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.

Mass Spectrometry Analysis of Intact Proteins from Crude Samples

“… 57 (2) Direct liquid extraction techniques, such as liquid extraction surface analysis (LESA), in which the sample is extracted from the surface by dispensing a solvent on its surface from a pipet tip. This creates a liquid junction between the tip and the sample surface, allowing analytes to dissolve and reaspire with a conductive pipet tip prior to ESI-MS. 2 (3) Flow-based techniques, such as nanospray desorption electrospray ionization (nano-DESI), in which a solvent bridge is created between two capillaries and the surface of interest. The main difference between nano-DESI and LESA is the configuration of the capillaries.…”

“…Taken together, ambient surface analysis techniques benefit from the fact that sample preparation or disruption procedures are not required, enabling preserving a more biologically relevant environment compared to MALDI imaging methods. 2 Moreover, the multiple charge states generated by electrospray ionization enables top-down analysis by a range of techniques, such as collision-induced dissociation (CID), electron capture dissociation (ECD), or electron transfer dissociation (ETD). 2 Another major advantage is the integration of ion mobility separation with the imaging workflows to analyze folded proteins and protein complexes in a spatially defined manner.…”

“… 2 Moreover, the multiple charge states generated by electrospray ionization enables top-down analysis by a range of techniques, such as collision-induced dissociation (CID), electron capture dissociation (ECD), or electron transfer dissociation (ETD). 2 Another major advantage is the integration of ion mobility separation with the imaging workflows to analyze folded proteins and protein complexes in a spatially defined manner. Nevertheless, the method is still limited by the ionization efficiency of protein assemblies, restricting the analysis of low-abundance and large species.…”

Protein identification by 3D OrbiSIMS to facilitate in situ imaging and depth profiling

“…However, routine analysis of proteins with this technique uses enzymatic digestion and the application of a matrix, which may modify the structure of the sample surface and consequently the spatial distribution of analytes in two and three dimensions 7 . Other methods used for protein characterization at surfaces are desorption electrospray ionization 8 and liquid extraction surface analysis MS 9 ; however, these techniques have limited lateral resolution (>100 μm) 10 .…”

High Performance Molecular Imaging with MALDI Trapped Ion Mobility Time-of-Flight (timsTOF) Mass Spectrometry

“…[31][32] Multiple types of ion mobility separations exist, including drift tube ion mobility (DTIM), [33][34] travelling-wave ion mobility spectrometry (TWIMS), [35][36] field asymmetric waveform ion mobility spectrometry (FAIMS) [37][38][39] and more recently, trapped ion mobility spectrometry (TIMS). [40][41] The three former techniques have been previously utilized in the imaging domain, studying metabolites, [42][43] lipids, 44 peptides [45][46] and proteins [46][47] , improving the separation, specificity and identification of species. In recent years, however, TIMS has shown unparalleled resolving power, proving extremely popular in theomics fields.…”