A Microfluidic-Fabricated Rod Sprayer for Nanoelectrospray Mass Spectrometry.

The nanoelectrosprayer is a key device in the hyphenation of nanoLC-ESI-MS, and its development plays a crucial role in pushing forward the mining depth of biological discovery and industrialization of omics science. In this work, a new type of nanoelectrospray emitter, a rod sprayer, was developed based on microfluidic manufacture. Due to its porous silica structure, the rod sprayer in effect worked as a multinozzle sprayer, which is composed of a bunch of micrometer sized spray channels. Without the need for sophisticated microfabrication equipment, a superclean environment, or a complicated assembling process, such sprayer rods can be facilely fabricated in a mass production style: 3,600 rods with excellent monodispersity have been fabricated in 1 h, and rod sprayers thus made have demonstrated excellent intraday, interday, and interbatch reproducibilities: RSD = 1.9, 4.9, and 6.1%, respectively. The rod sprayer can generate stable electrospray in a wide voltage range from 2.6 to 3.2 kV and flow rates from 50 to 1000 nL/min, covering typical flow rates of subnanoLC, nanoLC, to microLC, and work steadily even under complex matrix environments (e.g., Hank's balanced salt solution containing sodium, magnesium, and calcium ions) without clogging. Meanwhile, the rod sprayers exhibited 200-1800% ionization efficiency enhancement in comparison with commonly used tapered tip emitters, for small molecule drugs, peptides, and proteins, respectively, and provided a broadened linear dynamic range of 4 orders of magnitude. The excellent characteristics of the rod sprayer, together with its small size and mass production capacity, should provide a high quality, high durability, high consistency, and disposable use-supported nanoelectrospray solution for MS-based bioanalyses.

Performance of nanoflow liquid chromatography using core-shell particles: A comparison study.

Due to its unique structure, core-shell material has presented significantly improved chromatographic performance in comparison with conventional totally porous material. This has been well demonstrated in the analytical column format, e.g. 4.6 mm i.d. columns. In the proteomics field, there is always a demand for high resolution microseparation tools. In order to explore core-shell material's potential in proteomics-oriented microseparations, we investigated chromatographic performance of core-shell material in a nanoLC format, as well as its resolving power for protein digests. The results show core-shell nanoLC columns have similar van Deemter curves to the totally porous particle-packed nanoLC columns. For 100 µm i.d. capillary columns, the core-shell material does not have significantly better dynamics. However, both core-shell and totally porous particle-packed nanoLC columns have shown high efficiencies: plate heights of ~11 µm, equivalent to 90000 plates per meter, have been achieved with 5 µm particles. Using a 60 cm long core-shell nanoLC column, 72000 plates were realized in an isocratic separation of neutral compounds. For a 15 cm long nanoLC column, a maximum peak capacity of 220 has been achieved in a 5 hour gradient separation of protein digests, indicating the high resolving power of core-shell nanoLC columns. With a standard HeLa cell lysate as the sample, 2546 proteins were identified by using the core-shell nanoLC column, while 2916 proteins were identified by using the totally porous particle-packed nanoLC column. Comparing the two sets of proteomics data, it was found that 1830 proteins were identified by both columns, while 1086 and 716 proteins were uniquely identified by using totally porous and core-shell particle-packed nanoLC columns, respectively, suggesting their complementarity in nanoLC-MS based proteomics.

Direct Infusion ICP-qMS of Lined-up Single-Cell Using an Oil-Free Passive Microfluidic System.

When coupled online with mass spectrometry (MS), widely applied water-in-oil droplet-based microfluidics for single cell analysis met problems. For example, the oil phase rumpled the stability, efficiency, and accuracy of MS, the conventional interface between MS and the microfluidic chip suffered the low sample introduction efficiency, and the transportation rates sometimes unmatched the readout dwell times for transient signal acquisition. Considering cells are already "droplets" with hydrophilic surface and elastic hydrophobic membrane, we developed an oil-free passive microfluidic system (OFPMS) that consists of alternating straight-curved-straight microchannels and a direct infusion (dI) micronebulizer for inductively coupled plasma quadrupole-based mass spectrometry (ICP-qMS) of lined-up single-cell. OFPMS guarantees exact single cell isolation one by one just using a thermo-decomposable NH4HCO3 buffer, eliminating the use of any oil and incompatible polymer carriers. It is more flexible and facile to adapt to the dwell time of ICP-qMS owing to the adjustable throughput of 400 to 25000 cells/min and the controllable interval time of at least 20 ms between the lined-up adjacent single cells. Quantitative single-cell transportation and high detection efficiency of more than 70% was realized using OFPMS-dI-ICP-qMS exemplified here. Thus, cell-to-cell heterogeneity can be simply uncovered via the determination of metals in the individual cells.

Towards a high peak capacity of 130 using nanoflow hydrophilic interaction liquid chromatography.

Hydrophilic interaction liquid chromatography, HILIC, is a relatively new HPLC mode. Compared with other HPLC modes, HILIC is a high resolution chromatographic mode with high peak capacity for separations of complex mixtures. Although the separation mechanism is still not completely clear, HILIC has been widely used for analysis of hydrophilic compounds which are difficult for reversed phase chromatography to retain and separate. In this study, we fabricated and investigated nanoHILIC columns in terms of separation efficiency, van Deemter curves and more importantly, we focused on long packed capillary columns, and studied their extreme resolution for protein digests. Using meter long nanoHILIC columns packed with 5 µm particles, we realized a high peak capacity of 130. Based on nanoLC-MS, we compared the resolution and protein identification capabilities of nanoHILIC and nanoRPLC. The results indicate both nanoHILIC and nanoRPLC can provide high resolution for protein sequencing but neither mode is significantly better than the other. Among the 99 digest peptides identified, 17 were uniquely identified by nanoHILIC-MS and 20 were uniquely identified by nanoRPLC-MS and 62 were identified by both methods. Although at this moment in time, nanoRPLC is the most popular microseparation tool in proteomics, the excellent complementarity of nanoHILIC and nanoRPLC suggests their combined use in achieving deep-coverage in MS-based proteomics.