Investigation and reduction of sub-microgram peptide loss using molecular weight cut-off fractionation prior to mass spectrometric analysis

This work investigates the introduction of methanol and a salt modifier to molecular weight cut-off membrane-based centrifugal filters (MWCO) to enrich sub-microgram peptide quantities. Using a neuropeptide standard, bradykinin, sample loss was reduced over two orders of magnitude with and without undigested protein present. Additionally, a bovine serum albumin tryptic digestion was investigated. Twenty-seven tryptic peptides were identified from MALDI mass spectra after enriching with methanol while only two tryptic peptides were identified after MWCO separation using H₂O. The strategy presented here enhances recovery from MWCO separation for sub-μg peptide samples.