TY - JOUR
T1 - A simplified gradient solvent delivery system for capillary liquid chromatography-electrospray ionization mass spectrometry
AU - Ducret, Axel
AU - Bartone, Nick
AU - Haynes, Paul A.
AU - Blanchard, Alan
AU - Aebersold, Ruedi
PY - 1998/12/1
Y1 - 1998/12/1
N2 - We describe a simple solvent delivery system for gradient capillary HPLC at nanoliter per minute flow rates. The novel aspect of the system is that solvents are delivered one at a time, using a switching valve, into a relatively large-volume mixing chamber. Efficient mixing in the chamber causes the formation of a sigmoidal gradient from the initial solvent to the subsequent solvent, which is then delivered to a capillary column. The shape of the gradients formed can be predicted from a simple theoretical model. Gradients of different slope can be formed by varying either the size of the chamber or the system flow rate. The system is robust, reproducible, and simple to operate. We provide a detailed protocol of how to construct a low- cost capillary HPLC system consisting of two syringe pumps, a capillary mixing chamber, a capillary column, and a zero dead-volume microelectrospray interface. We demonstrate that the coupling of this HPLC system to a mass spectrometer enabled us to identify proteins at the low femtomole level in solution-phase digests and at the picomole level in digests of samples separated on SDS-PAGE gels. We believe that the strategy presented will be useful as a general method for the characterization of proteins and peptides by capillary HPLC-electrospray mass spectrometry.
AB - We describe a simple solvent delivery system for gradient capillary HPLC at nanoliter per minute flow rates. The novel aspect of the system is that solvents are delivered one at a time, using a switching valve, into a relatively large-volume mixing chamber. Efficient mixing in the chamber causes the formation of a sigmoidal gradient from the initial solvent to the subsequent solvent, which is then delivered to a capillary column. The shape of the gradients formed can be predicted from a simple theoretical model. Gradients of different slope can be formed by varying either the size of the chamber or the system flow rate. The system is robust, reproducible, and simple to operate. We provide a detailed protocol of how to construct a low- cost capillary HPLC system consisting of two syringe pumps, a capillary mixing chamber, a capillary column, and a zero dead-volume microelectrospray interface. We demonstrate that the coupling of this HPLC system to a mass spectrometer enabled us to identify proteins at the low femtomole level in solution-phase digests and at the picomole level in digests of samples separated on SDS-PAGE gels. We believe that the strategy presented will be useful as a general method for the characterization of proteins and peptides by capillary HPLC-electrospray mass spectrometry.
UR - http://www.scopus.com/inward/record.url?scp=0032403166&partnerID=8YFLogxK
U2 - 10.1006/abio.1998.2873
DO - 10.1006/abio.1998.2873
M3 - Article
C2 - 9866717
AN - SCOPUS:0032403166
SN - 0003-2697
VL - 265
SP - 129
EP - 138
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 1
ER -