Background: As an emerging field of omics, peptidomics is focused on analysis of endogenous native peptides (NP), which are produced from precursor proteins. The NP could be crucial for understanding physiological conditions or disease mechanisms. We developed an extraction protocol of NP from urine for LC-MS/MS based peptidomics. Since urine contains various substances, which are not so easy to remove, urine components eluted from C18 MonoSpin column still have something else other than NP.

Methods: Urinary proteins were isolated under a Methanol / Chloroform precipitation method and NP was then purified from the supernatant (SUP) by passing through 30K MWCO filter to remove high molecular weight components. Then, NP was precipitated by adding x5 volume of cold EtOH at -20C for overnight. After 30 minute centrifugation, NP in a pellet was washed by adding 80% EtOH and centrifuged for other 20 minutes. The NP pellet was lightly dried and dissolved in 8M urea buffer, then was reduced and alkylated by using DTT and IAA. In our protocol, C18 MonoSpin column was finally used for purification of NP. Hereby, we improved the purification method of NP by stepwise elution from C18 MonoSpin column with acetonitrile.

Results: Our results demonstrated the improved NP preparation protocol from urine provided stable and pure NP extraction than previous one. It makes sample loading easier for mass spectrometry analysis. NP measurement done by NanoDrop equipment provided a consistent scan shape as one shown by a standard peptide of BSA. Usage of ESI spry tip on mass spectrometry was enhanced due to removing of contaminants from the NP samples.

Conclusion: This protocol of NP preparation from urine could purify NP and remove other contaminants efficiently. The purified NP of high quality reduced troubles of LC-MS/MS instruments and made a comprehensive and unbiased creation of the native peptide profiles in urine possible and facilitated reproducible and accurate quantitation of the endogenous native peptides.