Fast, comprehensive two-dimensional HPLC separation of tryptic peptides based on high-temperature HPLC

Two-dimensional HPLC (2D-LC) has recently received considerable attention, and is being used as an alternative to 2D gel electrophoresis in proteomics research. The greatest impediment to the widespread use of 2D-LC is the long analysis time ranging up to days per analysis, making the technique impractical for many jobs. Here we focus on improving the speed of gradient separations since these are typically used as the second dimension in peptide separations by 2D-LC. Specifically we describe high-temperature, ultrafast HPLC conditions, along with the instrument modifications needed to reduce the analysis time of each complete second-dimension gradient separation to tens of seconds. Most importantly, this system is capable of generating a high peak capacity (1350) characteristic of comprehensive 2D-LC in a relatively shorter analysis time (20 min) with a sampling rate sufficient to minimize information loss with simpler instrumentation than currently used; this is equivalent to one unit of peak capacity per second.