Native Desorption Electrospray Ionization Liberates Soluble and Membrane Protein Complexes from Surfaces

Abstract

Mass spectrometry (MS) applications for intact protein complexes typically require electrospray( ES) ionization and have not been achieved via direct desorption from surfaces.D esorption ES ionization (DESI) MS has however transformed the study of tissue surfaces through release and characterisation of small molecules.Motivated by the desire to screen for ligand binding to intact protein complexes we report the development of an ative DESI platform. By establishing conditions that preserve non-covalent interactions we exploit the surface to capture ar apid turnover enzyme-substrate complex and to optimise detergents for membrane protein study.W ed emonstrate binding of lipids and drugs to membrane proteins deposited on surfaces and selectivity from am ix of related agonists for specific binding to aG PCR. Overall therefore we introduce this native DESI platform with the potential for high-throughput ligand screening of some of the most challenging drug targets including GPCRs.A range of applications,i ncluding 2D imaging of small molecules and metabolites released from tissue cross-sections, has become possible with the introduction of powerful DESI approaches when coupled with MS. [1,2] Thep rimary goal of DESI applications has been to focus on the small molecules released, for example in the real-time detection of tumour tissue during surgical procedures. [3,4] While DESI has also been adapted to study large biomolecules,v ia the mixing of ES droplets and solution in ap rocess known as liquid DESI, [5,6] it has not yet been applied to proteins deposited on surfaces and desorbed in solutions that retain their native state interactions.D espite considerable progress in applications of non-denaturing or native MS (nMS) of soluble [7][8][9] and membrane embedded proteins [10] the possibility of effectively "lifting" intact complexes from surfaces is desirable since many high throughput technologies then become accessible.[11] Moreover the lipid distribution in natural membranes is essentially planar and asymmetric with varying spatial and temporal arrangements in the vicinity of embedded protein complexes.[12] Thel ipid distribution and the desire for as urface technology that is also able to analyse membrane proteins motivated us to develop amodified DESI approach capable of releasing folded protein molecules from planar surfaces and to construct an interface that we could couple to ah igh-resolution Orbitrap MS optimised for high mass transmission [13] of membrane proteins. [14] We demonstrate the potential of this methodology in three ways 1) by capturing transient protein substrate products 2) by screening for optimal solution/purification conditions using picoMoles of membrane and soluble proteins and 3) by carrying out ligand binding experiments on planar surfaces.We modified the design of the original DESI set-up [1] and with our custom-built ion source,ESdevice and sample stage coupled our interface to an Orbitrap Q-Exactive (Figure 1a and Supporting Information Figure S1). We found that signal i...

Ambient ionisation mass spectrometry for in situ analysis of intact proteins

“…Noncovalent complexes between the ligand biotin and proteins avidin, bovine serum albumin, and haemoglobin were detected as shown in Figure B. Similar studies have recently been described for native DESI analysis of purified samples of intact proteins and protein assemblies; Ambrose et al describe detection of monomeric proteins such as apo lysozyme and bovine serum albumin, complexes of tetrameric alcohol dehydrogenase and tetradecameric GroEL using ammonium acetate solutions . They also show that native DESI is suitable for the analysis of membrane proteins, although some detergent sensitivity is exhibited, and for probing noncovalent protein interactions in the example of NAG‐5 bound to lysozyme.…”

“…Progress has since been made to mitigate these effects by use of solvent additives . In another approach, by modifying the DESI set‐up itself, DESI mass spectra of native protein complexes of up to 800 kDa in size (tetradecameric GroEL) spotted onto glass slides have recently been recorded . An alternative approach for analysing the surface layer of liquid samples generated ions of protein complexes of approximately 150 kDa .…”

Infrared Laser Desorption and Electrospray Ionisation of Non‐Covalent Protein Complexes: Generation of Intact, Multiply Charged Species

“…However, LILBID is still an infusion‐based technique and – similar to MALDI – produces mostly singly and doubly charged ions [10] . Desorption electrospray ionisation (DESI) has been successfully applied to non‐covalent protein complexes by the group of Robinson, demonstrating desorption of spray‐deposited protein complexes from a surface [11] . Additionally, liquid extraction surface analysis (LESA), a nano‐ESI based surface sampling method has also been shown to be compatible with native‐MS [12]…”

“…[10] Desorption electrospray ionisation (DESI) has been successfully applied to noncovalent protein complexes by the group of Robinson, demonstrating desorption of spray-deposited protein complexes from a surface. [11] Additionally, liquid extraction surface analysis (LESA), a nano-ESI based surface sampling method has also been shown to be compatible with native-MS. [12] Shiea et al [13] and others [14] demonstrated laser ablation electrospray ionisation (LAESI)-based analysis of various small proteins, such as myoglobin, cytochrome C and hemoglobin sub-units. These results were obtained with traditional electrospray solvents and additives, containing a high organic modifier percentage and high acidity.…”

Preservation of Protein Zwitterionic States in the Transition from Solution to Gas Phase Revealed by Sodium Adduction Mass Spectrometry

“…The analysis of proteins and their complexes is an important endeavor as nearly all biological processes involve regulated interactions between proteins and other biomolecules (e.g., DNA, RNA, cofactors, and other proteins) in both time and space . Because of its incomparable sensitivity, speed, and specificity, electrospray ionization mass spectrometry (ESI-MS) has emerged as an ideal tool for protein analysis. − The last two decades have seen the rapid development in an area of gas-phase structural biology, which aims to leverage the advantages of MS analysis for elucidation of a three-dimensional protein structure with the combination of ion mobility, fragmentation, and spectroscopy. Thus, it is of foundational significance to determine the extent of the native structure of a protein retained after desolvation. − It is now well-established that the nativelike compact conformer of a protein can be maintained after desolvation made evident by the relatively low charge states of a gaseous protein produced from nondenaturing aqueous solution by ESI. − A recent study that combined ESI-MS with ion mobility measurements and soft landing techniques and further made convincing the survival of the overall compactness of many proteins when desolvated. − …”