Nano volume fractionation strategy for dilute-and-shoot injections in off-line loss-less proteomic workflows for extensive protein identifications of ultra-low sample amounts.

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ID: 63876
2019
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Abstract
A proteomic workflow for a simple loss-less manual nano-fractionation (300 nL/fraction) for low µg sample amounts which avoids the need to dry down or transfer fractions to autosampler vials is shown to be feasible. It is demonstrated that the conventional procedure of drying samples down followed by reconstitution negatively affects the number of protein and peptide identifications. Furthermore, these losses seem to disproportionately affect hydrophobic peptides from the drying down and reconstitution step. By collecting and concatenating the fractions while the outlet of the column is submerged in a small predefined volume of 0.2% formic acid, the content of acetonitrile in the collecting vials was lowered such that it was compatible with direct injection for the online analysis. This additionally resulted in a time gain of approx. an hour for the total fractionation time. Acetonitrile concentrations up to 7.5% do not seem to compromise the chromatographic performance in the online analysis. Using as little as 2 µg digested HeLa lysate, approx. 7000 protein groups could be easily identified with 2 or more unique peptides. This was the case when fractionation was performed at pH 10 as well as at pH 5.5.
Reference Key
reubsaet2019nanojournal Use this key to autocite in the manuscript while using SciMatic Manuscript Manager or Thesis Manager
Authors Reubsaet, Léon;Sweredoski, Michael J;Moradian, Annie;Lomenick, Brett;Eggleston-Rangel, Roxana;Garbis, Spiros D;
Journal journal of chromatography a
Year 2019
DOI
S0021-9673(19)30891-X
URL
Keywords
concatenation loss-less proteomic workflow nano volume fraction collection peptide loss

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