An exponential dilution gradient system for nanoscale liquid chromatography in combination with MALDI or nano-ESI mass spectrometry for proteolytic digests

A simple and inexpensive nano high performance liquid chromatography system (nano-LC) employing the exponential dilution method for gradient separatio ns was built. The system was used to analyze a tryptic digest of Escherichi a coli uracil DNA glycosylase (Ung; M, = 25,563), a DNA-binding protein tha t initiates the uracil-excision DNA repair process by catalyzing the releas e of uracil from the deoxyribose phosphate backbone of DNA. Both on-line an d off-line approaches to analyzing peptides produced by in-gel digestion of Ung are demonstrated. The on-line approach uses nano-high performance liqu id chromatography (HPLC)/micro-electrospray MS to assign peptide masses. Th e off-line approach uses matrix-assisted laser desorption/ionization time-o f-flight mass spectrometry (MALDI-TOF MS) and nano-electrospray/ collision- induced dissociation (CID) tandem mass spectrometry, to analyze fractions ( 2-3 muL) collected manually from the nano-LC system. The nano-electrospray technique allows detailed fragmentation information to be obtained at diffe rent collision energies with only a marginal increase in sample handling du e to the nano-LC step. (C) 2001 American Society for Mass Spectrometry.