Nanospray sources

A number of different types of ESI sources, known as nanospray sources, have been designed that can operate at lower sample flow rates (10-200 nL min ). These generate smaller droplets and improve the signal intensity of the protein-ligand noncovalent complexes further, with the added benefit of reducing protein consumption up to 100-fold compared to standard ESI flow rates. Nanospray has also been reported to be more tolerant to nonvolatile cations in solution [37]. Recently, an automated fabricated chip nanospray source has been developed. This chip-based device has improved the ease-of-use for nanospray, while the design eliminates carryover effects as the spray is produced directly from an orifice on each sample well of the chip [38]. 

An ESI mass spectrometer equipped with a standard or a nanospray source and with the option for MSn may be used (Q-TOF, Ion Trap, etc.) as well as appropriate software. Ion Trap mass spectrometer (Esquire-LC, Bruker) equipped with a nanospray source was used. 

For nano-ESI-MS MS , the sample is loaded either in a previously rinsed gold/palladium-coated nanospray capillary (Promega) or in a PicoTip emitter (New Objective) and analyzed in a nanospray source. 

For static nanoESI-MS connect the static nanospray source (Protana, Odense, Denmark) to the mass spectrometer and use for each sample to be analyzed a new borosilicate spray capillary (ES380 Medium , PROXEON Biosystems AIS, Odense, Denmark see Note 3). 

Buffer Systems. Buffer systems should be preferentially volatile and of low concentration. Detergents are particularly detrimental to the ESI process. Volatile buffers should be prepared fresh every day to maintain a stable pH, EOF, and thus stable ion signal. Solvent evaporation, and/or buffer depletion in the reservoirs, will result in a change in pH, EOF, and ESI signal response. Thus, the buffers should be replenished on a regular basis. The EOF must be maintained in the direction of the ESI emitter, especially when nanospray sources are used (i.e., no liquid sheath or liquid junction fluids are delivered to the source). 

Interfaces Between Microfluidics and Mass Spectrometry, Fig. 2 Two nanospray sources (a) nozzle fabricated by micro milling of PMMA (Reproduced by permission of the Royal Society of Chemistry [4]) (b) nozzle formed by sandwiching a 2D parylene tip between cover plates (Reprinted with permission from Kameoka et al. [6]. Copyright 2002 American Chemical Society)... 

One of the limitations in the use of capillary columns is an irreproducible and unstable gradient in the nL/min flow range. A few years ago, the standard procedure to achieve reliable gradients was to use precolumn flow splitting [44], Any conventional or micro-LC pumping system could serve to deliver low flow rates by using an appropriate homemade or commercial flow splitter. Currently, LC pumps for capiflary colunms have become commercially available that can couple capillary colunms directly with a nanospray source [45]. 

Online intact protein separation was the same as for the Q-TOF LC-MS (above) for consistent protein retention times across platforms. For LC-MS/MS the eluent flow was split to a flow rate of 350 nL/min via the TriVersa NanoMate (Advion BioSci-ences, Ithaca, NY) chip-based nanospray source and analyzed with a LTQ-Oibitrap XL (Thermo Fisher, San Jose, CA) mass spectrometer. The instrument was operated in a top-three data dependent mode, with both MS spectra and collision-induced dissociation (CID) MS/MS spectra acquired at 60,000 resolving power in the Oibi-trap. CID collision energy was operated at 15 %. Each MS spectrum was composed of three microscans, and each MS/MS spectrum was the average of 10 microscans. To facilitate the analysis of intact proteins, the instrument was operated with the HCD gas off and the delay before image current detection shortened to 5 ms. 

Figure 2,2. Demonstration of how electrospray response of histidine changes very little as solution pH is adjusted from 3 to 11. Each panel displays data collected on a different triple quadrupole mass spectrometer (a) Quattro II (Micromass) with an electrospray source, (b) API 300 (Sciex) with a pneumatically assisted electrospray source, (c) API III (Sciex) with a pneumatically assisted electrospray source, (d) API III (Sciex) with a nanospray source. (Reprinted from Ref. 13, with permission.)...

One of the major attractions of ESI is its ability to serve as an interface between liquid chromatography and mass spectrometry. There are currently a number of low-flow HPLC systems on the market that are compatible with electrospray, microspray, and nanospray sources. Capillary HPLC systems are interfaced with electrospray conducted in the pL/min flow regime, while nanoflow systems can accommodate nL/min flow rates. When electrospray is coupled with conventional HPLC, it is necessary to accommodate a higher sample flow rate ( 0.1—2mL/min) than normal electrospray can tolerate. To facilitate operation at these higher flow rates, a technique called pneumatically assisted electrospray or ion spray is employed, in which sample nebulization by aflow of gas is used to stimulate a more... 

Different options are available for LC-MS instruments. The vacuum system of a mass spectrometer typically will accept liquid flows in the range of 10-20 p,L min-1. For higher flow-rates it is necessary to modify the vacuum system (TSP interface), to remove the solvent before entry into the ion source (MB interface) or to split the effluent of the column (DLI interface). In the latter case only a small fraction (10-20 iLrnin ) of the total effluent is introduced into the ion source, where the mobile phase provides for chemical ionisation of the sample. The currently available commercial LC-MS systems (Table 7.48) differ widely in characteristics mass spectrometer (QMS, QQQ, QITMS, ToF-MS, B, B-QITMS, QToF-MS), mass range m/z 25000), resolution (up to 5000), mass accuracy (at best <5ppm), scan speed (up to 13000Das-1), interface (usually ESP/ISP and APCI, nanospray, PB, CF-FAB). There is no single LC-MS interface and ionisation mode that is readily suitable for all compounds... 

Estimating the amount of a metabolite when an authentic reference standard is not available is still a challenge. Yu et al.191 described a procedure that uses the results of an in vitro metabolite identification based on a test compound that produces 14C-labelled metabolites essentially the 14C-labelled metabolites are used to provide a correction factor for the MS response when assaying samples that contain the same metabolite in a study that did not use the 14C-labelled test compound. Flop192 described another novel approach for metabolite quantitation based on the observation that the MS responses for most compounds are very similar to responses from nanospray ESI. Valaskovic et al.193 also reported equimolar MS responses for multiple compounds when the flow rate to the nanospray ESI source was set to about 10 nl/min. It is too soon to know whether these intriguing findings can be readily applied to discovery metabolite identification studies. 

Figure 14.5 Modified-ESI source for the direct infusion of undiluted ILs. A stainless steel wire is placed in the spray, leading to the optimal vaporization of the IL. Additionally, an orthogonal ESI source is used. Only a part of the IL ions is transferred into the MS, thus minimizing pollution of the source. (Modified from Dyson, R J. et al.. Direct probe electrospray (and nanospray) ionization mass spectrometry of neat ionic liquids. Chem. Commun., 2204, 2004. Reproduced by permission of the Royal Society of Chemistry.)...

Figure 31 Schematic diagram of the nanospray tip for the electrospray source. (Reprinted with permission from Ref 435a. 2003 American Chemical Society)...

The nano-electrospray (nanoES) source is essentially a miniaturized version of the ES source. This technique allows very small amounts of sample to be ionized efficiently at nanoliters per minute flow rates and it involves loading sample volumes of 1-2 pi into a gold-coated capillary needle, which is introduced to the ion source. Alternatively for on-line nanoLC-MS experiments the end of the nanoLC column serves as the nanospray needle. The nanoES source disperses the liquid analyte entirely by electrostatic means [27] and does not require assistance such as solvent pumps or nebulizing gas. This improves sample desolvation and ionization and sample loading can be made to last 30 minutes or more. Also, the creation of nanodroplets means a high surface area to volume ratio allowing the efficient use of the sample without losses. Additionally, the introduction of the Z-spray ion source on some instruments has enabled an increase in sensitivity. In a Z-spray ion source, the analyte ions follow a Z-shaped trajectory between the inlet tube to the final skimmer which differs from the linear trajectory of a conventional inlet. This allows ions to be diverted from neutral molecules such as solvents and buffers, resulting in enhanced sensitivity. 

FIGURE 1.10 Extracted ion chromatograms imlz 630) for Indinavir metabolites generated from a rat liver microsomal incubation at ( A) 200 aL/min through a conventional ESI source, and (B) 0.1 aL/min from a 1000 1 postcolumn nanosplitter with a 5- j.m-ID fused-silica nanospray emitter. (Reprinted from Gangl et al. [102], used with permission.)... 

The ES/MALDI-FT-ICR mass spectrometer of the Institute of Organic Chemistry at the University of Tubingen is from Bruker Daltonik GmbH, Bremen. The system is evacuated by efficient turbo molecular pumps which allows HPLC and GC coupling over long time periods. ES, NanoSpray and MALDI ionization sources allow individual adaptation to particular problems. Samples from combinatorial chemistry can be routinely analyzed with the 60°-ESI from Analytica of Branford Inc. (Branford, MA) via LC/ES-FT-ICR-MS as well as small amounts of valuable biological samples with nanoESI. 

In addition to this, we have investigated different ways to apply a HV on the liquid. Applying HV directly onto the silicon support of the nib turned out to give smoother and more reliable ionization conditions for nanospray purposes than using a metallic wire in contact with the solution. Moreover this route alleviates any problem linked to the deterioration of the conductive coating present on standard ionization sources, which often results in analysis degradation. Therefore, these nib-shaped sources should allow enhancement of analysis conditions in continuous mode for on-line analysis. 

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