you are looking at and dispel some myths and mis-information.
HPLC:
This is a method where a mixture of compounds are studied and analyzed and broken down to see how many different components it is made up of. This method does not identify the specific compound in hand at all. It does not give you the mass of the compound in daltons for you to cross match with what you are looking for. If you already have proof that you have something in that mixture it will show you purity but or not if there are several compounds are there. For example you can do an HPLC on a perfume to see how many different actual perfumes it is made up of etc. But for our purpose HPLC is pretty useless because it does not tell you anything useful. So when you see a sponsor post an HPLC of a so called product you need to be ware.
This is what a typical HPLC looks like. Notice the X axis is always in time and not weight so it does not tell you anything. The Y axis is the concentration but then again does not identify the product.
**broken link removed**
SDS-PAGE:
This is a sodium gel electrophoresis and used heavily in genetics, forensics and chemistry where proteins are separated based on their ELECTROPHORETIC mobility which is a function of their polypeptide chain length and its electric charge. This shows up as a band on the gel. This is pretty decent in identifying if a certain protein exists at a certain weight range we are looking for. For example if we know that IGF LR3 has a 9111 dalton weight we should expect a thick heavy band in the 9000 range. The major issues with this procedure is that it is very limited and not very accurate down to the dalton weight. You may see a band in the ~9000 range but you dont know if it is 8800 or 9200 etc. And this is a huge issue when you are actually identifying a specific product. It also DOES NOT tell you how much total of a compound you have to begin with.
This what a typical SDS-PAGE looks like. You can see bands showing up but there is nothing specific or accurate about it. For example, look at column 2. We can see a thick band showing up between 50-75kda but where does it actually fall? It is 58K or 63K? Who the hell knows. You also see lighter shadows around the thick band but doesnt tell you how bad the other impurities are.
MASS SPEC:
Mass spectrometry is where singular spectrum of the masses of the molecules comprising a sample of material are displayed. It really doesnt get much more accurate than this. It is basically used to determine the elemental composition of a sample based on the molecules mass weight. This will tell you not only if you have a pure sample but what the actual mass weight of the sample is and if there are other molecules that weight more or less than what you are looking for. Once again this will not tell you what is the total weight of your sample (as in total product weight). What i mean is that if i turn over a vial of IGF LR3 to the lab and they do a mass spec and it comes back with a perfect spike at 9111 daltons. All it means is that there was LR3 in there. It wont tell me if there was 0.1mg or 1mg or 10mg of LR3 in that sample. Hope that makes sense.
This is what a typical MALDI TOF mass spec looks like. You can see peaks at the different exact mass where the compounds were detected. There is no guess work.
**broken link removed**
So we have discussed the potential ways to identify a compound with a certain mass weight and discussed how mass spectrometry is the best method. But none of the above have verified what we actually have short of what is the mass weight of the molecule. Let me explain further. Since we have discussed IGF LR3 we will just stick with that. We know it has a mass weight of 9111 daltons. I can piece meal together enough crap protein in a chain in a lab to give you A SAMPLE that has a mass spec that will show up in the 9100 range. But does that mean just because you have a sample that weights close to 9111 that it is actually IGF LR3? HELL NO.
The only way is to do a full amino acid analysis. There is a two fold reason for this. One it will show if the percentage of amnio acids found in the sample match the % of amino acids that SHOULD be in the LR3. Because we know from the sequence how many glutamaine, leucine, aspartamate etc are in one chain of LR3. So when you do an amino acid analysis those percentages should match up. For example, if your sample shows up with an amino acid that doesnt even exist in the LR3 chain you know something is screwy.
The second reason for the amino acid analysis is that it will tell you within 10micrograms what the total weight of the protein you are testing for in that vial. What is the point of having a pure IGF LR3 but it really is 0.1mg vs 1mg or 10mg.
AMINO ACID ANALYSIS NOT WILL CONFIRM AND BACK UP WHAT YOU FOUND IN THE MASS SPEC BUT ALSO TELL YOU THE TOTAL WEIGHT OF PRODUCT IN GIVEN SAMPLE.
This is what a typical amino acid analysis looks like. I have taken out the name of the sponsor because this is not meant to be an advertisement and strictly educational. But you can see that we have the different amino acids listed on the left and what the known percentages should be vs what is the calculated from the sample. There is always a deviation allowed up to 1.0-1.5% considered very acceptable. You can see in this report that at the bottom there was a total of 952micrograms of LR3 found which is 0.952mg that is as close as to the 1.0mg advertised on the vial as you can get. You can also see that there was a slight amount of Histidine found in this sample. If you look at the sequence of IGF LR3 there is no Histidine found anywhere so to find it means that a very small sample had Histidine attached that were not cleaved off during the purification process. This is why amino acid analysis is so important.
Conclusion:
So what do we learn from all the above? No to be a sheep and just swallow everything that is fed to you. The only way any sponsor can prove to you what they are selling as far as a peptide/protein of any sort is to show you a proper Mass Spec report WITH a full amino acid analysis. One without the other is worthless. Just showing you a mass spec is worthless. Showing you HPLC reports and SDS PAGE reports shows that they really have no idea what they are doing. Please pass this information on as this should be a must read for everyone researching. Good luck.
And i am here to tell you that not ONE sponsor is innocent of this. When i did the recent LR3 analysis i had a chance to discuss the testing methods and procedures with many of the sponsors and was shocked that most did not even know what should be the actual procedure for testing their peptide or protein. Even the manufacturer that sells them peptide provides them with whatever BS tests that mean nothing. So i am going to explain some tests and graphs you see and what they mean and what they dont mean.
This is meant to be strictly educational so you guys understand what you are looking at and dispel some myths and mis-information.
HPLC:
This is a method where a mixture of compounds are studied and analyzed and broken down to see how many different components it is made up of. This method does not identify the specific compound in hand at all. It does not give you the mass of the compound in daltons for you to cross match with what you are looking for. If you already have proof that you have something in that mixture it will show you purity but or not if there are several compounds are there. For example you can do an HPLC on a perfume to see how many different actual perfumes it is made up of etc. But for our purpose HPLC is pretty useless because it does not tell you anything useful. So when you see a sponsor post an HPLC of a so called product you need to be ware.
This is what a typical HPLC looks like. Notice the X axis is always in time and not weight so it does not tell you anything. The Y axis is the concentration but then again does not identify the product.
**broken link removed**
SDS-PAGE:
This is a sodium gel electrophoresis and used heavily in genetics, forensics and chemistry where proteins are separated based on their ELECTROPHORETIC mobility which is a function of their polypeptide chain length and its electric charge. This shows up as a band on the gel. This is pretty decent in identifying if a certain protein exists at a certain weight range we are looking for. For example if we know that IGF LR3 has a 9111 dalton weight we should expect a thick heavy band in the 9000 range. The major issues with this procedure is that it is very limited and not very accurate down to the dalton weight. You may see a band in the ~9000 range but you dont know if it is 8800 or 9200 etc. And this is a huge issue when you are actually identifying a specific product. It also DOES NOT tell you how much total of a compound you have to begin with.
This what a typical SDS-PAGE looks like. You can see bands showing up but there is nothing specific or accurate about it. For example, look at column 2. We can see a thick band showing up between 50-75kda but where does it actually fall? It is 58K or 63K? Who the hell knows. You also see lighter shadows around the thick band but doesnt tell you how bad the other impurities are.
MASS SPEC:
Mass spectrometry is where singular spectrum of the masses of the molecules comprising a sample of material are displayed. It really doesnt get much more accurate than this. It is basically used to determine the elemental composition of a sample based on the molecules mass weight. This will tell you not only if you have a pure sample but what the actual mass weight of the sample is and if there are other molecules that weight more or less than what you are looking for. Once again this will not tell you what is the total weight of your sample (as in total product weight). What i mean is that if i turn over a vial of IGF LR3 to the lab and they do a mass spec and it comes back with a perfect spike at 9111 daltons. All it means is that there was LR3 in there. It wont tell me if there was 0.1mg or 1mg or 10mg of LR3 in that sample. Hope that makes sense.
This is what a typical MALDI TOF mass spec looks like. You can see peaks at the different exact mass where the compounds were detected. There is no guess work.
**broken link removed**
So we have discussed the potential ways to identify a compound with a certain mass weight and discussed how mass spectrometry is the best method. But none of the above have verified what we actually have short of what is the mass weight of the molecule. Let me explain further. Since we have discussed IGF LR3 we will just stick with that. We know it has a mass weight of 9111 daltons. I can piece meal together enough crap protein in a chain in a lab to give you A SAMPLE that has a mass spec that will show up in the 9100 range. But does that mean just because you have a sample that weights close to 9111 that it is actually IGF LR3? HELL NO.
The only way is to do a full amino acid analysis. There is a two fold reason for this. One it will show if the percentage of amnio acids found in the sample match the % of amino acids that SHOULD be in the LR3. Because we know from the sequence how many glutamaine, leucine, aspartamate etc are in one chain of LR3. So when you do an amino acid analysis those percentages should match up. For example, if your sample shows up with an amino acid that doesnt even exist in the LR3 chain you know something is screwy.
The second reason for the amino acid analysis is that it will tell you within 10micrograms what the total weight of the protein you are testing for in that vial. What is the point of having a pure IGF LR3 but it really is 0.1mg vs 1mg or 10mg.
AMINO ACID ANALYSIS NOT WILL CONFIRM AND BACK UP WHAT YOU FOUND IN THE MASS SPEC BUT ALSO TELL YOU THE TOTAL WEIGHT OF PRODUCT IN GIVEN SAMPLE.
This is what a typical amino acid analysis looks like. I have taken out the name of the sponsor because this is not meant to be an advertisement and strictly educational. But you can see that we have the different amino acids listed on the left and what the known percentages should be vs what is the calculated from the sample. There is always a deviation allowed up to 1.0-1.5% considered very acceptable. You can see in this report that at the bottom there was a total of 952micrograms of LR3 found which is 0.952mg that is as close as to the 1.0mg advertised on the vial as you can get. You can also see that there was a slight amount of Histidine found in this sample. If you look at the sequence of IGF LR3 there is no Histidine found anywhere so to find it means that a very small sample had Histidine attached that were not cleaved off during the purification process. This is why amino acid analysis is so important.
Conclusion:
So what do we learn from all the above? No to be a sheep and just swallow everything that is fed to you. The only way any sponsor can prove to you what they are selling as far as a peptide/protein of any sort is to show you a proper Mass Spec report WITH a full amino acid analysis. One without the other is worthless. Just showing you a mass spec is worthless. Showing you HPLC reports and SDS PAGE reports shows that they really have no idea what they are doing. Please pass this information on as this should be a must read for everyone researching. Good luck.
