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

• Published in: Journal of the American Society for Mass Spectrometry Vol. 12; no. 11; pp. 1205 - 1213
• Main Authors: Doneanu, Catalin E, Griffin, Donald A, Barofsky, Elisabeth L, Barofsky, Douglas F
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.11.2001; Springer Nature B.V
• Subjects: Chromatography; Chromatography, Liquid; Deoxyribonucleic acid; Desorption; Dilution; DNA; DNA - chemistry; E coli; Electrospraying; High performance liquid chromatography; Hydrolysis; Ionization; Ions; Lasers; Mass Spectrometry; Peptides; Peptides - chemistry; Proteins - chemistry; Scientific imaging; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Spectrophotometry, Ultraviolet; Spectroscopy; Trypsin; Uracil
• ISSN: 1044-0305; 1879-1123
• DOI: 10.1016/S1044-0305(01)00307-5

A simple and inexpensive nano high performance liquid chromatography system (nano-LC) employing the exponential dilution method for gradient separations was built. The system was used to analyze a tryptic digest of Escherichia coli uracil DNA glycosylase (Ung; M r = 25,563), a DNA-binding protein that initiates the uracil-excision DNA repair process by catalyzing the release of uracil from the deoxyribose phosphate backbone of DNA. Both on-line and off-line approaches to analyzing peptides produced by in-gel digestion of Ung are demonstrated. The on-line approach uses nano-high performance liquid chromatography (HPLC)/micro-electrospray MS to assign peptide masses. The off-line approach uses matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and nano-electrospray/collision-induced dissociation (CID) tandem mass spectrometry, to analyze fractions (2–3 μL) collected manually from the nano-LC system. The nano-electrospray technique allows detailed fragmentation information to be obtained at different collision energies with only a marginal increase in sample handling due to the nano-LC step.