If you wonder how to make the most out of IsoCor, have a look at the Tutorials.

## Wait, I needed to correct something?¶

If you don’t fully understand why you should correct your MS isotopic data, we invite you to have a look at Midani et al. 2017 excellent review.

Finally, practical examples are provided in the Tutorials.

## What are the alternatives to IsoCor?¶

You will find scripts for correction of HRMS data out there, but we won’t recommend any since, to the best of our knowledge, they implement algorithms that partly fail for high-resolution datasets (see IsoCor v2 publication).

Tool Has GUI? MS resolution? Tracers? Reference Comment
AccuCor No All 2H, 13C, 15N Su et al. 2017 Faulty at High-resolution
PyNAC No UltraHigh only 2H, 13C, 15N Carreer et al. 2013 none
IsoCorrectoR No Low & UltraHigh only All Heinrich et al. 2018 none
ElemCor Yes All 2H, 13C, 15N, 18O, 34S Du et al. 2019 Faulty at High-resolution
IsoCor v1 Yes Low only All Millard et al. 2012 none
IsoCor v2 Yes All All Millard et al. 2019 none

Note

If you would like your software to appear in this list, please get in touch with us.

## How many peaks should I measure?¶

For a compound with $$n$$ atoms of the tracer element, you should measure $$n+1$$ peaks.

## What mass fractions should I measure?¶

For a compound with $$n$$ atoms of the tracer element, you should measure the mass fractions corresponding to the compound having incorporated $$0, 1, ..., n$$ isotopic tracers.

## Can I perform correction in case of missing values?¶

Mass fractions cannot be corrected by IsoCor in case of missing measurement(s).

For instance, in a C2 compound for which M2 is not measured, one cannot estimate the contribution of the corresponding isotopologue – through (im)purity of the isotopic tracer – to M1 or M0. Hence, these mass fractions cannot be corrected for purity. As another example, in a C3 compound for which M2 is not measured, one cannot estimate its contribution – through natural abundance – to M3. Here again, these mass fractions cannot be corrected for natural abundance of isotopes.

To avoid misinterpretation of partially corrected data, we have prefered to not allow correction to be applied in case of missing values.

## How to add a new metabolite/derivative into the database?¶

Open the relevant database file with a rustic text editor (e.g. Notepad++) and add a new row in your file following the format described in Input data.

Database files are created by default at the first run of isocor in ‘/user/isocordb’. Additional metabolites and derivatives databases can also be defined, as described in Input data.

Take care not to modify the file format, nor its structure. A typical error comes from Excel replacing ‘.’ to ‘,’ in floats.

## What elemental formula should I declare into the database?¶

Obviously, errors in elemental formulas will result in erroneous isotopologue distributions; thus special care must be taken when defining these formulas. Details on the elemental formulas to be declared in IsoCor can be found in Tutorial section on formulas.

## Is it possible to correct multiply-charged molecules?¶

Yes, IsoCor takes into account the charge when constructing the correction matrix. The charge state of each metabolite should be declared in the corresponding database file, as detailed in Input data.

## Should I tailor natural abundance of isotopes for my experiment?¶

The abundance of each isotope in natural samples depends on their origin. For instance, marine organisms have been reported to have slightly less 13C than land plants [IUPAC2016]. Ideally, you should measure the exact abundance of each isotope present in an unlabeled sample prior to the labeling experiment. However, most of the time such an experiment would require too much resources for a negligible gain in precision, as we previously found [Millard2014]. The default values should be good enough for most users, unless you work with strongly exotic material.

## Where does the default values for natural abundance and mass come from?¶

From IUPAC [IUPAC2016].

## Should I correct the tracer purity for my experiment?¶

Yes, if you know it. The purity of your tracer should be available from your provider of labeled compound.

## What is the default value for the tracer purity?¶

By default, we assume a perfect tracer purity.

## Should I correct natural abundance of the tracer for my experiment?¶

Yes, you should correct for the presence of isotopes at natural abundance in unlabeled positions of non-uniformally labeled nutrients.

## How does IsoCor performs its corrections?¶

Please have a look at the examples in the Tutorials section. If you are looking for something more detailed, we invite you to review our source code from our git repository. Also, have a look at the logs in Verbose logs mode; all the intermediate results (correction vector used to construct the correction matrix, correction matrix, etc) will allow you to reproduce the results with pen and paper.

## How is computed the mean enrichment?¶

The mean isotopic enrichment of a metabolite refers to the mean content in isotopic tracer in the metabolite, expressed as the relative fraction of total atoms of its element in the metabolite. This information is particularly useful for the quantification of split ratios between two metabolic pathways resulting in different content of tracer. IsoCor calculates the mean enrichment ($$ME$$) using the formula $$ME = \frac{\sum^{n}_{i=1}M_{i}.i}{n}$$, where $$M_{i}$$ is the proportion of isotopologues with $$i$$ 13C atoms for a metabolite containing $$n$$ carbon atoms.

## What is the isotopic InChI?¶

The IUPAC International Chemical Identifier (InChI) is a textual identifier for chemical substances, designed to provide a standard way to encode molecular information and to facilitate the search for such information in databases and on the web.

The identifiers describe chemical substances in terms of layers of information. IsoCor generates an isotopic layer that specifies the isotopologue of the tracer element, following the extended representation proposed by the InChI Isotopologue and Isotopomer Development Team:

• Simple definition: /a(Ee#<+|->#…)

• Complete definition:

/a(<element><isotope_count><isotope_designation>[,<atom_number>])

• <element> - one or two letter Element code (Ee).
• <isotope_count> - number of atoms with the designated isotope (#).
• <isotope_designation> - isotope designation indicated by a sign (+ or -) and number indicating the unit mass difference from the rounded average atomic mass of the element. For example, the average atomic mass of Sn (118.710) is rounded to 119. We specify two 118Sn atoms as “/a(Sn2-1)”.
• Examples:
• 13C2-isotopologue of alpha-D-glucopyranose:

“InChI=1/C6H12O6/c7-1-2-3(8)4(9)5(10)6(11)12-2/h2-11H,1H2/t2-,3-,4+,5-,6+/m1/s1/a(C2+1),(C4+0)”

• 16O118O3-isotopologue of fumarate:

“InChI=1S/C4H4O4/c5-3(6)1-2-4(7)8/h1-2H,(H,5,6)(H,7,8)/p-2/b2-1+/a(O3+2),(O1+0)”

Warning

This is an experimental feature: isotopic inchis may be subject to change according to the evolution of the IUPAC specifications.

## I cannot start IsoCor graphical user interface, can you help me?¶

If you installed IsoCor following our standard procedure and that you are unable to start IsoCor by opening a terminal and typing isocor, then there is indeed something wrong. Do not panic, we are here to help! Please follow this simple procedure:

1. The first step of the debugging process will be to get a traceback, i.e. a message telling us what is actually going wrong:
• On Unix-based systems, you should already see it in the terminal you opened.
• On Windows, you will have to open IsoCor from your Anaconda prompt with python.exe -m isocor to display the traceback.
2. Read the traceback and try to understand what is going wrong:
• If it is related to your system or your Python installation, you will need to ask some help from your local system administrator or your IT department so they could guide you toward a clean installation. Tell them that you wanted “to use the graphical user interface of IsoCor, a Python 3.5 software” and what you did so far (installation), give them the traceback and a link toward the documentation. They should know what to do.
• If you believe the problem is in IsoCor or that your local system administrator told you so, then you probably have found a bug! We would greatly appreciate if you could open a new issue on our issue tracker. One of the developers will help you.

## I would like a new feature.¶

We would be glad to improve IsoCor. Please get in touch with us so we could discuss your problem.

 [IUPAC2016] (1, 2) Isotope-abundance variations and atomic weights of selected elements: 2016 (IUPAC Technical Report) https://doi.org/10.1515/pac-2016-0302
 [Millard2014] Isotopic studies of metabolic systems by mass spectrometry: using Pascal’s triangle to produce biological standards with fully controlled labeling patterns, 2014, Anal. Chem., 86(20):10288-10295, https://doi.org/10.1021/ac502490g