SUMMARY. Key words: in-gel digestion, filter plate, LC-MSMS, proteomics.

Transcription

1 J Electrophoresis 2005 ; 49 : 71 [Short Communication] Assessment of filter plates for multi-well in-gel digestion of proteins separated by polyacrylamide gel electrophoresis to identify them with LC-ESI/MSMS Kazuhisa Kameyama 1, Tomohiro Nanri 2, Yuko Yamanaka 2, Mikiko Arima 2 and Hiroshi Kawasaki 2 SUMMARY In-gel digestion is an important technique for proteome analysis using polyacrylamide gel electrophoresis. Although the identification by MSMS ion search is much reliable than PMF, MSMS analysis of peptides separated by nanolc directly coupled to MS is time consuming and clogging with particles from in-gel digest interrupts separation of peptide by nanolc. Removal of particles from the digest with filtration is essential for nanolc analysis, but it is laborious process, when a large number of samples should be processed. Simple and high-throughput method is necessary for in-gel digestion and the sample preparation of nanolc. The characteristics of filter plates are investigated and a method of in-gel digestion for nanolc-msms systems using inexpensive 96-well filter plate is presented. Key words: in-gel digestion, filter plate, LC-MSMS, proteomics. INTRODUCTION Polyacrylamide gel electrophoresis (PAGE) is an extremely powerful tool to quantify and resolve thousands of differentially expressed proteins within a cell. Mass spectrometric analysis of peptides generated by protease digestion can identify proteins in a spot or a band stained with dye. In-gel digestion bridges protein separation by PAGE and identification by mass spectrometry (MS). It is critical and laborious step for proteome analysis. Robots, which perform spot cutting, in-gel digestion and also spotting on a MALDI plate, are developed for the highthroughput proteomic analysis by peptide mass fingerprinting (PMF) 1, 2). PMF using matrix-assisted laser desorption ionization (MALDI) /time-of-flight (TOF) mass spectrometer is convenient and its throughput is high 3). It gives, however, ambivalent results for some spots. These spots should be identified by another method such as MSMS ion search. LC-MSMS analysis of the digest gives multiple MS spectra for single protein. Thus, the identification by MSMS ion search is much reliable than PMF is 4, 5). MSMS analysis of peptides separated by nanolc directly coupled to MS is time consuming and clogging with particles from in-gel digest interrupts separation of peptide by nanolc. Therefore, removal of particles from the digest with filtration is essential for nanolc analysis. Filtration is laborious process, when a large number of samples should be processed. We report here a method of in-gel digestion for nanolc- MSMS systems using inexpensive 96-well filter plate. The method of in-gel digestion using filter plate originally has been reported by Pluskal MG et al for PMF 6). Since PMF is highly affected by MS peaks from contaminations, the filter plates are strictly quality-controlled and very expensive. MSMS ion search, on the contrary, is relatively tolerant for the MS peaks from contaminations, so inexpensive filter plates can be used for the in-gel digestion to nanolc- MSMS analysis. We assessed the characteristics of inex- 1 Nihon-Millipore K.K. Mita Kokusai Bldg., 4-28, Mita 1-chome, Minato-ku, Tokyo , Japan. 2 Supra Molecular Biology, International Graduate School of Arts and Sciences, Yokohama City University, Maioka , Totsuka-ku, Yokohama , Japan. Correspondence address: Hiroshi Kawasaki; Supra Molecular Biology, International Graduate School of Arts and Sciences, Yokohama City University, Maioka , Totsuka-ku, Yokohama , Japan. Abbreviations: PAGE, Polyacrylamide gel electrophoresis; PMF, peptide mass fingerprinting; MALDI, matrix-assisted laser desorption ionization; TOF, time-of-flight; MS, mass spectrometry; CBB, Coomassie Brilliant Blue R-250; BSA, Bovine Serum Albumin; LC, liquid chromatography, ESI, electrospray ionization. (Received June 27, 2005, Accepted August 25, 2005, Published September 15, 2005)

2 J Electrophoresis 2005 ; 49 : 72 pensive filter plate. We describe a procedure of in-gel digestion suitable for using inexpensive filter plate. MATERIALS AND METHODS Bovine Serum Albumin (BSA) was purchased from Sigma-Aldrich Co. (MO, USA). MultiScreen-Solvinert hydrophilic PTFE (polytetrafluoroethylene) 96-well filter plate (MSRLN0410) and MultiScreen Vacuum Manifold (MAVM096R) was purchased from Millipore Co. (MA, USA). Peptide mixture for MSMS analysis (Trypsin digest of BSA) was purchased from KYA technologies Co. (Tokyo, Japan). Other reagents were purchased from Wako pure chemical Industries Ltd. (Osaka, Japan). 1) SDS-PAGE SDS-PAGE was carried out according to the method described by Laemmli 7). BSA was dissolved in the sample buffer and separated using slab gel at the amount of 7.5 pmole, 750 fmole, 75 fmole and 7.5 fmole for each lane. After staining with Coomassie Brilliant Blue R-250 (CBB), each band was cut. 2) In-gel digestion In-gel digestion was performed according to the procedure summarized in Table 1. The in-gel digestion procedure routinely used in our laboratory was modified and optimized for in-gel digestion with filter plate 8). 3) LC-ESI/MSMS analysis Digests were recovered from filter plates with filtration by centrifuge. They were applied directly to nanolc-ms, in which peptides were separated by capillary column (75 um 150 mm, Atlantis (Waters, MA, USA) or PepMap (LC packings, Amsterdam, the Netherlands)) using a gradient elution (2.5%/min of acetonitrile) with split flow by using CapLC system and were introduced to Q-Tof micro mass spectrometer using nanoesi interface with PicoTip spray tip. MSMS data of peptides were collected using survey mode of MassLynx. MS or MSMS spectra obtained were analyzed by ProteinLynx (Waters, MA, USA). RESULTS AND DISCUSSION There are several kinds of filter plate available commer- Table 1. Summary of the procedure of in-gel digestion using filter plate a) Cutting gel pieces Cut the gel pieces and put them in the wells b) Destaining Add 200 µl 100 mm NH 4 HCO 3 /60% acetonitrile Incubate 15 min at room temperature Add 200 µl acetonitrile Incubate 15 min at room temperature Repeat the steps for destaining above 3 times Wipe water drops beneath the plate with paper towel and dry the membrane under vacuum c) Digestion Add 25 µl 100 mm NH 4 HCO 3 containing 12.5 µg/ml Trypsin Incubate 5 min at room temperature Add 50 µl 100 mm NH 4 HCO 3 Incubate 3 hour at 37 C d) Extraction Collect the digest by centrifugation (1,000 G, 3 min) Add 50 µl 60% acetonitrile Incubate 20 min at room temperature Collect the solution by centrifugation (1,000 G, 3 min) Dry up and resolve the digests with 10 µl 0.1% formic acid/0.01% trifluoroacetic acid Table 2. Characteristics of Filter Plates MultiScreen Solvinert MultiScreen-GV MultiScreen-HA AcroPrep (GHP) UNIFILTER (FF) Manufacturer Millipore Millipore Millipore Pall Whatman Membrane material Hydrophilic PTFE/PP Hydrophilic PVDF Mixed Cellulose Esters Hydrophilic PP PP Membrane Pore Size 0.45 µm 0.45 µm 0.45 µm 0.45 µm 0.45 µm Base Plate Polyolefin copolymer Styrene Styrene Polypropyrene Polystyrene Chemical Compatibility Acetic acid, glacial Yes Limited Use No Yes Yes Trifluoroacetic acid Yes ND ND ND No Methyl Alcohol Yes Limited Use No Yes Yes Acetonitrile Yes No No Yes No

3 J Electrophoresis 2005 ; 49 : 73 cially. The characteristics of these filter plates are summarized in Table 2. Filter plate for in-gel digestion should be resistant for organic solvents such as methanol and acetonitrile and also have low peptide/ protein binding capacity. We selected MultiScreen 96-well filter plate (Millipore), since it is tolerant for organic solvent and reported to have quite low binding capacity for peptides. We investigated whether this plate can be used for in-gel digestion. Probably because the filter membrane of plates was coated with protectant, many peaks were observed in a filtrate by the analysis with LC-MS. However, no peaks were observed after washing of the membrane by filtrating 200 µl of acetonitrile twice or 200 µl of 0.05% TFA twice. Washing of the membrane with 200 µl of water twice could not decrease the peaks in the filtrate completely. Washing of filter membrane was usually done while the destaining process of gel pieces of in-gel digestion as summarized in Table 1. Figure 1 shows the peptide recovery from filter plates. Two MS chromatograms, which are BSA digests before or after filtration, respectively, were quite similar (Fig. 1a), but the recovery is dependent on peptides. Fig. 1b shows quantitation of representative peptides from BSA digest by LC-MS before or after filtration with the plate. Some peptides bind to the filter materials tightly. Recovery of peptide a) b) Fig. 1. Peptide recovery from the filter plate Various amounts of BSA digests were analyzed using MS mode of MassLynx with Q-Tof before and after filtration. a) MS chromatograms of BSA digest (1 pmole) before (lower) and after (upper) filtration with the plate. b) Quantitation of peptide before and after filtration. The peak areas of two peptides are shown. Triangle, before filtration; square, after filtration. Solid line, peptide (m/z=740.6, ETYGDMADC*C*EK) Broken line, peptide (m/z=722.7, YIC*DNQDTISSK) C*, carboxymethyl Cys

4 J Electrophoresis 2005 ; 49 : 74 a) b) Fig. 2. MSMS analysis and identification of proteins in-gel digested using the filter plate. a) Mass chromatrograms of the digests. Left, 7.5 pmole of the digest; Right, 75 fmole of the digest. Upper, MSMS chromatograms; Lower MS survey chromatograms. Numbers shown are m/z values of BSA peptides identified. m/z=495.81, ; m/z=653.39, ; m/z=820.51, ; m/z=571.89, ; m/z=700.36, ; m/z= , ; m/z=847.01, ; m/z=784.41, b) Screen copy of the search result by ProteinLynx. Lower window shows MSMS spectrum of a peptide (m/z=740.4), in which the assignment of sequence ions are shown. Almost all y-series ions are assigned.

5 J Electrophoresis 2005 ; 49 : 75 (ETYGDMADC*C*EK) from the plate was about 100%, but recovery of peptide (YIC*DNQDTISSK) was about 50%. In-gel digestion was carried out using the plate with the procedure shown in Table 1. The digests were analyzed by ESI-nanoLC-MSMS system. BSA was correctly identified by MSMS ion search with the peak list from LC-MSMS analysis of the digests containing 7.5 pmole, 750 fmole and 75 fmole of BSA. Figure 2a shows MS and MSMS chromatograms containing 7.5 pmole and 75 fmole of BSA. The elution positions of peptides, the MSMS spectrum of which were assigned to a peptide of BSA, are shown. Several MSMS spectra not assigned to BSA were obtained from 7.5 pmole digest, but they disappeared in 75-fmole digest. MSMS spectra after 30 min are commonly observed in ingel digests. They probably came from polymers in the gel. The sequence coverage of BSA identified was 19.3%, 13.7% and 8.4% for 7.5 pmole, 750 fmole and 75 fmole of BSA, respectively. As shown in Figure 2b, the quality of MSMS spectrum is good enough to assign peptide sequence; almost all y-series ions were observed in the MSMS spectrum obtained from 75-fmole digest. We could obtain MSMS spectra of peptides from the gel of 7.5 fmole BSA. Their quality was not enough for identification by MSMS ion search (Data not shown). Pluskal MG et. al reported that 250 fmole BSA in a gel piece was clearly identified by peptide mass fingerprinting using MALDI-TOF MS with 15 20% sequence coverage using qualitycontrolled and expensive filter plates for in-gel digestion 6). In this study, the samples containing 75 fmole proteins were clearly identified by MSMS ion search. The procedure summarized in Table 1 was applied to several samples separated by PAGE and we could get better MSMS spectrum enough to identify them (data not shown). We concluded that the inexpensive 96 well filter plate is usable for in-gel digestion of sub pmole protein with the protocol described here to identify proteins by MSMS ion search using LC- ESI-MSMS system. ACKNOWLEDGEMENTS We thank Hisashi Hirano (Yokohama City University) and Mariko Tosu (Nihon-Millipore K.K.) for supporting this study. REFERENCES 1) Canelle L, Pionneau C, Marie A, Bousquet J, Bigeard J, Lutomski D, Kadri T, Caron M, Joubert-Caron R. Automating proteome analysis: improvements in throughput, quality and accuracy of protein identification by peptide mass fingerprinting. Rapid Commun Mass Spectrom 2004;18: ) Houthaeve T, Gausepohl H, Ashman K, Nillson T, Mann M. Automated protein preparation techniques using a digest robot. J Protein Chem 1997;16: ) Thiede B, Hohenwarter W, Krah A, Mattow J, Schmid M, Schmidt F, Jungblut PR. Peptide mass fingerprinting. Methods 2005;35: ) Yates JR, Eng JK, McCormack AL, Schieltz D. Method to correlate tandem mass spectra of modified peptides to amino acid sequences in the protein database. Anal Chem 1995;67: ) Pappin DJ, Hojrup P, Bleasby AJ. Rapid identification of proteins by peptide-mass fingerprinting. Curr Biol 1993;3: ) Pluskal MG, Bogdanova A, Lopez M, Gutierrez S, Pitt AM. Multiwell in-gel protein digestion and microscale sample preparation for protein identification by mass spectrometry. Proteomics 2002;2: ) Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227: ) Iwafune Y, Kawasaki H, Hirano H. Electrophoretic analysis of phosphorylation of the yeast 20S proteasome. Electrophoresis 2002;23: