In terms of quantification, how do you know your peptide will be ionised efficiently.

BMS4137 formative and summative assessment – further guidance This document is designed to provide you with further guidance on writing your reports on “The use of LC-MS for the routine quantitative measurement of serum protein hormones in the clinical laboratory”. This guidance is largely based on the feedback I have provided for the formative version that many of you have submitted. General guidance 1. Read the question. The question asks specifically about routine quantitative measurement of serum protein hormones. You need to focus your report on the four keywords highlighted. Therefore, do not write about hormones that are not proteins 2. Read the question The question asks specifically about routine quantitative measurement of serum protein hormones. Therefore, do not write about the use of LC-MS to characterise protein structure. 3. Read the question The question asks specifically about routine quantitative measurement of serum protein hormones. Serum contains vast amounts of many hundreds of different proteins. Therefore, measurement of a specific protein from serum itself presents an obvious and significant challenge which must be acknowledged in your reports. 4. Be precise in your writing A number of you mention in your reports that LC-MS is very reliable or sensitive. These are very specific terms but you have used them in a very non-specific way to indicate that LC-MS is in some way “good”. A sensitive method is generally one where the limits of quantification are relatively low. In other words, a method is able to quantify low concentrations of a molecule relative to other methods. This is not generally the case for measuring protein hormones in serum. However, if you wish to assert that this is the case then you must provide some evidence to support this assertion. A reliable method is one which produces consistent results at a useful range of concentration. This is related to the precision of the assay. Again, if you wish to assert this then you must provide some evidence to support this. Challenge 1 and 2 – isoforms 1. Protein hormones may exist in many isoforms. Remember, there are many different types of isoforms. These may be due to post-translational modification, may be the result of alternative splice variants at the gene level or they may be dimers, trimers etc. 2. You should think about the implications of this range of isoforms. Remember, the point is to use LC-MS for the routine quantitative measurement of protein hormones in serum. Therefore, it is not relevant to point out that LC-MS may be able to detect different isoforms. The implication of these isoforms are many. You should bear in mind that these hormones are being measured in order to aid diagnosis and treatment or to inform prognosis. This raises a number of questions. For example, is detection of lots of isoforms likely to help diagnosis etc.? Which isoforms are bioactive and contribute to normal endocrine function? You will need to search the literature for relevant examples Challenge 3 – Enzymatic cleavage 1. Why is it done? It would be really useful if you explain why enzymatic cleavage is required for protein measurement. This related to their molecular weights. 2. What is done with the resulting peptides? You then need to identify an signature peptide which you will use for quantification. There are several features of this peptide which you should consider to allow it to be used as a measure of protein concentration. See link below for further information: https://www.labome.com/method/Quantitative-Bioanalysis-of-Proteins-by-MassSpectrometry.html 3. You may also consider the efficiency of enzymatic cleavage. Remember this is about quantitative measurement of protein hormones in serum. Serum contains many hundreds/thousands of proteins. This may well affect the efficiency 4. In terms of quantification, how do you know your peptide will be ionised efficiently. This may mean an internal standard is needed. How does this work? You will need to search the literature for relevant information Challenge 4 – extraction A major issue, as I mentioned several times already, is that you are trying to quantify levels of a single protein (hormone) in a mixture of many hundreds/thousands of other proteins. This is why an extraction or other type of sample preparation is used. You must consider then how you would try to separate your protein of interest from all these others. You may find some useful information about this in the attachment MS peptides extraction.pdf Challenge 5 – Clinical requirements A number of you have stressed the need for rapid turnaround of results. This is indeed an important consideration. However, there are other aspects to think about. This question relates to a number of issues that you should have already considered. These hormones are being measured in order to aid diagnosis and treatment or to inform prognosis. However, it is done, the methodology should enable this to happen and there are a number of aspects of LC-MS that make this problematic.. You will need to search the literature for relevant information