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The nanoelectrospray ion source (nanoES) has recently been developed and described theoretically. It is different from conventional electrospray sources and from other miniaturized electrospray sources by (i) its 1−2 μm spraying orifice achieved by pulling the spraying capillary to a fine tip, (ii) its very low flow rate of ∼20 nL/min and the small size of droplets it generates, and (iii) the absence of solvent pumps and inlet valves. The fabrication and operation of nanoES needles is described in detail. Solutions with up to 0.1 M salt contents could be sprayed without sheath flow or pneumatic assist. Improved desolvation in nanoES led to instrument-limited resolution of the signals of a glycoprotein and the ability to signal average extensively allowed the C-terminal sequencing of a 40 kDa protein. Extensive mass spectrometric and tandem mass spectrometric investigation of the components of an unseparated peptide …