Capillary scale monolithic trap column for desalting and preconcentration of peptides and proteins in one- and two-dimensional separations.
Monolithic columns based on poly-(styrene-divinylbenzene) (PS-DVB) were utilized both for preconcentration (in 10 mm x 0.20 mm I.D. format) and analytical separation (in 60 mm x 0.20 and 0.10 mm I.D. format) of peptides and proteins in column switching micro-scale high-performance liquid chromatography. A special holder for short monolithic preconcentration columns was designed and pressure durability tests approved long-term stability up to 400 bar. An 11-20% decrease in the average peak widths of nine peptides was obtained upon combining a preconcentration column with an analytical column as compared with a setup using an analytical column only. Trapping efficiency, especially for small and hydrophilic peptides, was optimized by using 0.10% heptafluorobutyric acid instead of 0.050% trifluoroacetic acid as solvent additive during sample loading. Using a 10 mm x 0.20 mm I.D. preconcentration column, loadabilities between 0.5 and 1.6 microg were determined by frontal analysis of proteins and bioactive peptides, respectively. A 100-fold concentration followed by direct on-line intact mass determination is demonstrated for diluted (3 micromolL(-1)) protein solutions. The applicability of the monolithic preconcentration column for multidimensional chromatography was tested by off-line two-dimensional separation, combining strong cation-exchange chromatography and ion-pair reversed-phase chromatography. Peptide identification data from digested protein mixtures demonstrated reproducibilities of 46-75% in triplicate analyses, and confident peptide identifications of low abundant peptides even in the presence of a 650-fold molar excess of high abundant peptides.
Schley
Christian
C
Department of Chemistry, Instrumental Analysis and Bioanalysis, Saarland University, 66123 Saarbrücken, Germany.
Swart
Remco
R
Huber
Christian G
CG
D053000:Analytic Sample Preparation Methods; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation* / Q000379:methods; D010455:Peptides / Q000302:isolation & purification*; D011137:Polystyrenes / Q000737:chemistry; D011506:Proteins / Q000302:isolation & purification*
D010455:Peptides; D011137:Polystyrenes; D011506:Proteins; C003771:Amberlite XAD-2 resin