NanoLC Specifications.docx

NanoLC Specifications/Configuration The Laboratory of Retinal Cell & Molecular Biology, NEI, requires a robust nanoscale Liquid Chromatography (nanoLC) system for high resolution mass spectrometry (MS) proteomics applications. This must be accessible to users of a wide range of technical proficiency. Despite its extremely high sensitivity, nanoLC is well-known for being a technically troublesome and demanding technique. Its high degree of complexity means that an extraordinary amount of troubleshooting often is required to make it work and this impacts its reproducibility and reliability. Much of this difficulty resides in the sequential use of extremely high-pressure pumps, extremely narrow columns and extremely low flowrate (mostly below 1 microliter/minute). All of these combined make nanoLC systems prone to blockage and downtime. Repeated downtime leads to variability and reduced reproducibility and the possibility of cross-contamination. Cross-contamination is an especially serious issue when the samples to be analyzed are so small. Therefore, an ideal system will also have enhanced measurement reproducibility and minimized cross-contamination. This can be accomplished by automating purification so that the steps of manually eluting samples, drying them down, and resuspending them are completely omitted. This is a novel approach only recently realized.

Required specifications/configuration:

a. Basic system: NanoLC system will incorporate integrated sample purification methodology.

b. Automated desalting/purification: Samples will be desalted offline and then eluted from each tip (arrayed in 96-well format) and run on the separation column in one integrated procedure. Use of disposable and single use trap column will reduce the sample cross contamination to <0.05%. Contaminating components will be trapped and discarded, peptides of interest will be transferred directly to the separation column without an intervening drying and resuspension step.

c. Low-pressure elution/gradient formation: Will occur at low pressure, ensuring reduced wear and tear. An off-set gradient will be preformed to allow stacking of peptides prior to final separation on the high-pressure analytical column.

d. High-pressure separation: Elution of the prestacked peptides on the high-performance analytical column will be achieved by short gradients of 3.2 to 88 minutes. Short gradients are especially attractive in combination with time-of-flight (TOF) instruments because of these systems’ very high scanning speed. The MS system that the nanoLC will be coupled to is a SCIEX 5600+ TripleTOF system with a very high scanning speed.

e. Optimized HPLC columns: Specialized narrow-bore C18 columns will be available for specific workflow methods.

f. Rapid and robust cycling: Will have minimal run-to-run times ranging from very short gradients efficient for clinical proteomics but with sufficient scope for longer analytical gradients to increase proteome coverage. This will open up high-throughput proteomics in areas like screening of host-cell proteins, protein interaction studies, and top-down proteomics.

g. Optimized, fixed methods: Will have the ability to run workflows ranging from 15 to 300 samples/day and allowing up to 93% MS utilization rate.

h. Mass Spectrometer Drivers: Analyst from Sciex will be integrated (as are drivers for systems of other vendors).