Protein Protocols Protein Protocols

696 Jensen and Wilm
10, 200; 2–490, 160, 12, 165. The first heating/pulling stage reduces the diameter of
the capillary to about 0.5 mm, while the second stage pulls the glass capillary apart,
producing two nanoelectrospray needles. These needles should have an opening of
1–2 µm. However, after pulling the opening diameter can be less than 100 nm and has
to be widened (see Methods). Nanolectrospray needles and ion sources are commercially
available from New Objective (Boston, MA) and MDS Proteomics (Odense,
Denmark) as well as from several MS instrument manufacturers.
Liquid injected into the needle is drawn to the tip by capillary force. To reduce the
flow resistance for a stable flow rate in the 10–25 nL/min range the narrow part of the
tip should not be longer than 500 µm (Fig. 1). Needles with very short constrictions
(50–100 µm) can be operated easily but with a higher risk of losing sample due to a
higher flow rate. Short needles are preferred for rapid mass measurements when abundant
sample is available, for example, recombinant proteins, synthetic peptides, or oligonucleotides.
Longer tips (200–500 µm) are used for tandem mass spectrometry
experiments when the longest possible operation time is desirable and when the sample
load volume will not exceed 1 µL. A major advantage of these types of nanelectrospray
needles is that they do not easily block due to the relatively short length of the needle tip.
Metal coating of the glass capillaries is achieved by metal (gold) vapor deposition in
a sputter chamber (Polaron SC 7610 sputter coater, Fisons Instruments, East Sussex,
UK). The needles are used only once so it is not a problem that the coating is not tightly
fixed to the glass and can be rubbed off. Methods to produce a more stable metal coating
include pretreatment with (3-mercaptopropyl)trimethoxysilane (9) or protecting
the metal layer by a second layer of SiO x (10). A stable gold coating is necessary when
a glass needle is used for several samples over a prolonged time. As the needle tip is
fragile it should be handled carefully when loading the sample and when mounting it in
the holder.
2. Methods
2.1. Protocol 1. Operation of the Nanoelectrospray Ion Source
Nanoelectrospray ion sources are now optional on a number of electrospray mass
spectrometers or they can be custom made for individual instruments. Note that
electrospray ion sources are operated at high voltages and care should be taken to avoid
electrical shock.
1. The geometrical location of the ion source.
The nanoelectrospray source is mounted directly in front of the orifice of the mass spectrometer,
usually at a distance of 1.5–2 mm from the orifice. Electrospray at a low flow
rate generates very small droplets with a diameter of 200 nm or less. Desolvation is therefore
achieved in a very short time and distance.
2. The voltage applied to the source.
To initiate the electrospray a minimal electrical field strength at the surface of the liquid
has to be reached (11). Conventional electrospray ion sources are operated with a 3 –5 kV
potential difference between the needle and counterelectrode (i.e., the orifice plate of the
mass spectrometer). The very small tip diameter of the nanoelectrospray needle allows a
spray cone to be established at a much lower electrical potential, typically 500–900 V.
3. The desolvation conditions in the interface region.
Because the charged droplets generated by the nanoelectrospray ion source are very small,
softer desolvation conditions in the interface (skimmer) region of the mass spectrometer