High-speed, high-resolution monolithic capillary LC-MALDI MS using an off-line continuous deposition interface for proteomic analysis.
High-speed, high-resolution LC separations, using a poly(styrene-divinylbenzene) monolithic column, have been coupled to MALDI MS and MS/MS through an off-line continuous deposition interface. The LC eluent was mixed with alpha-cyano-4-hydroxycinnamic acid matrix solution and deposited on a MALDI plate that had been precoated with nitrocellulose. Deposition at subatmospheric pressure (80 Torr) formed a 250-microm-wide serpentine trace with uniform width and microcrystalline morphology. The deposited trace was then analyzed in the MS mode using a MALDI-TOF/TOF MS instrument. Continuous deposition allowed interrogation of the separation with a high data sampling rate in the chromatographic dimensions, thus preserving the high resolution of narrow peaks (3-5-s peak width at half-height) of the fast monolithic LC. No extracolumn band broadening due to the deposition process was observed. Over 2000 components were resolved in a 10-min linear gradient separation of the model sample, and 386 unique peptides were identified in the subsequent MS/MS analysis. The continuous deposition interface allows the coupling of high-resolution separations to MALDI MS without degradation in separation efficiency, thus enabling high-throughput proteome analysis.
Chen
Hsuan-shen
HS
Barnett Institute and Department of Chemistry and Chemical Biology, Northeastern University, 341 Mugar, 360 Huntington Avenue, Boston, Massachusetts 02115, USA.
Rejtar
Tomas
T
Andreev
Viktor
V
Moskovets
Eugene
E
Karger
Barry L
BL
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D003373:Coumaric Acids / Q000737:chemistry; D019075:Electrophoresis, Capillary / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000295:instrumentation / Q000379:methods; D004867:Equipment Design; D008855:Microscopy, Electron, Scanning; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis / Q000302:isolation & purification; D040901:Proteomics / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000295:instrumentation / Q000379:methods*; D013696:Temperature; D015003:Yeasts / Q000737:chemistry
D003373:Coumaric Acids; D010455:Peptides; C007175:alpha-cyano-4-hydroxycinnamate