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J Biomol Tech. 2010 September; 21(3 Suppl): S33.
PMCID: PMC2918143

Increased Long Term Retention Time Stability in Scheduled MRM Peptide Assays Using Chip Based Chromatography

B. Young, E. Lin, and N. Hebert
Eksigent Technologies, Dublin, CA, United States



Overview: A chip based nanoLC platform is introduced for use as a high performance chromatographic tool for proteomics with the advantages of microfluidic design elements.Chromatographic performance on par with conventionally packed capillary columns is demonstrated. Excellent run-to-run reproducibility and low inter-column variation allow for scheduled MRM assays over long periods of time for large sets of samples with narrow time windows for each MRM. Introduction: Scheduled MRM assays are increasingly used for the verification of putative peptide biomarkers using nanoLC-MS. Scheduling the transitions allows for the analysis of more biomarkers in the same assay with improved quantitation. Excellent retention time reproducibility over long periods of time and multiple columns is required for the analysis of peptides using scheduled MRM's in large sets of samples. Although great improvements have been made in reproducible direct pumping nanoLC systems, inter-column reproducibility is difficult to achieve for capillary columns that operate at nanoliter flow rates. This is because both making capillary columns and connecting them reproducibly are difficult. Microfluidic devices have inherent advantages in chromatography as the microchannels are defined lithographically and are highly reproducible. We have reported recently on a microconnector which facilitates reproducible microfluidic connections at nanoliter flow rates with a very high degree of consistency, solving the problem of connecting the outside world to chip based columns. In this presentation, we will be reporting on our progress on a new microfluidic platform based on this microconnector for nanoliter separations on microchips, and demonstrate that robust, high quality separations facilitate the use of scheduled MRM based peptide quantitative assays for large numbers of samples. Materials/Equipment: NanoLC-Ultra nanoLC system with cHiPLC nanoflex (Eksigent Technologies, Dublin, CA) in direct injection mode. 15 cm × 75 μm ID ChromXP C18 3μm cHiPLC column (Eksigent Technologies) TSQ Quantum Access (Thermo Scientific, San José, CA) with New Objective (Woburn, MA) nanospray source. Bovine serum albumin reduced/alkylated and digested with trypsin. Summary of Results: We will demonstrate intra-and inter-run reproducibility of peptide retention times over long periods and large numbers of samples. Conclusions: The cHiPLC nanoflex platform enables the acquisition of high quality, highly reproducible data by establishing a robust chromatographic platform for nanoliter separations. The reduction in retention time variability and the ability to compare data across runs and columns enhances the attractiveness of scheduled MRM based peptide quantitation in clinical settings.

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