Plastic microchip liquid chromatography-matrix-assisted laser desorption/ionization mass spectrometry using monolithic columns.
A prototype array of monolithic liquid chromatography (LC) columns was prepared in a plastic microfluidic device for the off-line interface with matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The microfluidic channels were fabricated on a cyclic olefin copolymer (COC) plate by hot embossing. An array of methacrylate monolithic columns was prepared in the microfluidic channels by UV-initiated polymerization. The deposition system employed a pulsed electric field to transfer the effluents from multiple columns directly onto MALDI targets with a non-contact deposition method reported by Ericson et al. [C. Ericson, Q.T. Phung, D.M. Horn, E.C. Peters, J.R. Fitchett, S.B. Ficarro, A.R. Salmon, L.M. Brill, A. Brock, Anal. Chem. 75 (2003) 2309]. To characterize the off-line interface of the multiple-channel microchip LC and the MALDI-MS for the analysis of peptide mixtures, the separation efficiency and reproducibility tests in each column were carried out by separating a peptide mixture from tryptic digested proteins and depositing the multiple effluents simultaneously on the MALDI target plate. Using a MALDI-TOF mass spectrometer with a mass accuracy of +/-1 Da for peptide assignments of digested bovine serum albumin (BSA), amino acid sequence coverage of around 59% was obtained for the microchip LC-MALDI-MS compared to 23% obtained by the MALDI-MS method without LC separation. In sensitivity tests for the detection of low abundance proteins in the presence of high concentration protein mixtures, as low as 10 fmol/mul (S/N = 10) of a spiked peptide in 1 microg of digested BSA could be detected. In the analysis of a mixture of three digested proteins (BSA, myoglobin, and cytochrome c), more than twice the amino acid sequence coverage was obtained for the microchip LC-MALDI-MS compared to MALDI-MS alone.
Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, Charleston, 29425, USA.
Kyung Won
KW
Ro
Jian
J
Liu
Daniel R
DR
Knapp
D045304:Cytochromes c / Q000302:isolation & purification; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods*; D008855:Microscopy, Electron, Scanning; D009211:Myoglobin / Q000302:isolation & purification; D040901:Proteomics / Q000379:methods; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D009211:Myoglobin; D012710:Serum Albumin, Bovine; D045304:Cytochromes c