High-performance liquid chromatography-electrospray ionization mass spectrometry using monolithic capillary columns for proteomic studies.
The use of tetrahydrofuran/decanol as porogens for the fabrication of micropellicular poly(styrene/divinylbenzene) monoliths enabled the rapid and highly efficient separation of peptides and proteins by reversed-phase high-performance liquid chromatography (RP-HPLC). In contrast to conventional, granular, porous stationary phases, in which the loading capacity is a function of molecular mass, the loadability of the monoliths both for small peptides and large proteins was within the 0.40.9-pmol range for a 60- x 0.2-mm capillary column. Lower limits of detection obtained by measuring UV-absorbance at 214 nm with a 3-nl capillary detection cell were 500 amol for an octapeptide and 200 amol for ribonuclease A. Upon reduction of the concentration of trifluoroacetic acid in the eluent from the commonly used 0.1-0.2 to 0.05%, the separation system was successfully coupled to electrospray ionization mass spectrometry (ESI-MS) at the cost of only a small decrease in separation efficiency. Detection limits for proteins with ESI-MS were in the lower femtomole range. High-quality mass spectra were extracted from the reconstructed ion chromatograms, from which the masses of both peptides and proteins were deduced at a mass accuracy of 50-150 ppm. The applicability of monolithic column technology in proteomics was demonstrated by the mass fingerprinting of tryptic peptides of bovine catalase and human transferrin and by the analysis of membrane proteins related to the photosystem II antenna complex of higher plants.
Premstaller
A
A
Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens-University, Innsbruck, Austria.
Oberacher
H
H
Walcher
W
W
Timperio
A M
AM
Zolla
L
L
Chervet
J P
JP
Cavusoglu
N
N
van Dorsselaer
A
A
Huber
C G
CG
D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D010455:Peptides / Q000302:isolation & purification; D011506:Proteins / Q000302:isolation & purification; D020543:Proteome; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013056:Spectrophotometry, Ultraviolet
D010455:Peptides; D011506:Proteins; D020543:Proteome