In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS

Summary

This work presents a protocol for liquid handling and sample introduction to a microchannel for in situ time-of-flight secondary ion mass spectrometry analysis of protein biomolecules in an aqueous solution.

Abstract

This work demonstrates in situ characterization of protein biomolecules in the aqueous solution using the System for Analysis at the Liquid Vacuum Interface (SALVI) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). The fibronectin protein film was immobilized on the silicon nitride (SiN) membrane that forms the SALVI detection area. During ToF-SIMS analysis, three modes of analysis were conducted including high spatial resolution mass spectrometry, two-dimensional (2D) imaging, and depth profiling. Mass spectra were acquired in both positive and negative modes. Deionized water was also analyzed as a reference sample. Our results show that the fibronectin film in water has more distinct and stronger water cluster peaks compared to water alone. Characteristic peaks of amino acid fragments are also observable in the hydrated protein ToF-SIMS spectra. These results illustrate that protein molecule adsorption on a surface can be studied dynamically using SALVI and ToF-SIMS in the liquid environment for the first time.

Introduction

Hydration is crucial to the structure,¹ conformation,² and biological activity³ of proteins. Proteins without water molecules surrounding them would not have viable biological activities. Specifically, water molecules interact with the surface and internal structure of proteins, and different hydration states of proteins make such interactions distinct.⁴ The interaction of proteins with solid surfaces is a fundamental phenomenon with implications in nanotechnology, biomaterials and tissue engineering processes. Studies have long indicated that conformational changes may occur as a protein encounters a surface. ToF-SIMS has b....

Protocol

1. Cleaning and Sterilization of the SALVI Microchannel

1. Sterilization of the Microchannel in SALVI
   1. Draw 2 ml of 70% ethanol aqueous solution into a syringe, connect the syringe with the inlet end of SALVI, and slowly inject 1 ml of the liquid in 10 min. Remove the syringe at the end of injection. Next, connect the inlet and outlet of SALVI using a polyetheretherketone (PEEK) union. Alternately, use a syringe pump to conduct the same procedure. For example, set the flow speed at 100 μl/min.
   2. Keep the SALVI microchannel filled with 70% ethanol solution at room temperature for 4 hr.
2. Introduce Deionized (DI) W....

Results

A couple of representative results are presented to demonstrate the advantages of the proposed protocol. By using the SALVI microfluidic interface, the primary ion beam (Bi₃⁺) can directly bombard on the hydrated fibronectin film in DI water. Thus the molecular chemical mapping of the liquid surface can be successfully acquired.

Figure 1a and 1b show the positive ToF-SIMS m.......

Discussion

SALVI is a microfluidic interface that allows dynamic liquid surface and liquid-solid interface analysis by vacuum based instruments, such as ToF-SIMS and scanning electron microscopy (SEM). Due to the usage of small apertures to expose liquid directly in vacuum, SALVI is suitable for many finely focused spectroscopy and imaging techniques without any modifications;²² the portability and versatility of microfluidics make it a true multimodal imaging platform. Distinct features and significance of SALVI compare.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
ToF-SIMS	IONTOF	TOF.SIMS 5	Resolution: >10,000 m/Δm for mass resolution; >4,000 m/Δm for high spatial resolution
System for Analysis at the Liquid Vacuum Interface (SALVI)	Pacific Northwest National Laboratory	N/A	SALVI is a unique, self-contained, portable analytical tool that, for the first time, enables vacuum-based scientific instruments such as time-of-flight secondary ion mass spectrometry (ToF-SIMS) to analyze liquid surfaces in their natural state at the molecular level.
PEEK Union	Valco	ZU1TPK	for connecting the inlet and outlet of SALVI
5 Axes Sample Stage	IONTOF	N/A	Stage is self-made for mounting SALVI in ToF-SIMS
Barnstead Nanopure Water Purification System	Thermo Fisher Scientific	D11921	ROpure LP Reverse Osmosis filtration module (D2716)
Syringe	BD	309659	1 ml
Pipette	Thermo Fisher Scientific	21-377-821	Range: 100 to 1,000 ml
Pipette Tip	Neptune	2112.96.BS	1,000 µl
Centrifuge Tube	Corning	430791	15 ml
Fibronectin	Sigma-Aldrich	F1141	1 mg/ml
Ethanol	Thermo Fisher Scientific	S25310A	95% Denatured
Gibco PBS	Thermo Fisher Scientific	10010-023	pH 7.4