The Tool Buttons - HXMS Lab
Hydrogen-Deuterium Exchange

Hydrogen-deuterium exchange, coupled with MS is growing in popularity as a tool to investigate protein dynamics, interaction and
- to some degree - secondary structure.

The resulting scans can be difficult to interpret, because the mass of the deuterated precursor is heavier than its non-labelled counterpart by a factor that depends on the level of deuteraton.

A typical MS/MS scan of a deuterated peptide fragmented with ETD.
MassAI facilitates the interpretation of HXMS datasets by comparing datasets from both labelled and non-labelled samples in one go.

A non-labelled reference dataset is used to identify peptides present in the sample, while the deuterated dataset is used to determine the
level of deuteration.
Below is an example, where a non-deuterated control has been searched against a known protein sequence. This gives us both
peptide sequences as well as a reference fragmentation fingerprint that we can match the deuterated sample against
Once the initial search has been carried out, we open the HXMS lab from the TOOL BUTTON window.
Here we specify a number of datafiles from deuterated samples, in this case 3.

The default settings have been set to standard HDX, but in the example case, we have worked with a modification, where we can have 2 on
each residue. Therefore the value "Max labels per residue" has been changed to 2. Finally we press the "Analyse" button.
When the analysis is done, all matching H/D scan pairs are listed in the result table above.
Each pair is given an "alignment score", which signifies how well the deuterated and the reference scans pair up against each other. It is useful
to  sort the results by this value in descending order. The "DELTA" column shows the mass difference between the deuterated precursor and the
control precursor.
While knowning the level of deuteration on precursor level can be useful, it is often desirable to probe exactly which residues carry
deuterations. CID/HCD is known to scramble the deuterons throughout the peptide, making this approach less practical. ETD however, does
not scatter the deuterons, and this makes it attractive to look at the deuteration at residue resolution.

The MassAI HXMS Lab offers several ways to look at HDX at residue level.
The first is approached simply by LEFT clicking on any of the entries in the results list (above), or by navigating through the list with the
up/down keys on your keyboard.
This displays the annotated reference and deuterated scans mirrored around the X-axis. 
Each recognised fragment ion is annotated directly on the scan. The monoisotopic m/z value for both the deuterated and reference ion is
listed in the column to the right, along with the average m/z values for the isotope cluster. This is the value that is commonly the most
accurate when calculating the deuteration level at residue resolution.
To get a better overview of the deuteration level for each residue, press the LABELLING ATRESIDUE LEVEL button on
the main page of the HXMS lab. This brings up the following overview:
A
B
C
Panel A is simply a miniature version of the annotated scan, so that you can have it on the screen along with panels B and C
Panel B shows the deuteration level for each residue in the peptide, both from the b/c side and from the y/z side.
Like panel B, the C panel illustrates the deuteration level for each residue in the peptide, both from the b/c side and from the y/z side, in
three different ways:
1: Label by ion count. The deuteration level for each ion is displayed graphically.
Select numbering on the X-axis by ion count or by residue count, by using the radio buttons
2: Label by Residue. This display differs from the previous, as this chart displays the INCREASE in deuteration when we go from one
residue to the next, instead of the calculated deuteration level for the individual fragment ion.
The example above shows, that GLU 3 carries approx. two deuterons. (which is in agreement with this particular setup). The red column
signifies, that the observation was based on a y or z fragment ion. For GLU 12, we have both a blue b/c ion type column and a pale red
column. Both read a deuteration level of approx. 2. The pale red signifies that this particular observation is the first in the series, and that
the deuteration could have happened at an earlier residue in the y/z series. For the y-ion, this could have been E,A,N or R. Fortunately we
have the blue b-ion observation to support the notion, that the deuterated ion is in fact Glu 12.
Finally, we have a b/c type observation for Glu 9 with a deuteration level of approx.2. It is light blue, signifying that b9 is the first observed
b-ion, and hence, that deuteration could have happened anywhere between b1 and b9. What we observe here could therefore be either a
deuterated GLU 3 OR GLU 9.
3: Cumulated Label Level per Residue. This projection is the same as in 2, only this is an accumulation across all the scans with this
particular peptide. This helps to fill out any gaps in the fragment ions that may hapen in any one scan. Here we notice the many "first
observed" b-ions for GLU 3. Along with the dark blue b-ions for GLU 9 (which now have low deuteration levels), we can establish that the
peptide has two deuterated residues: GLU 3 and GLU 12, both of which carry two deuterons.