# Mass Spectrometry: A Rosetta Stone to Learn How Fungi Interact and Talk
## 3 Common Mass Spectrometry-Based Strategies Used in Mycology Studies
### Identification of Microbial Chemical Compounds
#### 3.2.1 Identification of VOCs of Fungal Origin  
At the moment, there are many publications related to fungal volatilomics, and to the best of our knowledge, there are two public VOC databases that include volatile information of more than 10,000 fungal species [45; 160; 167]. In order to monitor VOCs produced by micro-fungi, it is relevant to remember that VOCs are partitioned between gas and liquid phases under optimal growth conditions. Due to the large diversity of these compounds, there is no \"one\" analytical protocol to study them all. Gas chromatography coupled to mass spectrometry (GC-MS) is an extremely reliable and affordable method for the identification and quantification of VOCs. In general, VOCs are collected from a headspace, trapped on an adsorptive surface or solvent extracts, followed by thermal desorption or direct injection into the GC for separation, and then the individual VOCs from complex mixtures are identified by comparisons of the mass spectra with databases, internal standards and/or chromatographic retention indices [14; 168; 129; 169; 14; 170; 18]. Other mass spectrometry methods that have been used for the identification of VOCs include real-time measurements, which omit the use of a chromatography technique, such as proton transfer reaction-mass spectrometry (PTR-MS) that, for example, has been used in measuring volatile emissions of _Assocoryne scroides_ (100), _Hypoxylon_ sp. [99], _Musodor albus_[101] and _Nodulisporium_ sp. [102]. Another direct MS technique is selected ion flow tube-mass spectrometry (SIFT-MS) that has been adapted for the detection of volatile traces from medically important fungi such as species of _Aspergillus_, _Candida_, _Cryptococcus_, _Fusarium_ and _Mucor_[103; 104; 105]. Secondary electrospray ionization coupled to high-resolution mass spectrometry (SESI-MS) has also been used to analyze the VOCs produced during fermentation by the ale yeast _Saccharomyces cerevisiae_[106]. An exquisite advantage of the PTR-MS, SIFT-MS and SESI-MS online approaches is their relative simplicity [104; 170; 171], where small molecules can be directly detected from the volatile emissions of the samples of interest. Hence, the VOCs can be easily monitored in real-time and can continuously collect data about specific compounds [100]. However, online approaches have two major disadvantages when compared with offline methods (e.g., GC-MS; HPLC-MS): (i) a lack of resolution of isobaric compounds (i.e., compounds with identical mass-to-charge ratios), and ii) sensitivity to matrix effects [106; 172]. In general, MSs that are not coupled to an orthogonal separation system may provide ambiguous identification of VOCs [162; 165]. Though gas chromatography remains an excellent method for the separation of isobaric VOCs, this method is time-consuming. An alternative separation method of isobaric VOCs is ion mobility coupled to mass spectrometry (IMS). Ion mobility separation has a different principle [173; 174] than the one used in GC or LC-MS; hence it is able to resolve isobaric species and provide the rapid, sensitive and broad spectrum detection of trace organic components in moderately complex gas mixtures such as those found in _Aspergillus_ species [130]. The collection of volatile specialized metabolites can be performed by direct measurement, as in the case of PTR-MS, SIFT-MS and SESI-MS, or by using a direct collection system such as headspace techniques. Unfortunately, these collection strategies can be influenced by the volatiles that are also emitted by the cultivation media or by other sources in the environment during the analysis. These additional volatiles can distort the signal profiles that are detected on the mass spectrometer [103; 162; 164]. Therefore, the use of a suitable sample extraction strategy is recommended such as solid phase micro extraction (SPME), which involves a metallic fiber that has been coated with different adsorbent materials.