created: 24th October 1997, last updated: 13 July 1998,© 1997 ABRF
Millipore Corporation, 80 Ashby Road, Bedford, MA 01730, USA
e-mail address: Don_Sheer@Millipore.com
ABSTRACT
Mass Spectrometry has become the method of choice to identify and characterize cell-expressed biomolecules. This technology has evolved so rapidly that performing sample preparation in high throughput has become a rate-limiting step. The convenient MultiScreen® solvent resistant 96-well filter plate with Column Loader was demonstrated to provide efficient salt and detergent removal with excellent recovery in the required volume for subsequent sample work-up. The 96-well loading of dry powder or resin with Column Loader offers rapid and uniform sorbant packing of 25 and 45 µl 'mini-columns'. MultiScreen 96-well formed 'mini-column' performance was determined from well to well and plate to plate to assess variability with respect to sample recovery and reproducibility. Applications using silica and divinylbenzene are presented in order to demonstrate selective fractionation and efficient sample preparation for high throughput analyses.
INTRODUCTION
High-Throughput Screening (HTS) technology has rapidly evolved at most levels of assay development. However, HTS sample preparation still remains a challenge (1). The use of a 96-well format in combination with column loading for preparation of equivalent amounts of dry resin into 96 wells provides versatility in performing selective sample preparation. Similar to adsorptive column chromatography, optimization in sample recovery and reproducibility depends on the sample load, particle size and the selected HTS 'mini-column'(2). In order to demonstrate reproducibility and validation in the 96-well format as a sample preparation device, varying sized functional resins were loaded in solvent resistant MultiScreen plates using the 25 or 45 µl column loader. The 96 'mini-columns' containing dry resin were wetted and packed by centrifugation, sample bound, washed and eluted for direct analyses. Performance results are presented to summarize cytochrome c, BSA, and trypsinized cytochrome c recovery and reproducibility from well to well. The relative loading capacity of the 25 and 45 µl columns were compared and summarized in order to provide a guideline for resin selection and column bed volume. Applications were used to demonstrate simultaneous fractionation of 96 samples with efficient sample preparation for direct analyses.
MATERIALS AND METHODS
A. Materials
All assays were performed in MultiScreen-Resist LCR 0.4 µm Hydrophilic PTFE resistant plates (Millipore, Cat. # SE3R008M7) using the Centrifuge Ring (Cat. #S2ER088V7). The 96 'mini-columns' were simultaneously loaded with the 45 µl MultiScreen Column Loader (Cat. # MACL 096 45).
Figure 1. The MultiScreen Column Loaders bring the capability of performing sample preparation for varying amounts of sample using selected media and resins.
All centrifugations were performed on Jouan centrifuges at 2,500-3,000 x g for 5 minutes using the CR312 or CR412 models with E4 or 4/ST rotors, respectively.
The following adsorptive media was used for MultiScreen loading: Reversed Phase C18 at 300 Å -15 µm, 300 Å -40 µm (Amicon-Matrex), 300 Å 6-10 µm, and 300 Å 37-55 µm (Waters-Microbondapak); Styrenedivinylbenzene PLRP-S at 300 Å 15-20 µm (Polymer Labs); and Strong Cation Exchange at 300 Å -10-22 µm (Purolite).
B. Sample binding and washing with MultiScreen
Dry C18 silica or polymeric (i.e. divinylbenzene) resins were poured into the wells of the column loader, distributed with the beveled acrylic scraper (Figure 1), and loaded into MultiScreen as described.The resultant MultiScreen 'mini-columns' were packed by adding 300 µl of isopropanol (IPA) to each well followed by centrifugation at 3,000 x g for 5 minutes. Residual IPA was removed as columns were equilibrated in the respective binding solutions after receiving 200 µl of either 0.1% TFA (reversed phase resin) or 20 mM HCl (SCX resin) and centrifuged. Samples were loaded in 200 µl of appropriate binding solution and centrifuged. Loosely bound salts and detergents were removed with a 200 µl wash step using the respective binding solutions.
C. Sample elution in MultiScreen
Resin-bound analyte was recovered with 2 x 75 µl centrifugations containing 90% acetonitrile /0.1% TFA/water (RP) or 1.5 N ammonium hydroxide/50% methanol /water (SCX). Analysis of recovered sample was performed as described in the legends using visible (VMax plate reader; Molecular Devices, CA) or UV 214 absorbance (HPLC; Shimadzu, MD).
RESULTS
In order to evaluate reproducibility and consistency of the MultiScreen 'mini-columns' for a given sample loading, three different types of reversed phase media were packed into 45 and 25 µl columns. Cytochrome c loading was serially diluted from 50 µg as shown in Figure 2. Results showed that at lower sample loads (0.8 µg in 25 µl columns), recovery was approximately 60 to 65%.
Figure 2. Comparison of C18-like resin performance in MultiScreen as a function of cytochrome c loading in 45 or 25 µl 'mini columns'. Samples were loaded in 200 µl with 0.1% TFA and recovered in 2 x 75 µl elution by centrifugation using 90% acetonitrile / 0.1% TFA. Quantitation was determined against a standard curve by 405 nm absorbance (96-well plate reader) or UV 214 (reversed phase HPLC). Coefficients of Variation (%CV) were lower than 7% for all samples (N=36 for all loads).
To determine the day-to-day reproducibility in sample recovery as a function of sample loading, 45 µl packed columns of indicated resins received increasing amounts of cytochome c as shown in Figure 3. Standard deviation remained below 7% for sample loading and day to day column packing when using the 45 µl column loader.
Figure 3. Day to day variability for C18 'mini-columns' with increased cytochrome c loading. MultiScreen plates were prepared with 45 µl column loader and assayed on consecutive days as described (C.V.< 7%, for both days at all loads, N=36).
To compare small and large size proteins as a function of sample load and recovery, decreasing amounts of cytotochrome c and BSA were processed with C18 (15 µm, 200Å) using the 45 µl column loader shown in Table 1. The capacity of C18 for cytochrome c was about two fold higher than for BSA at loads >5 µg.
Table 1. Sample recovery as a function of cytochrome c and BSA loading with 45 µl packed 'mini-columns'. Samples were bound and washed in 0.1% TFA/water and eluted twice with 50 µl of 90% acetonitrile/0.1% TFA. Cytochrome c concentration was determined directly by absorbance at 410 nm in a spectrophotometer and BSA by UV at 280 nm or by BCA total protein assay at 562 nm (Pierce Chem. Co.) N=16.
Sample recovery as a function of particle size was studied in MultiScreen using 45 µl columns containing 15 and 40 µm-C18 resin shown in Figure 4. A significant increase in recovery was observed at higher sample loading (>25 µg) using the smaller particle size. There appears to be an advantage in using low cost resin (large particle size) at low sample loads (<25 µg) since cytochrome c binding significantly decreased at low sample loads with 40 µm C18 silica.
Figure 4. Comparison of cytochrome c loading as a function of particle size using C18 silica packed MultiScreen. 'Mini-columns' were prepared in separate plates as described in Materials and Methods. Relative amounts of cytochrome c were determined by absorbance at 405 nm using a plate reader, (at each concentration of cytochrome c, N=32).
To demonstrate the versatility of MultiScreen to accommodate other functional resins for a variety of applications, styrenedivinylbenzene, PLRPS (reversed phase) and Purolite (SCX) packed 45 µl columns were used to process complex peptide mixtures in Figure 5. Increasing amounts of cytochrome c tryptic peptides were bound, washed and eluted for sample recovery determination. Comparison of reversed phase and SCX resin capacity for small peptides were comparable, however the SCX exhibited about 15-20% increase at higher peptide loads.
Figure 5. Comparison of peptide binding and elution with PLRPS (reversed phase) and Purolite ( SCX) resins in MultiScreen. Tryptic digest of cytochrome c was bound in either 0.1% TFA (reversed phase) or 20 mM HCl (SCX) and washed once with these binding solutions. Elution was performed twice with 50 µl in either 90% acetonitrile/0.1% TFA (reversed phase) or 1.5 N ammonium hydroxide/50% acetonitrile/water (SCX). Relative amounts of recovered peptides were determined by absorbance at 405 nm and subsequently quantitated by reversed phase HPLC shown in Figure 6. (C.V. < 10% where N=16)
Selectivity and reproducibility of peptide recovery were investigated using 50 picomoles (~3.5 µg) of loaded trypsinized cytochrome c in 96 wells. Figure 6 shows HPLC analyses of 4 randomly selected wells following C18 processing. Sample fidelity was maintained since all peptides (excluding early eluting amino acids) were accounted for with highly reproducible chromatograms for the selected samples.
Figure 6. Chromatogram comparison plots of cytochrome c tryptic peptides following binding and desalting in C18-MultiScreen. Eluates were recovered, speed vacuum dried, and resuspended in 0.1% TFA for reversed phase HPLC analysis. Peptides were separated on a 150 x 2 mm C18-Amicon-Matrex 300Å-15 µm column using a 5-45% acetonitrile gradient over 40 minutes. Approximately 100 picomoles (~ 7 µg) were loaded onto the column using a Shimadzu HPLC-autosampler. Results were comparable for C18 silica and resin based PLRP-S (data not shown for C18 silica). Samples are as follows: A. Starting solution and peptide digest loaded onto MultiScreen. B, C, D and E. Random eluates following MultiScreen processing. Note: initial salt peak is reduced from MultiScreen treated samples while resolution is significantly improved for a variety of peptides at 22-28 minutes in gradient.
Discussion
The reported separations were performed by centrifugation in order to deliver the required uniformity for column packing, washing, and elution. For the small size resins studied, vacuum filtration is not recommended. Particle size and sample loading were optimized for sample recovery. Of the reversed phase matrices investigated, similar size resins exhibited comparable performance. Reproducible sample recovery of complex peptide mixtures was demonstrated after sample desalting with C18 and SCX. In comparison, SCX was more selective for hydrophilic peptides (data not shown). The combination of salt or detergent removal and stepwise batch separation (i.e. pH, hydrophobicity) can be performed with the 96-well format for downstream analysis (i.e. mass spectrometry, HPLC, capillary electrophoresis, metabolite screening and assay development). Results from this study demonstrate that consistent and reproducible chromatography with high throughput can be achieved with MultiScreen.
References