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J Biomol Tech. 2010 September; 21(3 Suppl): S46.
PMCID: PMC2918201

Automated 2D Peptide Separation on a 1D Nano-LC-MS System

P. Taylor,2 R. Constantineau,3 O. Vorm,3 and T. Kislinger1
1University of Toronto, Toronto, ON, Canada;
2Hospital for Sick Children, Toronto, ON, Canada;
3Proxeon, Odense, Denmark



Given the complexity of the mammalian proteome, high-resolution separation technologies are required to achieve comprehensive proteome coverage and to enhance the detection of low-abundance proteins. Among several technologies, Multidimensional Protein Identification Technology (MudPIT) enables on-line separation of highly complex peptide mixtures directly coupled with mass spectrometry-based identification. Here, we present a variation of the traditional MudPIT protocol. We combine highly sensitive chromatography on a nanoflow liquid chromatography system (nano-LC) with a two-dimensional pre-column in a vented column set-up. When compared to the traditional MudPIT approach, this nanoflow variation demonstrated superior first phase separation leading to the characterization of more proteins. In addition, the simpler instrumentation set-up and protocol were faster and easier to perform.To evaluate system performance, we analyzed a human multiple myeloma cell line proteome. Proteins were extracted, reduced and alkylated followed by trypsin digestion. The resulting peptide sample was purified and analyzed by 1D and 2D methods on a split-free nano LC-MS system (EASY-nLC, Proxeon, Odense, Denmark) coupled to LTQ-Orbitrap (Thermo-Fisher, Bremen, Germany). For the vented 2D LC approach, samples and the high salt (ammonium acetate) steps required to elute peptides from the strong cation exchange (SCX) material onto the reversed phase (RP) material were applied using the autosampler of the EASY-nLC. For each salt plug injection, eluted peptides were washed on the RP section of the two-dimensional pre-column, separated by gradient elution and subjected to MS/MS analysis.This study describes a simple automated approach to multidimensional peptide separation using SCX in the first dimension and RPC in the second dimension. We have compared this set-up to a traditional on-line MudPIT experiment and found that it characterizes more peptides, uses simpler instrumentation and hence is technically much easier to perform and automate. In a direct comparison, it was possible to identify around 20% more proteins using the described nanoflow vented column set-up instead of the traditional MudPIT approach.Importantly, the automated sample loading via the microplate autosampler enables the system to be operated ‘24/7’, thus increasing sample throughput. In contrast, a manual sample loading process used for traditional MudPIT procedure is, in practice, limited to one sample per day (at least for a 9-step set-up). Many samples can be analyzed using the same pre-column/ column set-up whereas a new column is used more frequently in the traditional approach. Samples that have been run on the same column have chromatograms that are more easily aligned and make post-acquisition analysis easier.Finally we present an abbreviated variant of this experiment which enables 3 times as many samples to be analyzed without significantly compromising data quality. Simple, fast nanoflow 2D-LC method for comprehensive peptide separation.

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