Fast, high-efficiency peptide separations on a 50-microm reversed-phase silica monolith in a nanoLC-MS set-up.
Proteomic studies have stimulated the development of novel stationary phases in miniaturised chromatographic columns that permit high linear flow velocities and exhibit high resolving power. In this work, a 50-microm reversed-phase silica-based monolith was chromatographically characterised for its use in proteomics applications using a nanoLC-MS set-up. It showed high efficiency for the separation of tryptic peptides under isocratic elution conditions (HETP(min)=5-10 microm at 2.4 mm/s). Flow rates up to 1.95 microL/min (18.4 mm/s) and gradient slopes up to an unusually fast 9% could be used. This resulted in rapid separations of peptide mixtures, with peak widths at half height of between 5 and 10 s. The 50-microm monolithic column was used to analyse depleted serum from a cervical cancer patient at a throughput of one sample per 30 min.
Rieux
Laurent
L
Pharmaceutical Analysis, University Centre for Pharmacy, University of Groningen, A. Deusinglaan 1, 9713 AV Groningen, The Netherlands.
Lubda
Dieter
D
Niederländer
Harm A G
HA
Verpoorte
Elisabeth
E
Bischoff
Rainer
R
D000595:Amino Acid Sequence; D001798:Blood Proteins / Q000737:chemistry / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D045304:Cytochromes c / Q000737:chemistry / Q000378:metabolism; D005260:Female; D006801:Humans; D008969:Molecular Sequence Data; D036103:Nanotechnology / Q000295:instrumentation / Q000379:methods*; D010446:Peptide Fragments / Q000097:blood / Q000737:chemistry / Q000302:isolation & purification*; D040901:Proteomics / Q000295:instrumentation / Q000379:methods; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation / Q000379:methods*; D013997:Time Factors; D014357:Trypsin / Q000378:metabolism; D002583:Uterine Cervical Neoplasms / Q000097:blood
D001798:Blood Proteins; D010446:Peptide Fragments; D045304:Cytochromes c; D014357:Trypsin