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Realised by ALMS™
developer of the AIDS-HIV Reference project
Abstract No.: ThP-077
Session: Biomarkers
Presentation date: Thu, Aug 31, 2006
Presentation time: 09:50 – 11:20

A Fast, Reproducible LC and Spotting System for Protein ID and Biomarker Discovery

Matthew Willetts1, Dominic Gostick1, Fadi Abdi1

1 Applied Biosystems, Framingham, United States

Correspondence address: Matthew Willetts, Applied Biosystems, 500 Old Connecticut Path, Framingham, 01701 United States.

Keywords: Biomarkers; Chromatography, Liquid -, High Pressure (HPLC); MS/MS, Liquid Chromatography; Protein Identification.

Novel aspect: Very reproducible chromatographic retention times coupled with dynamic exclusion allow improved protein identification in complex samples.

 

Introduction
LC-MALDI is a powerful technique for protein identification and biomarker discovery. Recent advances in monolithic column technology and highly reproducible gradient LC systems offer significant improvements in LC separations, allowing complex protein mixtures to be resolved in 15 30 min gradients, with peak widths of 6 seconds or less. In order to take full advantage of such chromatography a spotting device is needed that is able to deposit eluent in a fast and reliable manner. Highly reproducible chromatography allows greater sophistication in precursor selection as replicate injections can be compared very accurately in terms of mass and retention time. This permits reanalysis of peptides missed or those that had spectra of insufficient quality for identification during the first run.

Methods
Samples were loaded onto a 100 um CapRod™ (Merck, Darmstadt, Germany) at a flow rate of 2 uL/min. Separation was achieved using a 30 min reverse phase gradient. The column eluent was mixed 1:2 with alpha-cyano matrix and deposited onto a MALDI target plate at a rate of 1 fraction every 3 seconds. All chromatography and sample deposition was performed on an Applied Biosystems/MDS Sciex Tempo™ LC MALDI Spotting System. Mass spectra were acquired on an Applied Biosystems/MDS Sciex 4800 MALDI TOF/TOF™ Analyzer (Concord, ON). Chromatographic alignments were performed using MarkerView™ Software.

Preliminary Data
Replicate injections of a mixture of digested commercially sourced proteins were fractionated onto a target plate. Each separation was analyzed in MS mode with particular care being taken to ensure good quality data from every fraction. Typical peak widths were less than 5 seconds at half height. Retention times across the replicate injections differed by less than 2 seconds, indicating the highly reproducible flow rate and gradient capabilities of this system. In this poster we will show how accurate mass and accurate retention time pairs can be used with replicate sample injections to maximize the number and quality of protein identifications in complex proteomic samples.