Rapid Extraction Technique Prepares Urine Samples for Mass Spectrometry Analysis

While mass spectrometry is a powerful tool for biomarker determinations, the high salt content and the matrix of small molecules present in urine has reduced its applicability for urinary diagnosis. To correct this deficiency, investigators at Clemson University (SC, USA) packed micropipette tips with C-CP fibers. These fibers possess a unique geometry that includes eight channels that extend the entire fiber length (which can be miles on a spool). The fibers are nominally an oblong shape with diameters ranging from 35 to 50 micrometers, with the individual channels ranging in size from five to 20 micrometers.

Urine samples were passed through fiber-packed tubes by spinning them in a centrifuge for 30 seconds. Following centrifugation de-ionized water was run through the tubes for one minute to wash off salt and other contaminants. Hydrophobic proteins, which remained bound to the fibers, were extracted for MALDI-MS analysis with appropriate solvents during a 30 second centrifugation step.

Matrix-assisted laser desorption/ionization (MALDI) is a soft ionization technique used in mass spectrometry, allowing the analysis of biomolecules (biopolymers such as DNA, proteins, peptides, and sugars) and large organic molecules (such as polymers, dendrimers, and other macromolecules), which tend to be fragile and fragment when ionized by more conventional ionization methods. It is similar in character to electrospray ionization (ESI) in that both techniques are relatively soft ways of obtaining ions of large molecules in the gas phase, though MALDI produces far fewer multiply charged ions.

The C-CP fiber method was validated by measuring the urinary proteins beta-2-microglobulin, retinol binding protein, and transferrin. C-CP fiber tips offered several advantages including low materials costs, high throughput, microvolume processing, and the determination of sub-nanogram quantities of analyte; allowing determination of biomarkers that are otherwise undetectable in urine samples.

"You have got almost seawater coming out of you, and I am trying to find something far smaller than a needle in a haystack," said senior author Dr. Ken Marcus, professor of analytical chemistry at Clemson University. "The concentrations of these proteins would be one part in a billion."

The C-CP fiber method for urine sample purification was described in the March 18, 2015, online edition of the journal Proteomics-Clinical Applications.

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