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J Biomol Tech. 2007 February; 18(1): 40.
PMCID: PMC2291816

P115A-T High Speed Nanoscale UPLC Separations Combined with Off-Line MALDI MS/MS for Peptide Analysis


MALDI MS/MS is now a standard technique for protein identification and characterisation and this method has been shown to work well with mixtures of low complexity. In the analysis of biological samples considerable sample processing and chromatographic separation is crucial to limit the complexity and dynamic range presented to the MS. A common approach to protein prefractionation is 2D or 1D PAGE. The downside of this approach is that the complexity of the sample is still such that an LC-MALDI experiment is required, prior to analysis. With typical HPLC run times of 45 min to 1 h, the time required to analyze one top level sample can be prohibitive.

In this poster we describe the use of elevated flow rates combined with nanoscale columns packed with sub-2-μm particles for rapid peptide based separations using a nanoU-PLC system. Increasing the flow rate of the separation from approximately 300 nL/min to 900 nL/min and running a very rapid gradient over 8 minutes on a 75 μm × 15 cm column allows high quality peptide separations to be achieved with a sample to sample inject time of under ten minutes. The eluent is combined with matrix solution and spotted directly onto MALDI target plates. This combined with an orthogonal acceleration time-of-flight mass spectrometer equipped with a 200-Hz MALDI source, allows for the rapid characterisation of simplified protein mixtures, such as those obtained from 1D gel bands.

We will present data from standard tryptic digests of known proteins and mixtures of protein digests used in the development of this method and data from in-gel digests of 2D and 1D gel samples. This will be compared and contrasted against data acquired using conventional separations and mass spectrometric analysis.

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