Proteomic Expression Profiling and Identification of Serum Proteins Using Immobilized Trypsin Beads with MALDI-TOF/TOF Izabela D. Karbassi, Julius O. Nyalwidhe, Christopher E. Wilkins, Lisa H. Cazares, Raymond S. Lance, O. John Semmes, and Richard R. Drake* George L. Wright, Jr. Center for Biomedical Proteomics, Department of Microbiology and Molecular Cell Biology, Eastern Virginia Medical School, Norfolk, Virginia 23507 Received October 6, 2008
MALDI-TOF mass spectrometry is a widely used technique for serum protein expression profiling and biomarker discovery. Many profiling strategies typically employ chemical affinity beads or surfaces to decrease sample complexity of dynamic fluids such as serum or plasma. However, many of the proteins captured on a particular surface or bead are not resolved in the lower mass ranges where time-offlight mass spectrometers are most effective. Thus, a majority of reported protein expression profiling studies primarily interrogate the native low molecular mass constituents of the target sample. We report an expression profiling workflow that utilizes immobilized trypsin paramagnetic beads following an initial affinity bead fractionation step, thereby reducing large mass proteins to peptides that are better suited to analysis and sequencing determinations. Our bead-based trypsin approach resulted in more efficient digestion of complex serum protein extracts at short incubation times. This method was reproducible and readily adaptable to robotic sample handling and may be combined in tandem with other bead fractionation surfaces. When weak cationic and weak anionic bead surfaces were used, experimental conditions were optimized for tandem combinations of these beads with the immobilized trypsin step to produce an efficient serum fractionation strategy. A proof-of-concept pilot experiment using pooled human serum samples demonstrating reproducibility is presented, along with the sequence determination of selected tryptic peptides of serum proteins. Keywords: Biomarkers • immobilized trypsin • LIFT • MALDI-TOF • magnetic bead fractionation • profiling
Introduction Approaches to protein biomarker discovery continue to be spurred on by advances in mass spectrometry technology and improvements in clinical study design and bioinformatics strategies.1 A common approach has been to fractionate proteins in clinical samples using chemical affinity capture on beads or chip surfaces to reduce sample complexity. These approaches coupled with time-of-flight mass spectrometry, also termed expression profiling, can be automated for relatively high-throughput analysis of clinical samples.2,3 Serum expression profiling can be reproducible and portable across multiple laboratories, especially when rigorous study design and sample handling are combined with carefully controlled instrument calibration, automated sample preparation, and supervised bioinformatic data analysis.4-7 However, the difficulty in determining the protein identities of potential biomarker peaks, and the inability to interrogate low-abundance proteins, continues to hamper these expression profiling approaches.8-10 The application of TOF/TOF technology has brought with it the capability of protein identification through the generation * To whom correspondence should be addressed. Dr. Richard R. Drake, Eastern Virginia Medical School, Department of Microbiology and Molecular Cell Biology, 700 W. Olney Rd., Lewis Hall, Norfolk, VA 23517. E-mail:
[email protected]. Fax: 757-624-2255.
4182 Journal of Proteome Research 2009, 8, 4182–4192 Published on Web 07/15/2009
of MS/MS fragment ions.11 In particular for serum and plasma studies, new approaches are needed that incorporate the TOF/ TOF identification capabilities with strategies that address dynamic protein concentration ranges to further clinical biomarker assay development.1,6,12-14 A majority of reported MALDI-TOF based expression profiling studies only examine the native low molecular mass constituents (1-20 kDa) of serum or plasma. Thus, many proteins captured on a particular surface or bead are not effectively resolved on MALDI-TOF instruments due to their larger sizes (>20 kDa). In this regards, we report a workflow of magnetic bead-based chromatography surfaces and immobilized trypsin to generate peptide profiles reflective of the broader range of proteins captured in front-end purification and fractionation strategies applied to complex clinical fluids like serum or plasma. The generation of tryptic peptides increases the breadth of proteins detected and provides peak masses ideal for MALDI-TOF/TOF sequence identification. With the use of human serum samples, different modular and serial workflow combinations of chromatography beads with the immobilized trypsin beads are described, each of which can be performed manually or adapted to sample handling robotics systems. 10.1021/pr800836c CCC: $40.75
2009 American Chemical Society
Expression Profiling and Identification of Serum Proteins
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Figure 1. Linear mode MALDI-TOF spectra comparison of WCX fractionated serum samples after different trypsin digestion protocols. Serum samples were processed with 25 µL of trypsin beads for 30 min or with soluble trypsin (1:40 sample-to-trypsin ratio) for 30 min or overnight. The tryptic peptides were captured by MB-C18 beads, and eluted peptides were mixed 1:3 with CHCA matrix for MALDITOF analysis in linear mode.
Materials and Methods Serum Samples. A pooled human serum sample collected from over 360 donors4 was used for method development procedures. For the proof-of-concept tryptic analysis of clinical serum samples, pools of each group were generated. For the prostate cancer study, 120 serum samples from diagnosisconfirmed benign prostatic hyperplasia (BPH) and prostate cancer (PCa) patients were pooled in the following manner: 10 pools for BPH and 10 pools for PCa with each pool containing 6 samples. Patient demographics for BPH included a PSA range of 2.13-9.72 ng/mL (mean 6.04, median 6.40 ng/ mL) and a mean age of 67.8 years old (range 50-86). For PCa, there was a PSA range of 2.40-9.90 ng/mL (mean 5.82, median 5.64 ng/mL), Gleason score mean of 6.47, and a mean age of 63.7 years old (range 47-85). Magnetic Bead-Based Fractionation. Initial fractionation of serum was done with MB-WCX (weak cationic exchange) or MB-WAX (weak anionic exchange) paramagnetic beads as described by the manufacturer’s protocols (Bruker Daltonics, Bremen, Germany). Briefly, for MB-WCX fractionation, 20 µL of serum was mixed with 40 µL of binding solution supplied by the manufacturer and 20 µL of MB-WCX beads for 15 min (mixing every 5 min). A magnetic bead separator was used to concentrate the beads and for the wash/rinse processes. Unbound serum proteins were removed and the beads were washed 3 times with 100 µL of MB-WCX wash solution. Bound serum proteins were eluted with 10 µL of MB-WCX elution solution supplied by the manufacturer. Finally, 8 µL of HPLC
water and 1 µL of MB-WCX stabilization solution were added to the WCX eluate to give a final sample pH of ∼ 8. For the MB-WAX fractionation, 20 µL of MB-WAX beads were first washed with an activation solution and then equilibrated with a pH 5 binding solution supplied by the manufacturer. Twenty microliters of serum was subsequently incubated with 40 µL of the pH 5 binding solution along with the activated MB-WAX beads for 15 min (mixing every 5 min). Unbound serum proteins were removed and the beads were washed 3 times with 100 µL of MB-WAX pH 5 binding solution. Bound serum proteins were then eluted with 10 µL of MB-WAX elution solution supplied by the manufacturer and 11 µL of MB-WCX elution was added to the WAX eluate, to give a final sample pH of ∼ 8. These approaches may be done manually or with an automated method using a ClinProt liquid handling robot as per manufacturer’s instructions. For reduction and alkylation, 8 µg of the fractionated samples was reduced with 8 mM DTT in 25 mM ammonium bicarbonate (pH 7.8) at 56 °C for 1 h (24 µL total volume). If being done with the ClinProt robot, 10 µL of eluate was used for reduction as above. The reduced samples were then alkylated with 17 mM iodoacetamide in 20 mM ammonium bicarbonate total solution (29 µL total volume). Liquid and Magnetic Bead-Based Trypsinization. Sequencing grade trypsin (Roche, Basel, Switzerland) was resuspended in 50 mM ammonium bicarbonate/4% acetonitrile (ACN) to a final concentration of 40 ng/µL. For each reaction, 200 ng of trypsin was added to the reduced and alkylated samples Journal of Proteome Research • Vol. 8, No. 9, 2009 4183
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Karbassi et al.
Figure 2. Reflectron mode MALDI-TOF spectra comparison of WCX fractionated serum samples after different trypsin digestion protocols. Serum samples were processed with 25 µL of trypsin beads for 30 min or with soluble trypsin (1:40 sample-to-trypsin ratio) for 30 min or overnight. The tryptic peptides were captured by MB-C18 beads, and eluted peptides were mixed 1:3 with CHCA matrix for MALDITOF analysis in reflectron mode.
yielding a 40:1 serum protein to trypsin ratio (incubated for 30 min and overnight at 37 °C). Paramagnetic immobilized trypsin, EnzyBeads Trypsine (Agro-Bio, La Ferte Saint Aubin, France), was initially washed with 25 mM ammonium bicarbonate (pH 7.8). Twenty microliters of reduced/alkylated samples was trypsinized with 25 µL of beads as described by the manufacturer for 30 min at 37 °C. This is the equivalent of 3 units of enzyme activity per reaction, with 1 unit defined as the amount of EnzyBeads Trypsine required to hydrolyze 1 µmole of chromogenic substrate in 1 min at 25 °C. Digested peptides were removed from the beads that were held in place by a magnetic separator. Sample Cleanup and Concentration. Tryptic peptides were recaptured and concentrated with HIC-C18 paramagnetic beads (Bruker Daltonics, Bremen, Germany) as follows. Twenty microliters of the tryptic digest was incubated with 10 µL of HIC-C18 beads and 40 µL of HIC-C18 binding buffer. Bound 4184
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peptides were washed with the manufacturer’s wash solution and eluted in 10 µL of 50% ACN as per the manufacturer’s specifications. MALDI-TOF/TOF. Two microliters of the tryptic peptide sample after HIC-C18 cleanup was mixed with 4 µL of CHCA matrix solution (4 mL ethanol, 2 mL acetone, 0.008 g CHCA, and 0.1% TFA) and 1 µL of the mixture was spotted onto an AnchorChip plate. An UltraFlex III MALDI-TOF/TOF instrument (Bruker Daltonics, Bremen, Germany) was used to analyze peptides in linear and reflectron modes. Spectra were acquired from an average of 2000 laser shots. FlexAnalysis software in BioTools 3.1 (Bruker Daltonic) was used to baseline subtract, normalize spectra (using total ion current) and determine peak m/z values and intensities in the mass range of 1000-10 000 m/z for native serum peptides, or 800-4000 m/z for trypsin digested samples. A mass window of 0.5% was used for peak alignment. Peaks of interest were further analyzed on a separate
Expression Profiling and Identification of Serum Proteins
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platform using the LIFT function of a MALDI-TOF/TOF Ultraflex III instrument. BioTools 3.1 software and the MASCOT 2.2.03 search engine (www.matrixscience.com) were used to compare the TOF/TOF spectra against primary sequence databases (Swiss-Prot) to determine peptide sequence identities (search criteria: carbamidomethyl and oxidation modifications; 100 ppm mass tolerance MS; 0.5 Da MS/MS tolerance).
Results Integrating Trypsin Digestion into a Bead-Based Affinity Fractionation Workflow. The goal of this study was to introduce a trypsin digestion step following standard ion exchange fractionation of serum samples, so as to integrate a common off-line step. We hypothesized that inclusion of the trypsin digestion would facilitate more direct protein identifications of high molecular weight proteins by generating peptides in an optimal mass range (e4000 m/z) for detection and sequence identification by MALDI-TOF/TOF. This approach could also broaden the dynamic concentration and mass range of the proteins detected. Finally, by using commercially available magnetic bead surfaces, workflows could be developed that allow robotic sample processing and, therefore, opportunities for analysis of large numbers of samples such as those used in clinical proteomic studies. For the studies described herein, we utilized a trypsin product immobilized on paramagnetic beads, EnzyBeads Trypsine (Agro-Bio, La Ferte Saint Aubin, France), which can be used with a robotic sample handling system equipped with magnetic separation capabilities. We first compared the efficiency of the trypsin bead digestion of serum proteins to a standard soluble trypsin protocol. A pooled healthy serum sample was incubated with MB-WCX paramagnetic beads to reduce sample complexity, and the eluate proteins were used in subsequent digestions. Reduction and alkylation of the proteins eluted from the MB-WCX beads prior to either trypsin digest protocol significantly improved digestion efficiency (data not shown), and thus is included in all workflows described herein. The tryptic eluates were then fractionated by C18 magnetic beads, and spotted 1:3 with CHCA matrix for MALDI-TOF analysis. Representative spectra in linear (Figure 1) and reflectron (Figure 2) modes from the three tryptic digests (digested with trypsin beads for 30 min, digested with soluble trypsin for 30 min and digested overnight with soluble trypsin) were compared with the undigested MB-WCX eluate. In either mode, the trypsin beads were clearly more efficient after only 30 min incubation and produced a greater number of lower mass peptides as compared to the standard soluble trypsin at 30 min or overnight incubations. On the basis of separation and visualization of these sample digests on SDSpolyacrylamide gels, the greater number of lower mass peptides in the magnetic bead preparations is due to more complete digestion of the constituent proteins, including serum albumin, as shown in representative gel profiles of the digest methods in Figure 3. The lack of lower mass stained peptides/proteins in lane 3 of this gel is consistent with the inability to electrophoretically resolve and stain peptides below 4000 kDa, as also indicated by the presence of greater numbers of lower mass peptides following trypsin bead digestions (Figures 1 and 2). Reproducibility of Immobilized Trypsin Protocol. As our goal was to develop a useful workflow for evaluating large numbers of clinical samples, we next determined the reproducibility of the MB-WCX and trypsin bead digest workflow when applied to multiple aliquots of pooled serum. Six independent samples were processed manually for MB-WCX/trypsin/C18
Figure 3. SDS-PAGE separation of WCX fractionated serum samples after different trypsin digestion protocols. Serum proteins eluted from WCX beads (6 ug) were reduced, alkylated and digested with either soluble or immobilized trypsin as follows: WCX fractionated serum without trypsin digestion (lane 1); overnight soluble trypsin digest at 37 °C using a protein to trypsin ratio of 30:1 (lane 2); digestion with immobilized trypsin beads for 30 min at 37 °C (lane 3). Samples were denatured, separated on a NuPAGE 12% Bis-Tris gel (Invitrogen) and silver stained (Silver Stain Plus; Bio-Rad).
fractionation and spotted in triplicate for MALDI-TOF profiling. As shown in Figure 4, the spectral patterns were reproducible and reflected a similar degree of tryspin digestion per sample. The coefficients of variation (CV) of 16 representative peak intensities ranged from 5.60% to 13.65% (9.52% mean) for manual processing (Table 1). An automated robotic workflow was also examined in which the robot performed the WCX fractionation, then reduction and alkylation reactions on the four elution aliquots of the pooled sample were performed. Trypsin bead digestion and MB-C18 capture followed, with spotting on a MALDI plate in duplicate. The CVs of the same m/z peak intensities examined with the manual protocol ranged from 3.71% to 12.3% (7.95% mean) as listed in the left panel of Table 1. From these types of experiments, we determined that, under defined conditions, the amount of trypsin digestion was consistent and reproducible across multiple samples. Bead-Based Workflow with MALDI-TOF/TOF Identification of Tryptic Peptides. One of the goals of the study was to assess whether trypsin digestion of the MB-WCX captured serum proteins would lead to improved sequencing identification of the captured proteins within an expression profiling workflow that retained sample throughput capabilities. As a baseline comparison, native low mass peptides and proteins (
