Ion soft-landing into liquids: Protein identification, separation, and purification with retention of biological activity

Abstract

Protein ions, after mass spectrometric separation, can be soft-landed into liquid surfaces with preservation of their native structures. Retention of biological activity is strongly favored in glycerol-based surfaces but not in self-assembled monolayer solid surfaces. Soft-landing efficiency for multiply-charged hexokinase ions was found to be some four times higher for a glycerol/fructose liquid surface than for a fluorinated self-assembled monolayer surface. Soft-landing into liquid surfaces is also shown to allow (1) protein purification, (2) on-surface identification of the soft-landed material using MALDI, and (3) protein identification by in-surface tryptic digestion. Pure lysozyme was successfully isolated from different mixtures including an oxidized, partially decomposed batch of the protein and a partial tryptic digest. Liquid glycerol/carbohydrate mixtures could be used directly to record MALDI spectra on the soft-landed compounds provided they were fortified in advance with traditional MALDI matrices such as p-nitroaniline and ␣-cyano-4-hydroxycinnamic acid. Various proteins were soft-landed and detected on-target using these types of liquid surface. Soft-landing of multiply-charged lysozyme ions onto fluorinated self-assembled monolayer surfaces was found to occur with a limited amount of neutralization, and trapped multiply-charged ions could be desorbed from the surface by laser desorption. Initial data is shown for a new approach to protein identification that combines top-down and bottom-up approaches by utilizing protein ion soft-landing from a protein mixture, followed by tryptic digestion of the landed material and detection of characteristic tryptic fragments by MALDI. , has been applied in experiments ranging from studies using chemically inert and structurally organized self-assembled monolayers (SAMs) as substrates for ion storage [2], to studies of ion mobility through ice and vapor deposited films [3,4], to experiments aimed at developing a separation method in a form of preparatory mass spectrometry [5,6]. Simple organic cations [7,8] [12][13][14] have all been the targets of examination by ion soft-landing. The separation, purification, and storage of chemical species by a completely non-destructive, mass spectrometric approach are the objectives of the studies currently underway in our laboratory.In a preliminary report [5], we described a method which showed the potential for generating biologically active protein chips from protein mixtures by massselective ion soft-landing. After ionization of the mixture by electrospray (ESI), individual protein ions were mass-selected and soft-landed at predetermined locations on the surface. The results showed that the use of mass spectrometry as a separation method in biology provides high selectivity because components with different molecular formulas can be separated and softlanded. However, retention of the biological activity of a protein or other biomolecule in the course of this (or any mass spectrometry experiment) is a much more chall...

Collision induced unfolding of protein ions in the gas phase studied by ion mobility-mass spectrometry: The effect of ligand binding on conformational stability

“…In a particularly insightful experiment, the Cooks group employed the method, previously used for the analysis of viruses using mass spectrometry [24], of 'soft-landing' 8ϩ lysozyme ions into liquid surfaces after mass spectrometric separation [25]. The collected enzyme was shown to exhibit normal activity against hexa-N-acetylchitohexaose substrate, suggesting that the structure of lysozyme was either preserved in the gas phase, or perturbed in such a way as to allow the native fold of the enzyme to resume upon solvation.…”

Effect of the surface on charge reduction and desorption kinetics of soft landed peptide ions

“…SL is defined as intact capture of mass-selected polyatomic ions on substrates [6,7] and has already been successfully used to study charge retention by small closedshell ions [6 -9] and peptide and protein ions deposited onto self-assembled monolayer surfaces (SAMs) [10 -13]. Retention of biological activity (but not charge) by proteins deposited on liquid [10,14] and on plasma treated metal surfaces [15] has also been demonstrated.Recently we reported the first detailed study of the kinetics of desorption and charge reduction following SL of doubly protonated Gramicidin S (GS) onto the FSAM surface [16]. We utilized an in-line 8 keV Cs ϩ ion gun that allowed us to interrogate the surface in situ and in real time using secondary ion mass spectrometry (SIMS).…”

“…SL is defined as intact capture of mass-selected polyatomic ions on substrates [6,7] and has already been successfully used to study charge retention by small closedshell ions [6 -9] and peptide and protein ions deposited onto self-assembled monolayer surfaces (SAMs) [10 -13]. Retention of biological activity (but not charge) by proteins deposited on liquid [10,14] and on plasma treated metal surfaces [15] has also been demonstrated.…”

<em>In Situ</em> SIMS and IR Spectroscopy of Well-defined Surfaces Prepared by Soft Landing of Mass-selected Ions

“…To date, a multitude of techniques have been applied for this purpose including secondary ion mass spectrometry (SIMS) 19,[97][98][99][100] , temperature programmed desorption and reaction 50,52 , laser desorption and ionization 101 , pulsed molecular beam reaction 102 , infrared spectroscopy (FTIR and Raman) 98,103,104 , surface enhanced Raman spectroscopy 103,105 , cavity ringdown spectroscopy 106 , xray photoelectron spectroscopy 35,107 , scanning tunneling microscopy 33,[108][109][110][111] , atomic force microscopy [112][113][114] , and transmission electron microscopy 39 . However, to most accurately characterize surfaces prepared or modified by ion soft landing, it is crucial that the analysis be performed in situ without exposure of the substrate to the environment in the laboratory.…”