Capillary LC-MS2 at the Attomole Level for Monitoring and Discovering Endogenous Peptides in Microdialysis Samples Collected in Vivo
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- 作者：William E. Haskins ; Ziqiang Wang ; Christopher J. Watson ; Rebecca R. Rostand ; Steven R. Witowski ; David H. Powell ; and Robert T. Kennedy
- 刊名：Analytical Chemistry
- 出版年：2001
- 出版时间：November 1, 2001
- 年：2001
- 卷：73
- 期：21
- 页码：5005 - 5014
- 全文大小：432K
- 年卷期：v.73,no.21(November 1, 2001)
- ISSN：1520-6882
文摘
Fused-silica capillary LC columns (25-
m i.d.) with 3-m-i.d. integrated electrospray emitters interfaced to a quadrupole ion trap mass spectrometer were evaluated forhigh-sensitivity LC-MS2. Column preparation involvedconstructing frits by in situ photopolymerization of glycidylmethacrylate and trimethylolpropane trimethacrylate, preparing the electrospray emitter by pulling the columnoutlet to a fine tip with a CO2 laser puller, and slurry-packing the column with 5-m reversed-phase particles.Large-volume injections were facilitated by an automatedtwo-pump system that allowed high-flow rates for sampleloading and low-flow rates for elution. Small electrosprayemitters, low elution flow rates, and optimization ofgradient steepness allowed a detection limit of 4 amol,corresponding to 2 pM for 1.8 L injected on-column, fora mixture of peptides dissolved in artificial cerebral spinalfluid. The system was coupled on-line to microdialysissampling and was used to monitor and discover endogenous neuropeptides from the globus pallidus of anesthetized male Sprague-Dawley rats. Time-segmented MS2scans enabled simultaneous monitoring of Met-enkephalin, Leu-enkephalin, and unknown peptides. Basal dialysate levels of Met-enkephalin and Leu-enkephalin were60 ± 30 and 70 ± 20 pM while K+-stimulated levels were1900 ± 500 and 1300 ± 300 pM, respectively (n = 7).Data-dependent and time-segmented MS2 scans revealedseveral unknown peptides that were present in dialysate.One of the unknowns was identified as peptide I1-10(SPQLEDEAKE), a novel product of preproenkephalin Aprocessing, using MS2, MS3, and database searching.
m i.d.) with 3-m-i.d. integrated electrospray emitters interfaced to a quadrupole ion trap mass spectrometer were evaluated forhigh-sensitivity LC-MS2. Column preparation involvedconstructing frits by in situ photopolymerization of glycidylmethacrylate and trimethylolpropane trimethacrylate, preparing the electrospray emitter by pulling the columnoutlet to a fine tip with a CO2 laser puller, and slurry-packing the column with 5-m reversed-phase particles.Large-volume injections were facilitated by an automatedtwo-pump system that allowed high-flow rates for sampleloading and low-flow rates for elution. Small electrosprayemitters, low elution flow rates, and optimization ofgradient steepness allowed a detection limit of 4 amol,corresponding to 2 pM for 1.8 L injected on-column, fora mixture of peptides dissolved in artificial cerebral spinalfluid. The system was coupled on-line to microdialysissampling and was used to monitor and discover endogenous neuropeptides from the globus pallidus of anesthetized male Sprague-Dawley rats. Time-segmented MS2scans enabled simultaneous monitoring of Met-enkephalin, Leu-enkephalin, and unknown peptides. Basal dialysate levels of Met-enkephalin and Leu-enkephalin were60 ± 30 and 70 ± 20 pM while K+-stimulated levels were1900 ± 500 and 1300 ± 300 pM, respectively (n = 7).Data-dependent and time-segmented MS2 scans revealedseveral unknown peptides that were present in dialysate.One of the unknowns was identified as peptide I1-10(SPQLEDEAKE), a novel product of preproenkephalin Aprocessing, using MS2, MS3, and database searching.