Introduction: A multidimensional LC-LC-MS/MS method has been reported in the literature (Nature (1999) 17:676-682) as a powerful tool for large-scale protein analysis. The method provides high resolution, automation, high throughput and the ability to analyze a complicated mixture of proteins in a single run. In this paper, we use an automated two-dimensional (2D) capillary LC with tandem mass spectrometry (MS/MS) for the analysis of complicated protein mixtures. This 2D system utilizes two HPLC pumps, one strong cation exchange column, two reverse phase C 18 columns and an ion trap tandem mass spectrometer for the high throughput analysis of proteins.
Methods: Protein mixtures from cell lysates are digested and loaded onto a strong cation exchange column and then gradually released to a 0.18 mm diameter C18 column by elution with salt steps of increasing molarity. After loading, the peptide mixture is separate by a reverse phase HPLC and analyzed by a LCQ™ Deca XP mass spectrometer with a microflow electro-spray interface. The protein and peptide sequence is identified by comparison of theoretical and actual MS/MS spectra via TurboSEQUEST™ software.
Preliminary Data: Total cell extracts from human A431 cells were analyzed by both 1D and 2D LC-MS/MS. For a 1D separation, the gradient profile has a significant effect on the number of proteins that can be identified by their MS/MS spectra. A 30 minute gradient only identified 16 proteins. Extending the gradient profile to 480 minutes increases the number of proteins identified to 105. The use of 2D LC-MS/MS enabled the identification of many more proteins than 1D LC-MS/MS. The eight step salt gradient with 30 minute reverse phase separations (total run time of 5 hours) is able to identify 144 proteins. The 20 hour run provides the best results with 491 proteins identified. The 2D LC-MS/MS clearly provides more resolution and identifies more proteins than 1D LC-MS/MS.
When compared to the current two-dimensional electrophoresis-MS method, this on-line 2D LC-MS/MS system has advantages of higher identification capacity, higher sensitivity, higher throughput and a higher degree of automation. Thus, we reveal here an essential tool for proteomic analysis.
To view the complete poster, please download the PDF file below.
You can now also order additional proteomics resources, or learn about other mass spectrometry or proteomics solutions.
|
0 Items 