ABSTRACT
A blotting method that transfers analytes separated on wettable high-performance thin-layer chromatography (HPTLC) plates to a hydrophobic reversed-phase C8 HPLTC plate suitable for analysis with a liquid microjunction surface sampling probe electrospray ionization mass spectrometry system was described and demonstrated. The simple blotting procedure transfers the analyte from the wettable plate to the topmost surface of a rigidly backed, easy-to-mount hydrophobic substrate that already has been proven viable for analysis by this sampling probe/mass spectrometry system. The utility of the approach was demonstrated by the analysis of a four-component peptide mixture originally separated on a ProteoChrom® HPTLC cellulose sheet and then blotted onto the reversed-phase HPTLC plate.
Subject(s)
Chromatography, Thin Layer/instrumentation , Chromatography, Thin Layer/methods , Spectrometry, Mass, Electrospray Ionization/methods , Cellulose , Chromatography, High Pressure Liquid/methods , Chromatography, Reverse-Phase , Hydrophobic and Hydrophilic Interactions , Spectrometry, Fluorescence , WettabilityABSTRACT
An aerosol application procedure involving one or more commercially available silicone-based products was developed to create hydrophobic surfaces that enable analysis of otherwise wettable, absorbent surfaces using a liquid microjunction surface sampling probe/electrospray ionization mass spectrometry system. The treatment process resulted in a hydrophobic surface that enabled formation of the requisite probe-to-surface liquid microjunction for sampling and allowed efficient extraction of the analytes from the surface, but did not contribute significant chemical background in the mass spectra. The utility of this treatment process was demonstrated with the treatment of wettable high-performance thin layer chromatography plates, post-plate development, and their subsequent analysis with the sampling probe. The surface treatment process for different surface types was described and explained and the effectiveness of the treatment and subsequent analysis was illustrated using alkaloids from goldenseal (Hydrastis canadensis) root separated on a normal phase silica gel 60 F(254S) plate and peptides from protein tryptic digests separated on a ProteoChrom HPTLC Silica gel 60 F(254S) plate and a ProteoChrom HPTLC Cellulose sheet. This simple surface treatment process significantly expands the analytical surfaces that can be analyzed with the liquid microjunction surface sampling probe, and therefore, also expands the analytical utility of this liquid extraction based surface sampling approach.
Subject(s)
Spectrometry, Mass, Electrospray Ionization/instrumentation , Aerosols/chemistry , Alkaloids/analysis , Chromatography, Thin Layer/methods , Hydrastis/chemistry , Hydrophobic and Hydrophilic Interactions , Peptides/analysis , Peptides/isolation & purification , Plant Roots/chemistry , Silica Gel/chemistry , Spectrometry, Mass, Electrospray Ionization/methods , Surface Properties , WettabilityABSTRACT
The sampling, ionization and detection of tryptic peptides separated in one-dimension on reversed-phase high-performance thin layer chromatography (HPTLC) plates was performed using liquid microjunction surface sampling probe electrospray ionization mass spectrometry. Tryptic digests of five proteins [cytochrome c, myoglobin, beta-casein, lysozyme and bovine serum albumin (BSA)] were spotted on reversed phase HPTLC RP-8 F254s and HPTLC RP-18 F254s plates. The plates were then developed using 70/30 methanol/water with 0.1M ammonium acetate. A dual purpose extraction/electrospray solution containing 70/30/0.1 water/methanol/formic acid was infused through the sampling probe during analysis of the developed lanes. Both full scan mass spectra and data dependent tandem mass spectra were acquired for each development lane to detect and verify the peptide distributions. Data dependent tandem mass spectra provided both protein identification and sequence coverage information. Highest sequence coverages were achieved for cytochrome c and myoglobin (62.5% and 58.3%, respectively) on reversed phase RP-8 plates. While the tryptic peptides were separated enough for identification, the peptide bands did show some overlap with most peptides located in the lower half of the development lane. Proteins whose peptides were more separated gave higher sequence coverage. Larger proteins such as beta-casein and BSA which were spotted in lower relative amounts gave much lower sequence coverage than the smaller proteins.
