Coupling Isoelectric Focusing Gel Electrophoresis to Mass Spectrometry by Electrostatic Spray Ionization

Abstract

Gel electrophoresis has been used for decades as a high-resolution separation technique for proteins and protein isomers but has been limited in the coupling with MS because of low throughput and poor automaticity compared with LC-MS. In this work, we have developed an ambient ionization strategy, electrostatic spray ionization, for in situ ionization of proteins or peptides inside a surfactant-free polyacrylamide gel. The samples can be first separated by isoelectric focusing in a gel and then quickly in situ detected by scanning the gel with the electrostatic spray ionization mass spectrometry. With this strategy, nanograms of proteins or peptides inside a band are enough to be ionized for MS detection. This method for protein/peptide spots visualization is sensitive, providing sample molecular weight information while avoiding spot staining and chemical extraction procedures that can introduce contaminants and sample loss. Proof-of-principle results have demonstrated that the electrostatic spray ionization can produce sample ions from a complex background, and with a spatial resolution matching the isoelectric focusing, it is therefore a good choice to couple directly isoelectric focusing gel electrophoresis with mass spectrometry.

Combining high-throughput MALDI-TOF mass spectrometry and isoelectric focusing gel electrophoresis for virtual 2D gel-based proteomics

“…Electrostatic spray ionization (ESTASI), an ambient ionization method [35] was previously applied to analyses from IPG gels, although that work is not directly comparable, in so far as the IEF was performed under native conditions; i.e ., without the urea, thiourea, surfactants, and ampholytes important to denaturing IEF and, correspondingly, to 2D-PAGE and theoretically predictable isoelectric points. For ESTASI, a spatial resolution of ~3 mm was calculated, based on modeling the electric field for the ESTASI assembly; the resolution attainable experimentally was not reported.…”

Fingerprinting the tertiary structure of electroadsorbed lysozyme at soft interfaces by electrostatic spray ionization mass spectrometry

“…This means that a monolayer of lysozyme on the hemispherical organogel (0.45 pmol) is below the LOD for this technique (1.63 pmol). 13 As a result, detection by ESTASI-MS requires that multilayers of lysozyme are adsorbed; this is reflected in the high [lysozyme] aq (0.5 to 10 µM) and long t adsorb (30 min) required before MS signals appear following electroadsorption (Fig. 2).…”

Principles and applications of solid-substrate electrospray ionization mass spectrometry

“…最近, Zhang等 [95] 采用滴加试剂 同时采用静电诱导, 直接从每个样品点产生电喷雾电 离. 为实现大分子的分离, 常用的凝胶电泳材料在静 电条件下可以实现凝胶条带内蛋白的喷雾电离 [85] . 与 表面修饰材料类似, 商品化的固相微萃取(SPME)探针 也可以作为电喷雾电离基底直接分析富集的样品 [87] ,…”