Proteomics
There was an interesting paper recently:
A new type of Isobaric Labelling reagents? Do we really need more? Isn’t this what TMT and iTRAQ already do? Why is this important?
In Isobaric Labelling, each sample is modified with different versions of a chemical label which are near identical but have several heavier stable isotopes (+1 neutron) in different locations. Samples are then mixed. The masses of all versions of a same labelled peptide will be the same, so they will show up as one M/Z peak on the mass spec in MS1. However, the labels are designed to break off during peptide fragmentation, generating a set of reporter ions with different masses (offset by 1, 2, etc… neutrons). Both TMT and iTRAQ (maximum commercially available multiplicities of 11 and 8, resp.) operate on this same principle, with minute changes in the actual molecule allowing two different companies to essentially sell the same product[1]. In addition, TMT, normally limited to 6plex, achieves 11plexity using “isotopologues”, reporter ions that have the same total number of additional neutrons but localised in different atoms (typically nitrogen vs carbon). Amazingly, high resolution mass spectrometers can discriminate between these minute mass changes.
[1]Ahhh, the joys of Intellectual Property laws!
schematic of the structure of TMT-6plex
For more details about Isobaric Labelling click here
Isobaric labelling has several advantages over label-free analysis:
Because of its higher multiplicity compared with SILAC, ease of use and availability (compared with neuCode SILAC, which also has high multiplicity) and of the quality of the data provided, Isobaric Labelling is currently the most widely used type of relative quantitation in MS.
So what do EASI-tag labels bring to the table that we should care about them?
Ratios compression
Well, one longstanding issue with classic Isobaric Labels has been Ratios Compression. The problem is that fragmentation is required for relative quantitation, and in turn a pre-requisite to fragmentation is precursor isolation. Precursors are typically isolated using a 1 to 2 Th[1] wide isolation window. Narrower would result in too many losses. This width, however, is such that in many cases other peaks will be co-isolated. So we don’t actually have a single precursor, but (usually) a main precursor contaminated with other peptides. If we fragment all this, we will get a complex MS/MS, which we can still manage to match, and we will get reporter ion intensities which will be a mix of those of all co-fragmented peptides. This can be thought off as something like blurring the measurements. Because the majority of peptides are not affected in most cases, the tendency will be for the most extreme ratios to be decreased. Hence, a global “compression” of ratios.
[1]1 Thompson = 1 Da/Z
Now, two main strategies have been developed for dealing with this:
Enter EASI-tags…
EASI-tags
The issue with ratios compression comes from the fact that fragmentation of any peptide generates the same reporter ions. So if I am doing TMT-6plex and co-fragment 2 peptides, I get 6 reporter ion peaks whose intensities are an average of those contributed by both parents.
EASI-tags provides the following two crucial improvements:
Because for EASI-tags label fragmentation occurs at low energy, the reporter ions generated still contain the un-fragmented parent peptide, attached to a remainder of the labels. Thus, each peptide generates a specific series of reporter ions. Co-isolated peptides will generate different, easily distinguishable series of peaks. For all intents and purposes, EASI-tag spells the end of ratios compression.
Importantly, a single fragmentation step is sufficient to generate both reporter ions (quantitation) and fragment ions (identification). Thus, while for TMT/iTRAQ an MS3-capable instrument is required (at least for methods nearly-eliminating ratio compression), EASI-tag samples can be run on Q-Exactive instruments.
Because I am nice, I made this little summary – which should be mostly accurate. You’re welcome.
Note: charge things can get complicated, so I am only showing an easy case with charge 2+.
Charged (= detectable) bits are in yellow. I indicated in blue where the two things we are interested, i.e. identification and quantitation, happen.
Interference issues
There are, inevitably, some issues. A peptide is not a single peak, but an isotopic envelope. Most peptides have charge 2 or 3. A 2+ (resp. 3+) peptide will be an isotopic envelope of several peaks separated by 0.5 Th (resp. 0.333…Th). The isotopic envelope is the result of the existence of rarer, heavier isotopes in nature.
In TMT and iTRAQ, co-isolation of several peaks of the envelope does not matter, because they all have the same ratio and will simply contribute equally to all channels. For EASI-tags however, the M/Z difference between two consecutive reporter ions is the same as that between two peaks of the original envelope, which will cause interference. Let us look at a concrete example:
Assuming we have a 6plex EASI-tag labelled sample. We will name our labels a, b, c, d, e and f. If we co-fragment peaks 1 and 2 of the isotopic envelope (let us call them IE1 and IE2), then we will get 7 reporter peaks, each contributed by:
a) Interference for a 2+ peptide if using a 1.4 Th wide isolation window. b) The interference is removed for the same peptide by shifting the isolation window left. Note that usually you would shift a window for identification to the right to avoid co-isolating other precursors. Here, however, different precursors would rarely generate interfering reporter ions so we do not have to worry about them.
This will make precise quantitation tricky. Luckily, the authors show how using an isolation window shifted to the left can avoid these issues for 2+ and 3+ peptides, i.e. the vast majority of peptides. For higher charge peptides, the problem will have to wait for better quadrupoles allowing for narrower isolation windows.
This interference issue also means that, sadly, EASI-tag will not be compatible with DIA.
Currently, EASI-tags are not – that we know – commercially available. The big unknowns which will decide whether it becomes adopted will be:
