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J Biomol Tech. 2010 September; 21(3 Suppl): S55.
PMCID: PMC2918167

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

S. Hahner, A. Resemann, M. Macht, and D. Suckau
Bruker Daltonik GmbH, Bremen, Germany



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.

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