An Improved NanoLC-MALDI Approach for the Analysis of Complex Proteomic Samples.

RP-109 Sample complexity continues to be one of the most challenging factors for in-depth proteomic analyses. LC-MS/MS based methods have been established for the analysis of such complex protein mixtures. The effectiveness of these methods, however, is defined by a number of analytical key factors, these are: acquisition speed, resolution, mass accuracy, sensitivity, dynamic range, long term robustness and availabilty of suitable software for efficient downstream data analysis. We describe here an improved nanoLC-MALDI approach which is based on the following parts: i)a new MALDI-TOF/TOF system that allows for the accelerated acquisition (laser repetition rate 1000Hz) of MALDI-MS and -MS/MS spectra providing a so far unseen level of resolution (up to R>40000) and mass accuracy (typically 5ppm, external calibration), ii)a new proteomics database software for highly efficient organization and analysis of large amounts of proteomics MS and MS/MS data. The capabilities of this new approach are demonstrated utilizing as a sample for benchmarking a well characterized trypsin digested E.coli cell lysate. Analysis of 500ng E.coli digest, using the new instrumentation, results in the identification of more than 650 unique E.coli proteins (global peptide FDR 1%). Laser repetition rate of 1000Hz enabled significantly increased MALDI acquisition speed of 3s per MS spectrum and 5s or less per MS/MS spectrum. In total, more than 9700 MS/MS spectra were acquired, with nearly 50% of this data leading to peptide assignments. As an additional feature, the new MALDI-TOF/TOF system described here provides a unique, IR laser based technology for reliable self-cleaning of the MALDI ion source within 15min, which is of geat benefit to minimize instrument down-time and to increase sample throughput.