More sensitive and quantitative proteomic measurements using very low flow rate porous silica monolithic LC columns with electrospray ionization-mass spectrometry
The sensitivity of proteomics measurements using liquid chromatography (LC) separations interfaced with electrospray ionization-mass spectrometry (ESI-MS) improves approximately inversely with liquid flow rate, making attractive the use of smaller inner diameter LC columns. We report the development and initial application of 10 µm i.d. silica-based monolithic LC columns providing more sensitive proteomics measurements. The implementation provides robust performance and suitability for automated proteome analyses due to integration with a micro solid phase extraction pre-column for ease of sample injection and clean-up prior to the reversed phased LC separation. Greater than 10-fold improvement in sensitivity was obtained compared to analyses using more conventional capillary LC, enabling e.g. the identification of >5000 different peptides by MS/MS from 100-ng of a Shewanella oneidensis tryptic digest using an ion trap MS. The low nL/min LC flow rates provide more uniform signal intensities for different peptides, and provided improved quantitative measurements compared to conventional separation systems without the use of internal standards or isotopic labeling. The improved sensitivity allowed LC-MS measurements of immunopurified protein phosphatase 5 that were in good agreement with quantitative western blot analyses.
- Research Organization:
- Pacific Northwest National Lab. (PNNL), Richland, WA (United States). Environmental Molecular Sciences Lab. (EMSL)
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- AC05-76RL01830
- OSTI ID:
- 885460
- Report Number(s):
- PNNL-SA-47984; 6699; 400412000; TRN: US200616%%651
- Journal Information:
- Journal of Proteome Research, 5(5):1091-1097, Vol. 5, Issue 5
- Country of Publication:
- United States
- Language:
- English
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CHROMATOGRAPHY
FLOW RATE
IMPLEMENTATION
LIQUID FLOW
PEPTIDES
PERFORMANCE
PHOSPHATASES
PROTEINS
SENSITIVITY
SILICA
SPECTROSCOPY
quantitative proteomics
ion suppression
label free approach
microSPE-nanoLC-ESI-MS
silica-based monolithic column
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Environmental Molecular Sciences Laboratory