Chromatography separates a venom into its components, but it does not say what the separated peaks are. To establish the identity of a component, you need to know its mass and, if possible, its amino acid sequence. Mass spectrometry can provide both pieces of information together and is one of the core tools for peptide identification in venom research.
Principle of the method
A mass spectrometer does not weigh molecules directly. It first converts them into electrically charged particles, that is, ions; it then separates these ions according to their behaviour in electric or magnetic fields. The quantity measured is not the mass itself but the mass-to-charge ratio, written as m/z. The result is a spectrum with m/z on the horizontal axis and signal intensity on the vertical axis.
| Component | Function | Common examples |
|---|---|---|
| Ion source | Gives the molecule a charge without breaking it apart | Electrospray, matrix-assisted laser desorption/ionization |
| Mass analyser | Separates ions by their m/z value | Time of flight, quadrupole, ion trap |
| Detector | Counts the separated ions | Electron multipliers and similar systems |
Soft ionization methods have been developed for large, fragile molecules such as peptides. In electrospray, the solution is sprayed from a fine needle under high voltage; as the droplets evaporate, the molecules pass into the gas phase in a charged state. Because this approach can be coupled directly to liquid chromatography, separation and measurement can be carried out in a single flow. In the matrix-assisted laser method, by contrast, the sample is dried together with an auxiliary substance that absorbs light and is ionized with short laser pulses.
Charge states and mass information
A peptide ionized by electrospray usually carries more than one proton. The same molecule therefore appears in the spectrum not as a single line but as a series of peaks corresponding to different numbers of charges. Starting from this series, the software calculates back to the uncharged mass of the molecule; this process is called deconvolution.
On instruments with high measurement precision, the fine structure arising from the molecule's natural stable isotopes can also be resolved. This detail is useful in two ways: the spacing between adjacent peaks directly shows the charge number, while the shape of the isotope pattern gives an idea of whether the signal really belongs to a peptide.
The accuracy of the measurement depends on regular calibration of the instrument. Calibration is performed with standard mixtures of known mass; in some studies, drift is corrected throughout the run using an internal standard introduced continuously with the sample. When the calibration was performed is also part of the reported result.
Mass information also carries clues about the state of the disulfide bridges. Because hydrogen is lost from the molecule when the bridges form, a folded peptide is slightly lighter than its reduced form; when both forms of the same sample are measured, this difference can be seen. The role of the bridges in the structure was discussed in the article on disulfide bridges.
MS/MS: getting to the sequence
Mass alone is often not enough to identify a peptide definitively, since different sequences can have the same mass. This is why tandem mass spectrometry, MS/MS for short, is used.
The approach consists of three steps. In the first analyser, the ion of interest is selected; it is then fragmented in a controlled way by collision with inert gas molecules in a collision cell; finally, the m/z values of the resulting fragments are measured. The peptide chain mostly breaks at the bonds linking the amino acids, so the fragments form a ladder, each one amino acid apart from the next. The mass difference between two adjacent fragments gives the identity of the amino acid between them; when the fragment series is followed from start to finish, part or all of the sequence can be read.
The reading is not always perfect. Some pairs of amino acids have masses that are very close or identical; leucine and isoleucine cannot be distinguished under standard conditions. In addition, not every bond in the chain breaks equally easily, so the fragment signal may remain weak for some regions. These gaps are usually filled using different fragmentation methods or samples prepared with different enzymes.
Two basic approaches
- Measuring the whole molecule: The peptide is measured intact, without fragmentation. For small, bridge-stabilized venom peptides, this is a quick way to obtain a profile.
- Measuring after enzymatic digestion: The protein is first cut into small pieces by an enzyme that cleaves at specific bonds, and the pieces are then measured. This is preferred for large proteins and enzyme content.
The data obtained are compared against databases that collect known sequences. The software matches the measured masses and fragment patterns with the values expected from recorded sequences and produces a confidence score for each match. The probability that a match is due to chance is estimated by methods such as repeating the search against reversed sequences.
Limits in new species
A database search depends on the sequence in question being present in the records. In the venom of a little-studied or newly described species, however, the sequences of many components have not yet been recorded. In this case, two routes are followed: the sequence is read directly from the spectrum, or a search library is built from gene sequence data obtained from the species' venom gland. The second approach has become widespread in venom research because it combines transcriptome and proteome data.
In such cases, the result is often a placement within a family rather than a definitive name: the component is classified according to how closely it resembles a known peptide family. The fact that the peptide identified in our reference batch report is given with a class name, as α-KTx 3.6, is an example of this approach; the general features of the family were described in the article on α-KTx peptides.
Alongside other methods
Mass spectrometry is not a method that works on its own. The sample is usually separated by chromatography first; the separation approach was covered in the article on HPLC, and the method that shows the molecular weight distribution on a gel in the article on SDS-PAGE. These methods ask different questions of the same sample; when mutually confirming results come together, the identification becomes stronger.
The methods applied in the report for our reference batch and the values obtained are published on the analysis page. It should be noted that the results are batch-specific and that the measurements are repeated for each batch.
