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 (for the columns having the same separation efficiency, linear velocity, and porosity), making attractive the use of smaller inner diameter LC columns. We report the development and initial application of 10 m i.d. silica-based monolithic LC columns providing more sensitive proteomics measurements. A 50-m-i.d. micro solid-phase extraction precolumn was used for ease of sample injection and cleanup prior to the reversed-phase LC separation, enabling the sample volume loading speed to be increased by ∼50-fold. 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 aShewanella oneidensistryptic digest using an ion trap MS. The low nL/min LC flow rates provide more uniform responses 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.