A robust micro-electrospray ionization technique for high-throughput liquid chromatography/mass spectrometry proteomics using a sanded metal needle as an emitter.
A 60 microm internal diameter (i.d.) stainless-steel needle was adapted to the orthogonal ESI probe ( microESI) of a commercial ion trap mass spectrometer, and used for capillary liquid chromatography/tandem mass spectrometry (LC/MS/MS) protein identification experiments. The modification allows for the use of nitrogen sheath gas which helps in the nebulization at LC flow rates exceeding 500 nL/min and eliminates problems caused by liquid junctions commonly used to initiate nanospray ionization (NSI). A comparison is made between the performance of a 75 microm i.d. column with a 15 microm pulled glass tip using a liquid junction, and that of a 150 microm i.d. column using the new microESI device. The combination of the 150 microm i.d. column and microESI gave sensitivity close to that of NSI (250 attomoles horse heart myoglobin digest on column), and proved to be more robust than the standard pulled glass tips of similar i.d. No evidence of metal needle catalyzed oxidation of methionine was observed during analysis of the tetrapeptide MRFA under a range of test conditions. Phosphorylated peptides in a beta-casein tryptic digest were also successfully identified using the microESI interface with a steel needle. In addition it was found that a mild sanding of the metal needle tip improved spray performance.
Guzzetta
Andrew W
AW
ThermoFinnigan, LC-MS Product Development Group, 355 River Oaks Parkway, San Jose, CA 95134-1991, USA.
Thakur
Rohan A
RA
Mylchreest
Iain C
IC
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006736:Horses; D008715:Methionine / Q000737:chemistry; D009211:Myoglobin / Q000032:analysis; D036103:Nanotechnology / Q000379:methods*; D010084:Oxidation-Reduction; D010455:Peptides / Q000032:analysis / Q000737:chemistry; D010766:Phosphorylation; D040901:Proteomics; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation / Q000379:methods*
D009211:Myoglobin; D010455:Peptides; D008715:Methionine