Nanoelectrospray tandem mass spectrometry is a widely used technique for the identification of peptide sequences from 2-D gel-separated protein spots. This method involves excision of the protein spot of interest from the gel, followed by digestion with a proteolytic enzyme, typically trypsin, to produce a mixture of peptides.
The resulting peptides are then analyzed by nanoelectrospray tandem mass spectrometry, which involves the ionization of peptides in a small volume of solution and subsequent fragmentation in the mass spectrometer. The resulting mass spectra can be used to identify the amino acid sequence of the peptides and, subsequently, the protein from which they originated.
Database searching software is commonly used to match the observed peptides to a protein database. This allows for the identification of the protein present in the original sample. The accuracy of peptide sequencing by this method is greatly improved by the use of high-resolution mass spectrometry and the availability of comprehensive protein databases.
Peptide sequencing of 2-D gel-separated protein spots by nanoelectrospray tandem mass spectrometry is a powerful tool for the identification and characterization of proteins in complex mixtures. This technique has been used in a variety of fields, including proteomics, drug discovery, and biomarker identification.
1. Filling Nanospray Needles for Static Nanospray
The two main methods for loading samples into nanospray needle tips, using fusedsilica needles and gel-loading tips, are described below.
1.1 Fused-Silica Needles Connected to a Conventional Syringe
a) Substitute a fused-silica needle for a stainless-steel needle of a conventional syringe (with a maximum volume of 10 or 25 μL) from Hamilton and other suppliers.
b) Centrifuge the sample in a bench-top centrifuge to remove particulates, and load supernatant into the syringe.
c) Insert the filling needle into the distal end of the nanospray needle.
d) Push the needle as far in as possible without damaging either the filling needle or the nanospray needle tip. It is not critical to reach right into the end of the tapered region, since capillary action will fill the tip.
e) Slowly inject the liquid into the PicoTip. Rapid injection can lead to air bubbles, or foaming of the sample. This foaming is especially problematic with concentrated protein and peptide samples. To prevent this, inject sample (1–5 μL) over a period of approx 5 s.
f) Slowly withdraw the filling needle from the PicoTip. The tip will fill by capillary action. Air bubbles may appear along the shank of the tip, but as the sample sprays from the tip, capillary action will provide a continuous feed and should eliminate the air bubbles in the taper region.
1.2 Gel-Loader Tips
Gel-loader disposable pipet tips with an o.d. less than or equal to 0.35 mm are also used to load samples into nanospray needle tips.
a) Centrifuge the sample in bench-top centrifuge to remove particulates and load supernatant into the smallest-o.d. gel-loader tip available for your particular make of pipet.
b) Insert the pipet tip as far as possible into the distal end of the nanospray needle tip (emitter) and deliver 1–5 μL of the liquid slowly while removing the pipet tip. A typical gel-loader tip is not sufficiently long to extend all the way to the tip of the emitter. The sample will fill only the back end of the tip, but capillary action will bring the sample into the tip.
c) Wait a few minutes for this filling action to take place, if necessary.
d) Inspect for proper filling with transmitted light (rather than reflected light) microscope at ×50–100.
2 Optimizing Nanospray
Applied voltage is perhaps the most important parameter for stable, efficient operation. To prevent an arc or corona discharge, do not use a turn-on voltage above 500 V unless a stable electrospray ionization (ESI) has been previously established.
a) Position tip with respect to the center of the ion-transfer tube using x and y adjustments on the xyz stage. This should be within 1 mm of center.
b) Using the z adjustment knob, position emitter approx 2 mm away from ion transfer tube.
c) Start tuning at a low voltage, under 1 kV, and increase the operating potential in 100 V increments until stable operation is achieved. In low-flow electrospray, the flow rate and the applied electric field are interdependent. For a given tip size, stable electrospray ionization can occur over a wide range of flow rates, but only over a narrow range of field strength (50 V or less). Raising the flow rate requires a higher field strength, and vice versa.
d) Tune for an even electrospray ionization plume of droplets, by observing the magnified image of the spray pattern on the video monitor.
e) Flow can be maintained by increasing pressure slightly with the back-pressure syringe.
f) Full optimization will depend on the MS instrument type, and will require different optimization parameters for dynamic nanospray.
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Reference
- Walker, J. M. (Ed.). (2005). The proteomics protocols handbook. Humana press.
