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 separations was built. The system was used to analyze a tryptic digest of Escherichia coli uracil DNA glycosylase (Ung; Mr = 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 microL) 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.
Doneanu
C E
CE
Department of Chemistry, Oregon State University, Corvallis 97331-4003, USA.
Griffin
D A
DA
Barofsky
E L
EL
Barofsky
D F
DF
D002853:Chromatography, Liquid; D004247:DNA / Q000737:chemistry; D006868:Hydrolysis; D013058:Mass Spectrometry; D010455:Peptides / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013056:Spectrophotometry, Ultraviolet; D014357:Trypsin
D010455:Peptides; D011506:Proteins; D004247:DNA; D014357:Trypsin