Emmanuelle Claude (Presenter)
Authorship: Mark Towers(1); Philippa Hart(1); Emrys Jones(1); Tamas Karancsi(2); Emmanuelle Claude(1)
(1)Waters Corporation, Wilmslow, UK; (2)Waters Research Center, Budapest, Hungary;
The detection of large biomolecules such as peptides and proteins directly from tissue sections by DESI-MSI has proven to be extremely challenging. Here we describe a newly developed method combining a series of optimized parameters and conditions (gas pressure, solvent composition and flow rate) which allow the extraction of large biomolecules from the tissue in droplet form. These droplets are desorbed/ionized using a heated MS capillary into multiply charged ions Detection of these multiply charged ions is enhanced using Ion Mobility which enables separation from the intense endogenous ion species and chemical background.
Desorption electrospray ionisation mass spectrometry imaging (DESI-MSI) is typically known for the mapping of small molecules such as lipids and metabolites, in singly charged form, directly from tissue sections. Whilst the detection of spotted protein standards from target plates has previously been shown(1), the detection of large biomolecules directly from tissue sections by DESI-MS has presented difficulty. Recently however, it has been demonstrated that when using Ion Mobility Separation (IMS), doubly and triply charged gangliosides can be mapped directly from murine brain sections(2).
Here we report a newly optimised method allowing the mapping of multiply charged proteins (intact or enzymatically digested) directly from tissue sections by DESI-MS utilising IMS to aid in visualisation.
All experiments were carried out on a SYNAPT HDMS G2-Si Q-ToF (Waters) with a Prosolia2D DESI stage. A modified inlet capillary with a heated sheath connected to an adjustable power supply was used; voltages between 0 and 12V were applied to generate a variable heating effect. Optimisations were performed assessing tissue pre treatment, spray solvent flow rate, nebulising gas pressure and solvent composition.
For tryptic peptides, Promega procine trypsin was applied to tissue sections using a Suncollect (Sunchrom) automated sprayer and incubated overnight for digestion.
DESI-MSI and MSMS experiments were setup, acquired and analysed using Masslynx and HDI 1.4, with the additional use of DriftScope for processing of the ion mobility dimension.
Rat liver sections were first taken through a series of ethanol based washes to remove salts, followed by a chloroform wash to deplete lipids. The tissue sections were analysed IMS allowing the ions to be separated in the gas phase under an electric field before entering the Time-Of-Flight.
Through the enhanced ionisation offered by the heated inlet capillary, the evaluation of optimal conditions (the spray solvent flow rate, nebulising gas pressure and solvent composition ) was found. A highly abundant protein charge series (from +9 to +21) could be observed, further enhanced with IMS when the data was mined in the Driftscope software package. After extracting the dominant trend line and using a maximum entropy algorithm it was possible to putatively identify four Haemoglobin subunits: Alpha -1 and Alpha-2 (with an aspartic acid substituted with alanine), Beta 1 and Beta 1 with serine substituted with threonine. Additional trend lines were found to relate to background solvent peaks, residual lipids and more importantly lower abundant small proteins / large peptides. These small proteins / peptides were observed with charge states from +1 to +8, the majority of which could only be resolved from the background through the use of ion mobility.
Further work was carried out to evaluate the application of the new DESI MSI IMS set-up when proteins were tryptically digested directly from tissue. Up to +4 tryptic peptides were mapped directly from a mouse brain followed by MS/MS experiments for identification.
Conclusions & Discussion
We have demonstrated advances in the mapping of intact and digested proteins directly from tissue sections by DESI MSI with ion mobility separation.
References & Acknowledgements:
1: Shin YS, Drolet B, Mayer R, Dolence K and Basile F; Desorption Electrospray Ionization-Mass Spectrometry (DESI-MS) of Proteins, Anal Chem, 2007, May 1; 79(9); 3514-3518
2: 1. Škraškova K, Claude E, Jones EA, Towers M, Ellis SR, Heeren R; Enhanced capabilities for imaging gangliosides in murine brain with matrix-assissted laser desorption ionisation and desoprtion electrospray ionisation mass spectrometry coupled to ion mobility separation, Methods, 2016, 104: 69-78.
IP Royalty: no
|Planning to mention or discuss specific products or technology of the company(ies) listed above:||