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I have found that many studies use the mRNA concentration as a “proxy” for protein activity because there should be correlation between mRNA levels and proteins expression levels. How is protein activity quantified? Which quantity is used? What are protein expression levels?

I am doing some statistical data analysis using this dataset. I would like to understand why they have been interested in measuring mRNA concentration after the cells gamma irradiation (and in general it seems to me that biologists in many experiments are interested in that). I found in this site that discussion about correlation between mRNA concentration (so gene expression level) and "protein expression level". Therefore I wanted to know what the latter is. Moreover, in the article related to my dataset is written

Very few DNA repair genes displayed significant differential expression in P. furiosus following gamma irradiation.

And then

the data we reported here suggest that DNA repair proteins in P. furiosus and several other archaea are constitutively expressed and that they may be present in the cell at a level sufficient to maintain the integrity of the cell’s genetic material.

So putting together what the scientists conclude with what MattDMo has answered to me and what is discussed here , I think that I can conclude that "protein expression level" is a measure of the amount of protein actually translated from the mRNA, that then (in this case) will effectively act to repair DNA. Therefore, "protein expression level", as well as "protein activity", indicates the proteins translated from mRNA and that will actively act to repair DNA damages (in this case that we are talking about DNA repair proteins and DNA repair genes).

I have to say that I do not really know well what "DNA repair proteins are constitutively expressed" means, but I think that is that DNA repair proteins have already been translated from the mRNA and therefore they are "present in the cell at a level sufficient to maintain the integrity of the cell’s genetic material" (as they say soon after).

All this makes sense to me if I think about my first objective that was to understand the reason for measuring mRNA concentration. We are interested in it because there should be correlation between mRNA concentration (gene expression level) and the proteins actually translated from mRNA from that specific gene, in order to know which kind of proteins will effectively act in the DNA repair processes (in this case).

I wonder if all this interpretation is correct...

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  • $\begingroup$ I have deleted my comments as they are now embodied in an answer. I have changed your title to be more general and for reasons I make evident in my answer. $\endgroup$ – David Sep 28 '20 at 11:40
  • $\begingroup$ I have now added a postscript addressing the specific circumstances of your work, which are of less general interest than main topic of my answer. (Good questions on answers on Stack Exchange should be of general interest, not a dialogue with the poster.) In summary, "all this interpretation" is not correct. Further, measuring the mRNA concentration is done because it reflects the extent to which a gene is expressed as mRNA. The topic of interest is gene expression — of which the amount of protein produced is just a part. And the term "protein activity" is meaningless and is not to be used. $\endgroup$ – David Sep 30 '20 at 11:37
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What is Protein Expression Level?

This was the original title of the post, which I edited myself because I regard the answer as trivial, but the question as more substantial. To deal with the trivial first:

‘Level’ is not a scientific unit, and can only be used unambiguously as a scientific term in its English sense in relation to liquids, e.g. “The level of mercury in the thermometer had fallen.”, “The land is 10 metres above sea level.”

It is used by some people in informal speech or writing to indicate an undefined quantitation, and because of its very ambiguity is to be strongly discouraged in scientific communication.

So neither I nor anyone else can say what ‘protein expression level’ is without finding out what the originator of the phrase intended (if indeed he knew) in any individual instance: quantity or concentration of protein, or the rate of its synthesis.

Quantitation

Let us consider some general aspects of a cellular molecule that may require quantitation: Amount, Rate of Synthesis, Rate of Degradation and, where relevant, Biological Activity.

The quantitation of molecules at its most basic is the number species, or, more practically, their total mass (grams), where possible related to their molecular mass (i.e. moles). The rate of their synthesis or degradation is expressed in terms of the change of their amount in unit time.

In order to compare different systems a reference is needed for the quantitation. This may be per unit volume, per cell, per g cellular protein, per g DNA etc. (However for comparison of similar systems or within one system the reference is often omitted.)

The biological activity of molecules only has meaning if the molecules are, indeed, biologically active (e.g. enzymes). It is expressed in units related to that activity.

Examples of Quantitation for Proteins

The actual units used in quantitation are determined by how one is able to measure the parameter of interest.

Relative amount of protein: You might detect a protein by the intensity of staining of a band on a gel, or by the extent of precipitation using a corresponding antibody. These would have to be calibrated against standards in order to convert raw experimental measurements to g or mole protein. Typical units of relative amount are g/g total protein, g/g DNA.

Rate of synthesis or degradation of protein. You might detect the synthesis of protein by the rate of incorporation of radioactive amino acids into non-radioactive protein, and the rate of degradation by the liberation of radioactive amino acids from pre-labelled protein. Typical units of synthesis would be mg amino acid incorporated per min per mg total protein. (This can be converted to molecules synthesized or degraded per minute, if necessary.)

‘Protein Activity’: The use of this term is not recommended. In chemical terms the activity of a molecule is a measure of its “effective concentration”, and in relation to proteins would only be the concern of biophysicists and the like. Other biologists would associate the term with the biological activity a protein such as an enzyme might have, but as many proteins are structural the term ‘activity’ cannot be applied to proteins generally. Where it is of interest it would be quantified in terms of the nature of the activity, e.g. an enzyme is quantified in units related to the amount of substrate converted to product in a given time.

Gene Expression

Quantitation of Gene Expression using Oligonucleotide Microarrays

The two extremes in quantifying gene expression are the detailed study of the expression of one particular gene encoding a protein for which information and tools are available; and general study of the expression of many genes using modern methods that allow the study of many molecules simultaneously. Such methods included mass spectrometry (for small metabolites), two-dimensional gel electrophoresis (for proteins) and microarrays or RNAseq (for mRNA). In general in the latter case one would examine the effect of some agent or condition on the whole spectrum of gene expression.

Let us examine microarray technology as that is the main concern of the poster.

Microarray technology measures the relative amounts of mRNA.

The methodology involves amplifying a mixture mRNA species by reverse transcription to cDNAs, which are labelling with a fluorescent dye. The cDNA is hybridized to immobilized oligonucleotides based on the sequences of the genes in the organism, and the strength of the fluorescence signal is assumed to be proportional to the amount of individual mRNA species in the sample. However the data supplied to the user is the relative image intensity within the experiment, rather than units that allow one to calculate the actual amount of mRNA.

How does these relative amounts of mRNA relate to gene expression?

  1. The relative amounts of mRNA are a relative measure of the rate of synthesis of proteins (3 in dig.), if one assumes that the rate of synthesis of each protein is similarly proportional to the amount of its mRNA (a in dig.) which would be limiting. This is a reasonable assumption in most cases given mRNA is degraded more rapidly than protein. (They are not a measure of the amount of protein — b in dig.)

  2. The relative amounts of mRNA cannot be taken as a measure of the rate of transcription of a mRNA from its gene (1 in dig.) because of the relatively greater effect of mRNA degradation (2 in dig.) on the steady state concentration of mRNAs, and the fact that different mRNAs have different half-lives.

Postscript for the Poster

I have tried to make this answer general, to be of use to more people. As the poster is not a biologist she may still have difficulty in understanding the background to the biological experiments that generated the data she is analysing.

The biological system of interest is gene expression, which encompasses the whole series of events from transcription of genes to mRNA and their translation into protein. The background to this is that there are some genes that are (almost) always expressed, irrespective of physiological circumstances because they are needed to maintain the structure and everyday functions of the cell. This is termed constitutive expression, examples being expression of the genes for cytoskeletal actins or ribosomal proteins. Other genes are expressed (‘switched on’) only when needed (and may be termed inducible). The question here would appear to be whether the genes for the enzymes involved in DNA repair are expressed all the time (constitutively) to deal with normal ‘wear and tear’ of DNA, or whether their expression only occurs in response to some insult knwn to damage DNA, such as gamma irradiation.

As I mentioned, as an experimental scientist, one approach that can be adopted here is to look at the expression of one or two well characterized proteins that are known to be involved in DNA repair. However, there may be proteins involved in this process that you are unaware of, so the modern methods of looking at the expression of all the genes in an organism (if the DNA sequence is known) — oligonucleotide microarrays or, better, RNASeq. These methods measure relative quantities of mRNA in a cell. This is not a proxy for amount of protein or rate of its synthesis (the term ‘protein activity’ is meaningless and is not and should not be used), it is what it is, but is also a reflection of the expression of the genes that encode the mRNAs. No expression, no mRNA.

You can think of the microarray and RNASeq approaches as a fishing expeditions. If you find mRNAs that are only present after the cell receives a stimulus or an insult, expression of the mRNA has occurred (whether or not the amounts are directly proportional to the rate of synthesis). If the mRNA is synthesised you can assume it is translated into the protein it encodes. In the case of gamma irradiation you might assume any mRNA that shows a large increase in quantity encodes a protein that involved in protecting the cell from the radiation. This will be of scientific interest, especially if it is not what one anticipated.

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  • $\begingroup$ I have a doubt about the relative amounts of mRNA: they are somehow the log10 ratio between the irradiated sample and the reference sample . So if the ratio is 0, there should be no changes in gene expression (because it is log10(1)=0); if it is greater than 0, it means that the gene expression (amount of mRNA) is increased after irradiation (up-regulation); finally, if it is lower than 0, it means that the mRNA production decreases (down-regulated gene) ? $\endgroup$ – Manuela Oct 11 '20 at 19:15
  • $\begingroup$ This is what I have deduced by reading the Wikipedia explanation (indicated by you) and the comments about RadA in fig1a of the article . I think that if I have understood well this case with not irradiated samples and irradiated, I can extend this reasoning to other cases like unhealthy and healthy cells cultures. $\endgroup$ – Manuela Oct 11 '20 at 19:32

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