Separating protein complexes into discreet, identifiable components and monitoring changes in proteins which are expressed at low levels are 2 challenges which face most who are working to further characterize their protein of interest. Mass spectrometry can address both of these issues due to the discriminatory nature of a protein’s intrinsic mass property. However, chromatography is often needed as an initial filtering tool to avoid overly complex spectra (which can lead to questionable identifications). Optimal sensitivity is an important goal when considering mass spectrometry for proteomic studies. Generally, electrospray ionization is a sensitive interface between liquid chromatography and the mass spectrometer. As liquid effluent flows from the separation column to the mass analyzer, the liquid is nebulized and rapidly vaporized. The more efficient this process is, the cleaner the signal will be, due to lower chemical noise. A “nanospray” interface allows for low flow rate which has several benefits. First, there is less sample consumption due to smaller injection volume requirements. Second, less solvent consumption is needed due to lower flow rates. Third, less aggressive nebulization means are necessary to get the sample to vaporize. Fourth, less solvent delivery means less noise and thus potentially higher signal-to-noise.
Nanospray interfaces have come to the foreground in the mass spectrometry industry; this is especially true when proteomics is the target application. Nano-electrospray flows are much slower than conventional electrospray (between 1 and 300 nL/min). Low flow rates require special electrospray emitters which taper down to a very narrow orifice (1 to 30 µm) which Proxeon supplies; similar emitters can also be obtained from other manufacturers. Mass spectrometry manufacturers typically offer their own version which will interface with their instrument. Proxeon offers nanospray source configuration kits that can be purchased for different commercially-available mass spectrometers (the Sciex API-3000 along with the Thermo LCQ-Deca and LTQ ion traps are used in this laboratory). We have found that Proxeon’s nanospray interface has distinct advantages over those supplied by other companies. The routine use of small emitters requires special manipulation and visualization features which help insure that the best flow rate accompanies proper nebulization. The Proxeon system is also a completely open interface (atmospheric pressure) which allows for easy manipulation, tip placement and observation by both direct visualization and through closed circuit monitors (available as a complete kit through Proxeon). The nanoSpray stage can be manipulated in three dimensions, as is typical. Set up is relatively easy but can be considered involved to a novice. For an Applied Biosystems instrument, it involves the reconfiguration of the front end voltage plugs and exchange of the orifice plate with one provided by Proxeon. This set up provides optimal proximity to the orifice during effluent sampling. As is typical for nanoSpray interfaces, some practice is required to be able to find the right stage position, but Proxeon’s open configuration makes this task easier. There are two modes that can be employed: 1) off-line which employs emitters into which the sample is placed, and 2) Micro-to-nanoflow LC-coupling through an electrode-housed tee and fluid split line. Both worked very well once configured to our analytical needs.
Some caution must be exercised when using these independently manufactured sources, however. Because they are not made to be ‘read’ by the host instrument, typical voltages may not be properly set, leading to arcing or improper ion steering through the sample orifice. The Proxeon manuals do a good job of suggesting proper settings to ensure successful ion transmission while avoiding potential damage to emitters and the instrument. It is our experience that taking the time to gain optimal ionization yields excellent, reproducible results. This is independent of the source manufacturer.
Ronald A. Miller
Research Biochemist
Merck Research Laboratories
Department of Alzheimer's Disease Research