Over the past couple of years, the National Reference Collection team at CABI in Egham have done a lot of work into developing a rapid and inexpensive method for discriminating between plants, insects, bacteria, fungi, and other biological materials at species level, and even between regional biotypes within a species.

This blog is a summary from the team of what they have been up to, along with links to some of the papers that they have published over the past 12 months. Most of these papers cover novel method development, illustrating how this technique can be used for field work, even in situations where samples are collected in one country for analysis in another).

If you think that there are ways that you can work with the team, please contact M.Reeve@cabi.org.

Brief overview and references

Matrix-assisted laser-desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an incredibly simple technique that prepares singly-charged and unfragmented proteins in the gas phase. From these, using fairly simple instrumentation, a mass-spectrum ‘fingerprint’ for the sample can be generated. There are several MALDI-TOF MS sample-preparation methods currently available, but not all are applicable to some forms of fungal biomass and few are well suited to the analysis of plant or insect material.

Faced with this limitation, we developed a simplified method¹ that combines all the traditional steps in a single reagent, generates spectra with good intensity and peak richness, costs no more (and generally less) than current methods, and is not constrained in terms of throughput by the availability of centrifuges.

Our method takes less than 30 seconds per sample and is a single-tube process. Using methodology developed in¹, we have demonstrated clear discrimination between closely related plant species and between regional biotypes within invasive plant species², particularly using novel methodology that we have developed for high-resolution sample discrimination by comparison between ‘blind-test’ MALDI-TOF MS spectra and reference-sample spectra.

MALDI-TOF MS analysis does however require relatively fresh biological material that contains proteins that have not yet undergone significant degradation. For field-work collection of samples, this presents a serious limitation.

In order to overcome this, we have developed a simple and inexpensive method for practical storage of field-sample proteins³ in which biological material is simply crushed onto filter paper and dried. The dried and protein-impregnated filter paper can then be soaked in an alcoholic solution suitable for the inactivation of microorganisms of concern and again dried for storage.

After dry storage, the ‘fingerprint’ proteins may be eluted from the paper using methodology developed in the first paper. Unlike untreated leaf material (which degrades very rapidly), high-quality spectra can be obtained, with and without alcohol treatment, even after storage for one month at up to 40°C! We have also used this method to discriminate between regional biotypes within biological-control-insect species⁴.

Adapting the methodology developed in the first paper, we have additionally demonstrated clear discrimination between regional biotypes within invasive plant species⁵ using seeds, and we have also carried out studies comparing seed varieties resulting from intensive plant-breeding activities⁶.

For cases where viable seeds may not be sent across national boundaries, we have developed methods for simple and local preparation of proteins immobilised onto paper prior to shipping for subsequent MALDI-TOF MS analysis⁷.

For filamentous fungi, significant variation in MALDI-TOF MS spectra can unfortunately be observed for growth under different conditions on agar plates, as well as over time for growth on the same medium. For this reason it is desirable to have a means of growth in which the resulting spectra remain as consistent as possible over time.

To this end we have investigated numerous methods for fungal growth⁸ and have developed a novel method employing filter-paper-supported growth in a novel MALDI-TOF MS-compatible rich medium within sealed 1.5 ml Eppendorf tubes. This method gives slightly better identifications and lower spectral variance compared to agar-plate controls and will enable growth-time-point-specific databases for fungal identification.

Finally, analytical techniques currently available for the characterisation of mixtures of microorganisms are generally based upon next-generation sequencing. Motivated to develop practical and less-expensive methods for characterising such mixtures, we have proposed, as an alternative, the use of MALDI-TOF MS.

A number of potential approaches employing this technique for such characterisation are possible, but many contain impediments to their successful employment⁹. As a consequence, our rationale has been to capitalise instead on the powerful algorithms currently available for spectral comparison.

Following this rationale, a first priority is to ensure the generation of MALDI-TOF MS spectra from mixtures of microorganisms that contain manageable peak complexities, and which can also be handled by existing spectral-comparison algorithms, preferably with the option to archive and re-run sample preparations and to pipette replicates of these onto MALDI-TOF MS sample plates.

The second priority is to ensure that database entry is comparably facile to sample preparation so that large databases of known microorganism-mixture MALDI-TOF MS can readily be prepared for comparison with the spectra of unknown mixtures.

We have now addressed the above priorities and have generated illustrative MALDI-TOF MS spectra in order to demonstrate the utility of this approach. In addition, we have investigated methods aimed at chemically modulating the peak complexity of the MALDI-TOF MS spectra and methods for the convenient and stable storage and shipping of protein samples from microorganism mixtures based upon previously published work^3,4,6.

References with links (all open access)

1. Reeve MA, Buddie AG, Pollard KM, Varia S, Seier MK, Offord LC, Cock MJW. A highly-simplified and inexpensive MALDI-TOF mass spectrometry sample-preparation method with broad applicability to microorganisms, plants, and insects. J Biol Methods 2018;5(4):e103. http://dx.doi.org/10.14440/jbm.2018.261
2. Reeve MA, Pollard KM, Kurose D. Differentiation between closely-related Impatiens and regional biotypes of Impatiens glandulifera using a highly-simplified and inexpensive method for MALDI-TOF MS. Plant Methods 2018:14:60. https://doi.org/10.1186/s13007-018-0323-6
3. Reeve MA and Buddie AG. A simple and inexpensive method for practical storage of field-sample proteins for subsequent MALDI-TOF MS analysis. Plant Methods 2018:14:90. https://doi.org/10.1186/s13007-018-0358-8
4. Reeve MA and Seehausen ML. Discrimination between Asian populations of the parasitoid wasp Ganaspis brasiliensis using a simple MALDI-TOF MS-based method for use with insects. Biology Methods and Protocols, 2019;4(1). https://doi.org/10.1093/biomethods/bpz002
5. Reeve MA and Pollard KM. Discrimination between regional biotypes of Impatiens glandulifera using a simple MALDI-TOF MS-based method for use with seeds. Plant Methods 201915:25. https://doi.org/10.1186/s13007-019-0412-1
6. Reeve MA. MALDI-TOF MS-Based Analysis of Seed Proteins from Catalogue Varieties of Solanum lycopersicum/Lycopersicon esculentum. Horticulturae 2019, 5(3), 48; https://doi.org/10.3390/horticulturae5030048
7. Reeve MA and Pollard KM. MALDI-TOF MS-based analysis of dried seed proteins immobilized on filter paper, Biology Methods and Protocols, 2019;4(1). https://doi.org/10.1093/biomethods/bpz007
8. Reeve MA and Bachmann D. A method for filamentous fungal growth and sample preparation aimed at more consistent MALDI-TOF MS spectra despite variations in growth rates and/or incubation times. Biology Methods and Protocols, 2019;4(1). https://doi.org/10.1093/biomethods/bpz003
9. Reeve MA and Bachmann D. MALDI-TOF MS protein fingerprinting of mixed samples. Submitted to Biology Methods and Protocols

Mike Reeve BA, MA, D.Phil, Innovations Manager, CABI

Mike’s background is in industrial technology innovation, having spent 16 years working for Amersham (now part of GE Healthcare) and nine years with Sharp Laboratories of Europe prior to joining CABI.

Since switching from his main field (DNA preparation and sequencing) with the arrival of the Capability-4 MALDI-TOF MS instrument, Mike has focused on method development and new applications for this incredibly-versatile instrument. This has been an excellent opportunity to simplify existing methods and to extend the scope of analysis to include field-work, even with samples from different countries.

The CHAP MALDI-TOF MS really was a case of the right technology, in the right place, and at the right time – something Mike is keen to cite as key to the work over the last couple of years, along with excellent collaborators for the publications that have emanated from this work.

In addition to running a small group and his role in contracts management at CABI, Mike still spends as much time as possible in the laboratory and his current research interest is the adaptation of MALDI-TOF MS for the study of mixtures of microorganisms, which he firmly believes holds many opportunities for further research and applications within CHAP.

If you have any questions specifically about our National Reference Collection capabilities or would like to discuss any of our other capabilities, please send us an email at enquiries@chap-solutions.co.uk

Please note, the opinions expressed in this article are the author’s own and do not necessarily reflect the views or opinions of CHAP.