Introduction: A 0.003 inch ID stainless-steel needle was adapted to an ESI probe and used for capillary LC/MS/MS protein identification experiments. The modification allowed for the use of nitrogen sheath gas which helped in the nebulization at LC flow rates exceeding 500nl/min and eliminated problems caused by liquid junctions. The combination of a 150µm ID column and the new µESI gave near nanospray sensitivity (250 attomoles horse heart myoglobin digest on column), and proved to be more robust than the standard pulled glass-tips of similar ID. This new form of robust µESI allowed for extended continuous analysis.
Methods: The LC eluent was introduced into the ion trap mass spectrometer using the µESI interface. The µESI interface consists of a 0.003 inch ID stainless-steel needle inserted into a 26 gauge needle housed in a standard orthogonal ESI nozzle assembly. A 150µm ID column was directly attached to the grounding-union bracket on the µESI flange body, and a piece of 30µm ID fused silica connected the outlet of the column to the µESI inlet. A voltage of +2.8-3.2kV was applied to the tip of the µESI interface (positive ion mode) and 15au of nitrogen was used as a sheath gas.
Preliminary Data: Sensitivity tests were conducted for two different protein digests, namely horse heart myoglobin at 2.5 fmol and 250 amol, and BSA at 5 fmol and 20 fmol. Only two peaks were detected in the 250amol run, enough for protein identification. Clearly, even at 250 amol injected on column, the peaks that were detected had a S/N of 4:1, while those at 2.5 fmol injected had a S/N of 25:1, more than enough for triggering data-dependency, which is the crux of the protein identification experiment. These results indicate that low fmol to high amol levels of detection can be achieved using the µESI interface with increased simplicity of operation and robustness.
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