Ambient ionisation mass spectrometry for in situ analysis of intact proteins

Abstract

Ambient surface mass spectrometry is an emerging field which shows great promise for the analysis of biomolecules directly from their biological substrate. In this article, we describe ambient ionisation mass spectrometry techniques for the in situ analysis of intact proteins. As a broad approach, the analysis of intact proteins offers unique advantages for the determination of primary sequence variations and posttranslational modifications, as well as interrogation of tertiary and quaternary structure and protein-protein/ligand interactions. In situ analysis of intact proteins offers the potential to couple these advantages with information relating to their biological environment, for example, their spatial distributions within healthy and diseased tissues. Here, we describe the techniques most commonly applied to in situ protein analysis (liquid extraction surface analysis, continuous flow liquid microjunction surface sampling, nano desorption electrospray ionisation, and desorption electrospray ionisation), their advantages, and limitations and describe their applications to date. We also discuss the incorporation of ion mobility spectrometry techniques (high field asymmetric waveform ion mobility spectrometry and travelling wave ion mobility spectrometry) into ambient workflows. Finally, future directions for the field are discussed.

Keywords: DESI; Flowprobe; LESA; ambient mass spectrometry; intact proteins; ion mobility spectrometry; nanoDESI; surface sampling.

Figure 1

Ambient ionisation techniques for protein analysis. A, Liquid extraction surface analysis. B, Flowprobe. C, Nano desorption electrospray ionisation. D, Desorption electrospray ionisation

Figure 2

Peptide fragmentation nomenclature.50 b, y, and a minor proportion of a‐type fragments are produced by collision‐induced dissociation; electron capture and electron transfer dissociation generate chiefly c and z‐type ions, with some a and y ions

Figure 3

Liquid extraction surface analysis MS of human nonalcoholic steatohepatitis tissue. A, Liquid extraction surface analysis followed by bottom‐up LC MS/MS analysis: Numbers of proteins identified following extraction by use of three different solvents and the sequence coverage obtained for fatty acid binding protein (FABP1) extracted in ammonium bicarbonate. B, Top‐down CID spectrum and sequence coverage showing protein identification of FABP1; further identified proteins are marked in the full‐scan mass spectrum below. Adapted and reproduced from J. Sarsby, N. J. Martin, P. F. Lalor, J. Bunch and H. J. Cooper, Journal of the American Society for Mass Spectrometry, 2014, 25 (11) p 1953‐1961. DOI: 10.1007/s13361‐014‐0967‐z. Published by Springer US under the terms of the Creative Commons Attribution Licence (http://creativecommons.org/licenses/by/4.0/)

Figure 4

Ambient mass spectrometry imaging of intact proteins from mouse brain tissue via A, nano desorption electrospray ionisation, B, liquid extraction surface analysis, and C, raster‐mode Flowprobe. Adapted and reproduced with permission from A, C. Hsu, P. Chou and R. N. Zare, Analytical Chemistry, 2015, 87 (22); p 11171‐11175. DOI: 10.1021/acs. analchem. 5b03389, Copyright 2015 American Chemical Society, B, R. L. Griffiths, E. K. Sisley, A. F. Lopez‐Clavijo, A. L. Simmonds, I. B. Styles and H. J. Cooper, International Journal of Mass Spectrometry, 2017, In Press. DOI: 10.1016/j.ijms. 2017.10.009. Published by Elsevier under the Creative Commons Attribution Licence (CC‐BY) (http://creativecommons.org/licenses/by/4.0/), and C, R. L. Griffiths, E. C. Randall, A. M. Race, J. Bunch and H. J. Cooper, Analytical Chemistry, 2017, 89 (11); p 5683‐5687. DOI: 10.1021/acs. analchem. 7b00977. Published under the Creative Commons Attribution Licence (CC‐BY). Published 2017 by American Chemical Society

Figure 5

Liquid extraction surface analysis mass spectrometry of E. coli K‐12. A, Representative full‐scan mass spectrum of a colony stored at 4°C, sampled at the location marked in red. The m/z region containing most of the observed protein peaks is shown below. B, CID mass spectrum of ions centred at m/z 923.51, charge state +10 (marked with a star in the full‐scan mass spectrum). The protein was identified as the DNA‐binding protein HU‐β. Adapted and reproduced from E. C. Randall, J. Bunch, and H. J. Cooper, Analytical Chemistry, 2014, 10504‐10510. DOI: 10.1021/ac503349d. Published under the Creative Commons Attribution Licence (CC‐BY). Published 2014 by American Chemical Society

Figure 6

Contact liquid extraction surface analysis mass spectra of three representative bacterial species: Escherichia coli K‐12, Pseudomonas aeruginosa PS1054, and Staphylococcus aureus MSSA476. Adapted and reproduced from K. I. Kocurek, L. stones, J. Bunch, R. C. May, and H. J. Cooper, Journal of the American Society for Mass Spectrometry, 2017, 28 (10); p 2066‐2077. DOI: 10.1007/s13361‐017‐1718‐8. Published by Springer US under the Creative Commons Attribution 4.0 International Licence (http://creativecommons.org/licenses/by/4.0/)

Figure 7

Schematics of ion mobility separation techniques. A, High field asymmetric waveform ion mobility separation. B, Travelling wave ion mobility separation

Figure 8

Example mass spectra and ion images of mouse brain tissue demonstrating the benefits of incorporating field asymmetric waveform ion mobility separation into liquid extraction surface analysis and Flowprobe MS workflows. Reproduced from (A) R. L. Griffiths, A. M. Race, A. J. Creese, J. Bunch, and H. J. Cooper, Analytical Chemistry, 2016, 88 (13), p 6758‐6766, DOI: 10.1021/acs. Analchem. 6b01060, published under the Creative Commons Attribution Licence (CC‐BY), published 2016 by American Chemical Society and (B) C. L. Feider, N. Elizondo, and L. S. Eberlin, Analytical Chemistry, 2016, 88 (23) p 11533‐11541, DOI: 10.1021/acs. Analchem. 6b02798. Copyright 2016 American Chemical Society

Figure 9

Native liquid extraction surface analysis (LESA) MS. A, Native LESA MS of tetradecameric GroEL (~800 kDa). B, Native LESA MS of biotin binding to haemoglobin. C, Native LESA MS of tetrameric haemoglobin extracted from dried blood spots. D, Native LESA MS imaging of mouse brain tissue with selected ion images. Adapted and reproduced from (A) and (B) V.A. Mikhailov, R. L. Griffiths, and H. J. Cooper, International Journal of Mass Spectrometry, 2017, (420), 43‐50. DOI: 10.1016/j.ijms. 2016.09.011. Published by Elsevier under the Creative Commons Attribution Licence (CC‐BY) (http://creativecommons.org/licenses/by/4.0/), (C) N. J. Martin, R. L. Griffiths, R. L. Edwards, and H. J. Cooper, Journal of the American Society for Mass Spectrometry, 2015, (8), 1320‐7. DOI: 10.1007/s13361‐015‐1152‐8. Published by Springer US under the Creative Commons Attribution 4.0 International Licence (http://creativecommons.org/licenses/by/4.0/), and (D) R. L. Griffiths, E. K. Sisley, A. F. Lopez‐Clavijo, A. L. Simmonds, I. B. Styles, and H. J. Cooper, International Journal of Mass Spectrometry, 2017, In Press. DOI: 10.1016/j.ijms. 2017.10.009. Published by Elsevier under the Creative Commons Attribution Licence (CC‐BY) (http://creativecommons.org/licenses/by/4.0/)