Anal. Chem. 2002, 74, 4725-4733
A Direct Nanoflow Liquid Chromatography-Tandem Mass Spectrometry System for Interaction Proteomics Tohru Natsume,†,‡ Yoshio Yamauchi,† Hiroshi Nakayama,† Takashi Shinkawa,§ Mitsuaki Yanagida,†,| Nobuhiro Takahashi,†,| and Toshiaki Isobe*,†,§
Integrated Proteomics System Project, Pioneer Research on Genome the Frontier, Education and Science Agency, Japan, c/o Department of Chemistry, Graduate School of Science, Tokyo Metropolitan University 1-1, Minami-ohsawa, Hachioji, Tokyo 192-0397, Japan, Department of Applied Biological Science and Department of Biotechnology, United Graduate School of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu-shi, Tokyo 183-8509, Japan, and Department of Chemistry, Graduate School of Science, Tokyo Metropolitan University, 1-1, Minami-ohsawa, Hachioji, Tokyo 192-0397, Japan
One of the strategies of functional proteomics, research aiming to discover gene function at the protein level, is the comprehensive analysis of protein-protein interactions related to the functional linkage among proteins and analysis of functional cellular machinery to better understand the basis of cell functions. Here, we describe the direct nanoflow LC (DNLC) system, which is equipped with a fritless high-resolution electrospray interface column packed with 1-µm reversed-phase (RP) beads and a novel splitless nanoflow gradient elution system to operate the column. Using RP-DNLC at an extremely slow flow rate, 50 nL/min). The system is pressure-resistant (∼100 kg/cm2) and can be automated for reproducible high-throughput analysis. A combination of this LC system with data-dependent collision-induced dissociation tandem MS (MS/MS) on a Q-TOF hybrid mass spectrometer plus automated data processing facilitated identification of ∼100 protein components in a low-femtomole amount of a functional multiprotein complex that could be prepared from one dish of cultured cells. EXPERIMENTAL SECTION Materials. Standard laboratory chemicals were obtained from Sigma (St. Louis, MO), and modified trypsin (sequence grade) was from Boehringer-Mannheim (Framingham, MA). HPLC-grade acetonitrile and formic acid were purchased from Waken Chemical (Tokyo, Japan). Achromobacter protease 1 and reversed-phase packing material (Mightysil C18, particle size 1 µm) were provided by Dr. T. Masaki and M. Hosoda (Kanto Chemical, Tokyo), respectively. The reversed-phase material was slurry-packed into an ESI column without any pretreatment. Direct Nanoflow LC System. Assembly of the DNLC system is shown in Figure 1a. The LC system consists of a constant-flow nanoflow pump with a pressure limit of ∼300 bar (KYA Technologies, Tokyo), which delivers solvent to the ESI column a gradient device that we call a revolving nanoconnection (ReNCon) system, a high-pressure LC pump module (feeding pumps A and B in Figure 1a, HP1100 G1312A, Hewlett-Packard) to generate and to feed stepwise gradients to the ReNCon device, and an injection valve (Cheminart C2-0006, Valco, TX) for sample loading. The (15) Davis, M. T.; Stahl, D. C.; Swiderek, K. M.; Lee, T. D. Methods: Companion Methods Enzymol. 1994, 6, 304-314. (16) Figeys, D.; Aebersold, R. Anal. Chem. 1998, 70, 3721-3727. (17) Le Bihan, T.; Pinto, D.; Figeys, D. Anal. Chem. 2001, 73, 1307-1315. (18) Davis, M. T.; Stahl, D. C.; Lee, T. D. J. Am. Soc. Mass Spectrom. 1995, 6, 571-677.
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ReNCon gradient device consists of 10 channel solvent reservoirs made of PEEK tubings (8-cm length, 1.6-mm o.d., 254-µm i.d., inside volume 4.0 µL) connected between a 10-port manifold (Z10M1, Valco) and a 10-position switching valve (C5-1000, Valco) with finger-tight fittings. All these modules are assembled through a two-way six-position switching valve (Figure 1a) that serves to switch the flow path of the system, one for solvent feed to the ReNCon device and another for analysis (Figure 1b). In the feed position, programmed step gradients for RP-LC are generated by the high-pressure pump module and are supplied to reservoirs of the ReNCon device via an inlet on the 10-port manifold (Figure 1b). Thus, each reservoir is filled with different RP solvents of increasing acetonitrile concentrations by rotating the 10-position switching valve automatically at programmed time intervals. For routine analysis, reservoirs 1 and 2 were filled with 0.1% formic acid, 3 with 10% acetinitrile in 0.1% formic acid, 4 with 20%, 5 with 30%, 6 with 40%, 7 with 50%, 8 with 60%, line 9 with 70%, and 10 with 80%, respectively. After the filling process, the ReNCon device is connected to the nanoflow pump by switching the six-way valve to the analysis position. Further description of the DNLC system is given in the Results and Discussion section. Preparation of Tryptic Digest of Human Serum Albumin (HSA). HSA (5 mg) was dissolved in 2 mL of 0.5 M Tris-HCl (pH 8.5) containing 7 M guanidium hydrochloride and 10 mM EDTA, and dithiothreitol was added to a 3 mM final concentration while the solution was gently bubbled with N2 gas. After 2 h, 40 mg of iodoacetamide was added and the solution was left in the dark for 1 h at room temperature. The mixture was then dialyzed extensively against 10 mM ammonium bicarbonate buffer (pH 8.0), and the S-carbamoylmethyl HSA was recovered by lyophilization. Tryptic digestion was done at 37 °C for 8 h in 10 mM ammonium bicarbonate buffer (pH 8.0) at a HSA-to-trypsin ratio of 50:1. Preparation of Tryptic Digest of Total E. coli Extracts. Lyophilized E. coli proteins were dissolved in 8 M urea, 200 mM NH4HCO3, and 20 mM CaCl2. After a 4-fold dilution with distilled water, modified trypsin was added at a final substrate-to-trypsin ratio of 50:1. The mixture was then incubated at 37 °C for 15 h. The digested peptides were desalted on an RP column, lyophilized, and redissolved in distilled water containing 1% formic acid. Isolation and Digestion of a Nucleolin (NCL)-Binding Complex. Expression of flag-tagged NCL and immunoprecipitation to isolate the NCL-binding complex were performed as described.19 Briefly, after 48 h of transfection of flag-tagged NCL, human 293EBNA cells were harvested, washed with phosphatebuffered saline (PBS), and lysed in 1 mL of lysis buffer. For immunoprecipitation, the resulting cell lysate was then incubated overnight at 4 °C with 20 µL of M2-agarose. The protein-bound agarose beads were washed extensively with lysis buffer, and the proteins were eluted with flag peptide. For sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, 0.5 µg of the eluted proteins was loaded on an 11% SDSpolyacrylamide gel, followed by silver staining. For LC-MS/MS analysis, 0.2 µg of the isolated complex was precipitated using 20 µL of mixed methanol and chloroform (1:1 v/v). After vacuumdrying, the precipitate was digested with 5 µL of Achromobacter protease I (40 pM, substrate-to-enzyme ratio 50: 1) dissolved in (19) Yanagida, M.; Shimamoto, A.; Nishikawa, K.; Furuichi, Y.; Isobe, T.; Takahashi, N. Proteomics 2001, 1, 1390-1404.
Figure 1. Assembly (a) and flow diagram (b) of the direct nanoflow LC (DNLC) system. The system consists of a high-pressure syringe pump for constant nanoflow solvent delivery, a gradient device consisting of 10 channel solvent reservoirs for step elution solvents to carry out RP-LC, a high-pressure pump mixing module for solvent feed to the gradient device, and a flitless ESI column packed with 1-µm RP beads. These modules are assembled around the two-position six-port valve, which switches the flow path of the system from the solvent feed to the analysis position or vice versa.
Tris buffer (50 mM Tris-HCl, 6 M urea, 0.005% n-octyl glucopyranoside, pH 9.0) overnight at 37 °C. The digestion mixture was then directly injected onto the nano ESI column via a 5-µL sample loop. Nano ESI Column. The nano ESI column was prepared as described14 but with modifications. Briefly, a fused-silica capillary (150 µm i.d. × 375 µm o.d.) was pulled using a laser puller (Sutter Instruments Co., Novato, CA) to obtain a tip size of 0.3-0.5-µm i.d. (measured under a scanning electron microscope). The column was packed with a reversed-phase material (Mightysil C18, particle size 1 µm, Kanto Chemical) to a length of 2.5 cm and then connected to the LC line with microfingertight fittings via a metal union (Valco). High voltage for ionization (0.9-1.5 kV) was
applied to the metal union, and the eluate from RP-LC was sprayed on-line to a Micromass hybrid quadrupole time-of-flight (Q-Tof2) mass spectrometer (Micromass, Manchester, U.K.). LC-MS/MS. Reversed-phase separation and concentration of the digested peptide mixture were done on the DNLC in conjunction with an ESI column, as described above, and at flow rates of 500-25 nL/min. Samples were injected at a flow rate of 500 nL/min for 10 min (5 µL), and then the flow rate was decreased to the values indicated in the text. After back pressure of the column became stable (within 5-10 min), the peptides were eluted by a 60-min linear gradient from 0 to 70% B solvent, generated by the ReNCon system described above. Analytical Chemistry, Vol. 74, No. 18, September 15, 2002
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Figure 2. General scheme of the DNLC system. The solvent reservoirs of the ReNCon gradient device are connected between a 10-port electrical switching valve and a manifold. By sequentially rotating the electrical valves for programmed time duration, each solvent was transferred step by step to the fused-silica capillary, where a linear gradient is generated by diffusion of solvent boundaries during transfer to the column.
Analysis of MS/MS Data. MS and MS/MS spectra were obtained in a data-dependent mode. Up to four precursor ions above the intensity threshold of 10 counts/s were selected for MS/MS analyses from each survey scan. All MS/MS spectra of the E. coli cell lysate and NCL-binding complex were searched for against protein sequences of NCBInr, using batch processes of a Mascot software package.20 Criteria for match acceptance were as follows: (1) when the match scores were exceeded over 10 above the threshold, identifications were accepted without further consideration. (2) When scores were lower than 10 or identifications were based on single matched MS/MS spectra, we manually inspected the raw data for confirmation prior to acceptance. (3) Peptides assigned by less than three y series ions and those with a +4 charge state were all eliminated regardless of scores. RESULTS AND DISCUSSION Installation of the DNLC System. To maximize the resolution and sensitivity of the LC-MS/MS analysis, we designed a nano ESI column with a spray tip with an extremely narrow end (internal diameter of >0.5 µm). This column design accelerated ionization of peptides because smaller droplets formed during the spray ionization10 and also allowed for use of the high-resolution RP separation media of a very small particle size (1-µm diameter) without frit. However, this fritless nano ESI column forced us to develop a splitless solvent delivery system because the column had a relatively high back pressure (10-50 kg/cm2) that was hardly acceptable for the split-flow method using the conventional LC system. To obtain a stable solvent flow, we constructed a novel direct nanoflow solvent delivery system. The most difficult problem in constructing such a delivery system was to design the device to make a linear gradient on a nanoliter scale. (20) Perkins, D. N.; Pappin, D. J.; Creasy, D. M.; Cottrell, J. S. Electrophoresis 1999, 20, 3551-3567.
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Figure 3. Gradient curves generated by the DNLC system. Solvent reservoirs of the ReNCon gradient device were filled with solvents of different acetone concentrations (indicated below) and operated as described under the Experimental Section at various flow rates shown in the figure. The profiles were obtained by monitoring the acetone concentration at 275 nm on a UV detector (250-nL light path, UV/visible VIS-201, Linear) directly connected to the ReNCon device. The solvents used were (A) water and (B) 1% acetone in acetonitrile. Each port contained the following: ports 1 and 2, 0% B; 3, 10% B; 4, 20% B; 5, 30% B; 6, 40% B; 7, 50% B; 8, 60% B; 9, 70% B; 10, 80% B.
The design of the gradient device, which we call the ReNCon system, is shown in Figure 1a. The device consists of 10 channel solvent reservoirs (inside volume, 4 µL each) connected by means of a 10-port electrical switching valve and a manifold. Each reservoir was filled with step elution solvent for RP-LC supplied from two separate reservoirs for initial and final solvents using an automated high-flow-rate mixing module (Figure 1b; see also the Experimental Section). Figure 2 shows a schematic diagram of the DNLC system equipped with a single pressure-driven pump
Figure 4. Base peak chromatogram of the tryptic digest of HSA (100 fmol). Chromatography was done as described under the Experimental Section and at a flow rate of 100 nL/min. Ten major peptide ions used to estimate half-widths and a peptide ion used to estimate the detection limit of the system are indicated by open arrowheads and an a closed arrowhead, respectively.
for constant nanoflow solvent delivery, the ReNCon gradient device, and a nano ESI column. By rotating the 10-position electrical valves of the gradient device sequentially for programmed time duration, each solvent in 10 channel reservoirs was transferred step by step to the fused-silica capillary connected to the ESI column, where a linear gradient is generated by diffusion of solvent boundaries during transfer to the column (Figure 2). Figure 3 shows the gradient profile derived from the system at three different flow rates ranging from 300 to 40 nL/ min. This system produced almost linear profiles of a solvent gradient in a wide range of flow rates, in particular, at extremely low flow rates less than 100 nL/min (Figure 3). Analysis of the Tryptic Digest of Human Serum Albumin. To validate resolution and sensitivity of the DNLC-MS/MS system we analyzed the tryptic digest of HSA. The tryptic digest of reduced and S-carbamoylmethyl HSA was applied to the DNLC system, and the eluate was directly sprayed into a Q-Tof hybrid mass spectrometer to generate MS and MS/MS data. The DNLC system exhibited an excellent peak resolution and reproducibility, as illustrated by the base peak chromatogram obtained with a 100-fmol HSA digest (Figure 4). Here, the average half-width of 10 major peaks indicated by open arrowheads was 7.0 s (standard deviation, 0.7), and variations in retention times of these peaks were less than 0.5 min in three repeated analytical runs (data not shown). The detection limit of this system was estimated to be 120 amol at a signal-to-noise ratio (S/N) of 2, based on a fragment with m/z ) 682.383+ (indicated by a closed arrowhead in Figure 4) detected using a single-ion monitor. Analysis of MS data suggested that 65 peptides were eluted from the ESI column, which covered most of the HSA sequence. In the MS/MS analysis on the same sample, the 44 peptides were assigned to HSA, based on a database search, which corresponded to a 66% coverage of
the total sequence (401 among 609 total residues, Table 1). Thus, the DNLC-MS/MS system is useful for sensitive identification of proteins and for analyzing posttranslational modifications in proteins available in limited amounts. Idenification of E. coli Proteins in Crude Cell Lysate. To determine the feasibility of the DNLC-MS/MS system, we attempted to identify E. coli proteins by analysis of a small aliquot (1 µg) of peptide mixture generated by tryptic digestion of a total cell lysate. Figure 5 shows the number of identifications as a function of flow rate of this system. At 500 nL/min, the analysis identified only 30 E. coli proteins. The number of identifications, however, increased dramatically with decrease in a flow rate, and finally, at 50 nL/min we identified 136 proteins. It should be noted that the total analysis time was almost the same among these experiments as when the ReNCon device produces essentially the same gradient profile within programmed time duration, regardless of flow rate. Therefore, the observed increase in the number of proteins identified under a decreased flow rate would be because the peptide samples were introduced into MS at a higher concentration by lowering the flow rate of LC and thereby increased sensitivity of the MS/MS analysis. Retrieval of the data indicated that the identification of 136 proteins was based on 215 unique peptides assigned by analyses of 482 MS/MS spectra acquired in a data-dependent mode (∼1.6 peptide assignments/ protein on average). Thus, we anticipated that the DNLC-MS/ MS system would be comparable to the non-gel-based analysis of many biological complexes isolated from cells in terms of number of identifications (∼100 proteins) and sample consumption (submicrogram). Identification of Proteins in a Premature Mammalian Ribosome. To demonstrate the potential of this system in interaction proteomics, we isolated a preribosome complex from Analytical Chemistry, Vol. 74, No. 18, September 15, 2002
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Table 1. Results of DNLC-MS/MS Analysis of Tryptic Digests of S-Carbamoylmethyl HSA (100 fmol)a obsd mass
charge
Mr (calc)
∆
sequence
start-end
501.81 543.27 569.78 717.80 767.41 749.82 506.95 725.79 653.34 537.80 554.94 492.77 438.28 671.86 840.12 626.86 516.29 575.34 686.32 722.35 696.31 613.84 845.44 820.44 542.33 467.29 528.33 564.88 547.35 464.27 709.03 637.68 500.83 644.71 849.09 679.85 656.41 682.40 867.14 633.70 772.74 892.14 830.79 812.44
2 3 2 2 3 2 3 2 2 2 3 2 2 2 3 2 3 2 2 2 3 2 3 2 2 2 2 2 3 2 3 3 2 3 3 2 2 3 3 3 3 3 3 2
1001.55 1626.73 1137.52 1433.55 2299.10 1497.60 1517.77 1449.55 1304.64 1073.54 1661.74 983.48 874.50 1341.63 2517.23 1251.65 1545.81 1148.61 1370.58 1442.65 2085.87 1225.60 2533.23 1638.78 1082.59 932.52 1054.58 1127.69 1638.93 926.49 2124.02 1909.94 999.60 1931.04 2544.17 1357.62 1310.73 2044.09 2598.30 1897.99 2315.09 2673.33 2489.29 1622.78
0.05 0.05 0.03 0.02 0.10 0.03 0.06 0.02 0.03 0.05 0.05 0.05 0.05 0.07 0.09 0.06 0.04 0.06 0.04 0.04 0.04 0.06 0.08 0.08 0.06 0.04 0.06 0.05 0.09 0.04 0.05 0.07 0.05 0.06 0.09 0.06 0.06 0.09 0.10 0.09 0.09 0.07 0.05 0.08
(K) TPVSDRVTK (C) (K) ADDKETCFAEEGKK (L) (K) CCTESLVNR (R) (R) ETYGEMADCCAK (Q) (K) NYAEAKDVFLGMFLYEYAR (R) (K) TCVADESAENCDK (S) (K) LDELRDEGKASSAK (Q) (R) ETYGEMADCCAK (Q) (K) ECCEKPLLEK (S) (K) LDELRDEGK (A) (R) YKAAFTECCQAADK (A) (K) TYETTLEK (C) (R) LSQRFPK (A) (K) AVMDDFAAFVEK (C) (R) MPCAEDYLSVVLNQLCVLHEK (T) (R) FPKAEFAEVSK (L) (K) LKECCEKPLLEK (S) (K) LVNEVTEFAK (T) (K) AAFTECCQAADK (A) (K) YICENQDSISSK (L) (K) VHTECCHGDLLECADDR (A) (R) FKDLGEENFK (A) (R) MPCAEDYLSVVLNQLCVLHEK (T) (K) DVFLGMFLYEYAR (R) (K) YLYEIARR (H) (K) LCTVATLR (E) (K) KYLYEIAR (R) (K) KQTALVELVK (H) (K) KVPQVSTPTLVEVSR (N) (K) YLYEIAR (R) (K) AAFTECCQAADKAACLLPK (L) (R) RPCFSALEVDETYVPK (E) (K) QTALVELVK (H) (K) SLHTLFGDKLCTVATLR (E) (K) EFNAETFTFHADICTLSEKER (Q) (K) AVMDDFAAFVEK (C) (R) HPDYSVVLLLR (L) (K) VFDEFKPLVEEPQNLIK (Q) (K) QNCELFEQLGEYKFQNALLVR (Y) (R) RHPYFYAPELLFFAK (R) (K) NYAEAKDVFLGMFLYEYAR (R) (K) RMPCAEDYLSVVLNQLCVLHEK (T) (K) ALVLIAFAQYLQQCPFEDHVK (L) (K) DVFLGMFLYEYAR (R)
491-499 585-598 500-508 106-117 342-360 76-88 206-219 106-117 301-310 206-214 185-198 376-383 243-249 570-581 470-490 247-257 299-310 66-75 187-198 287-298 265-281 35-44 470-490 348-360 162-169 98-105 161-168 549-558 438-452 162-168 187-205 509-524 550-558 89-105 525-545 570-581 362-372 397-413 414-434 169-183 342-360 469-490 45-65 348-360
a The table incorporates the observed mass and charge of the peptide ion, together with the calculated mass of each ion and their difference (∆). The table also indicates the amino acid sequence assigned to each peptide and its position in the total HSA sequence.
Figure 5. Cumulative numbers of unique proteins identified in a digested E. coli total cell extract using DNLC-MS/MS performed under various flow rates of LC. Numbers below the columns are total numbers of unique proteins identified. An identical amount of digested proteins (1 µg) was used for all experiments.
cultured NCL-transfected NT293EBNA cells using a pull-down experiment and flag-tagged nucleolin as bait. It has been reported that NCL binds to the nucleolar precursor of the ribosomal 4730
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complex and participates in the regulation of ribosome biogenesis and maturation.19 The preribosome complex isolated as the NCL immunoprecipitate contained more than 70 protein bands visualized by silver-stained SDS-PAGE (Figure 6a). In a previous report,19 we identified 40 ribosomal proteins and 20 nonribosomal components, such as RNA helicase involved in rRNA processing, using conventional in-gel digestion followed by MS analysis. This complex (∼0.2 µg) was subjected to Achromobacter protease I digestion without gel separation, and the resulting peptide mixture was analyzed using the DNLC-MS/MS system (Figure 6b). With the automated data processing, we identified 64 ribosomal proteins and 28 nonribosomal components, the total number being 92. This was 32 more than had been identified using the gel-based MS analysis. Table 2 compares the ribosomal proteins identified in each experiment. The non-gel-based DNLC-MS/MS analysis identified 15 more ribosomal proteins of the large 60S subunit and 9 more of the small 40S subunit, respectively, and covered all the ribosomal proteins found in the case of the previous gel-
Figure 6. Identification of NCL-binding proteins. (a) SDS-PAGE (11% gel) profile of proteins coprecipitated with flag NCL (silver staining). (b) Total ion chromatograms for DNLC-MS/MS analysis. Four precursor peptide ions were selected for parallel data-dependent collision-induced dissociation. The unfractionated digest of NCL-binding proteins (0.2 µg) was directly analyzed automatically in a data-dependent mode. Elution: 60-min linear gradient from 0.1% formic acid to 70% acetonitrile in 0.1% formic acid at a flow rate of 50 nL/min.
based analysis. Many additional preribosomal components were identified using the LC-based technique, including small ribosomal proteins that might be missed during gel electrophoresis (Table 2). While the mature ribosome consists of 78 distinct proteins present in equimolar amounts, it is unclear if the NCL-binding nucleolar precursor particle is a complex of stoichiometric constituents.19 Therefore, some ribosomal proteins might be low in abundance in intermediate particles and may not be detectable using gel-based identification technology. We also note that the gel-based analysis is relatively time- and labor-intensive and requires multistep manual handling, whereas the LC-based technique described here is an automated 1-h process. Other approaches for direct nanoflow LC-MS/MS analysis reported to date include those utilizing the microfabricated device driven by an electroosmotic pump16 or by gas pressure17 or a device equipped with a loop to store preformed gradients.18 The DNLC reported herein is an excellent alternative to these methods and has the advantage that it can operate high-resolution LC columns regardless of column back pressure or pressure changes during the chromatography and which frequently occurs during the injection of crude biological samples in chaotrophic reagents
such as 8 M urea. Analysis made using the DNLC system can easily be automated for reproducible, high-throughput protein identification of protein complexes that are compatible to largescale protein interaction analysis.21,22 In addition, DNLC can deliver solvents at a much wider range of flow rate (1000-25 nL/min) than previous systems. This is advantageous in biological experiments because one can quickly apply to the system a relatively large volume (5-10 µL) of biological samples at a high flow rate (21) Gavin, A. C.; Bosche, M.; Krause, R.; Grandi, P.; Marzioch, M.; Bauer, A.; Schultz, J.; Rick, J. M.; Michon, A. M.; Cruciat, C. M.; Remor, M.; Hofert, C.; Schelder, M.; Brajenovic, M.; Ruffner, H.; Merino, A.; Klein, K.; Hudak, M.; Dickson, D.; Rudi, T.; Gnau, V.; Bauch, A.; Bastuck, S.; Huhse, B.; Leutwein, C.; Heurtier, M. A.; Copley, R. R.; Edelmann, A.; Querfurth, E.; Rybin, V.; Drewes, G.; Raida, M.; Bouwmeester, T.; Bork, P.; Seraphin, B.; Kuster, B.; Neubauer, G.; Superti-Furga, G. Nature 2002, 415, 141-147. (22) Ho, Y.; Gruhler, A.; Heilbut, A.; Bader, G. D.; Moore, L.; Adams, S. L.; Millar, A.; Taylor, P.; Bennett, K.; Boutilier, K.; Yang, L.; Wolting, C.; Donaldson, I.; Schandorff, S.; Shewnarane, J.; Vo, M.; Taggart, J.; Goudreault, M.; Muskat, B.; Alfarano, C.; Dewar, D.; Lin, Z.; Michalickova, K.; Willems, A. R.; Sassi, H.; Nielsen, P. A.; Rasmussen, K. J.; Andersen, J. R.; Johansen, L. E.; Hansen, L. H.; Jespersen, H.; Podtelejnikov, A.; Nielsen, E.; Crawford, J.; Poulsen, V.; Sorensen, B. D.; Matthiesen, J.; Hendrickson, R. C.; Gleeson, F.; Pawson, T.; Moran, M. F.; Durocher, D.; Mann, M.; Hogue, C. W.; Figeys, D.; Tyers, M. Nature 2002, 415, 180-183.
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Table 2. Comparison of Proteins Identified Using DNLC-MS/MS and Gel-Based Analysisa protein
accession (GI)
MW
pI
remarka
ribosomal protein L3 ribosomal protein L4 ribosomal protein L5 ribosomal protein L6 ribosomal protein L7 ribosomal protein L7a ribosomal protein L9 ribosomal protein L10 ribosomal protein L10a ribosomal protein L11 ribosomal protein L12 ribosomal protein L13 ribosomal protein L13a ribosomal protein L14 ribosomal protein L15 ribosomal protein L17 ribosomal protein L18 ribosomal protein L18a ribosomal protein L21 ribosomal protein L23a ribosomal protein L26 ribosomal protein L27 ribosomal protein L27a ribosomal protein L30 ribosomal protein L35 ribosomal protein L36 ribosomal protein L8 ribosomal protein L19 ribosomal protein L22 ribosomal protein L23 ribosomal protein L24 ribosomal protein L28 ribosomal protein L29 ribosomal protein L31 ribosomal protein L32 ribosomal protein L34 ribosomal protein L35a ribosomal protein L37a ribosomal protein L39 ribosomal protein, large, P0 ribosomal protein, large, P1 ribosomal protein, large, P2
Large Ribosomal Subunit Proteins 2 119 106 45 469.4 1 350 681 47 759.7 4 506 655 34 447.9 1 350 762 32 728.1 133 021 29 225.9 4 506 661 29 995.8 417 677 21 863.6 5 174 431 24 577.0 6 325 472 24 859.5 6 226 544 20 252.5 4 506 597 17 818.8 4 506 599 24 291.6 730 451 23 577.4 1 710 488 23 289.8 730 532 24 086.1 4 506 617 21 397.2 4 506 607 21 634.6 730 538 20 762.5 1 172 991 18 564.9 132 848 17 695.1 292 435 17 288.3 4 506 623 15 797.8 4 506 625 16 561.5 4 506 631 12 784.1 6 005 860 14 551.6 7 661 638 12 234.7 4 506 663 28 024.9 4 506 609 23 466.2 4 506 613 14 787.1 4 506 605 14 865.6 4 506 619 17 779.1 11 424 039 15 747.6 1 082 766 17 667.1 4 506 633 14 463.0 4 506 635 15 859.9 4 506 637 13 305.0 4 506 639 12 494.6 4 506 643 10 275.4 4 506 647 6 406.7 4 506 667 34 273.7 4 506 669 11 514.0 4 506 671 11 665.0
10.16 11.11 9.76 10.59 10.66 10.61 9.96 10.11 9.94 9.64 9.48 11.65 10.94 10.94 11.56 10.18 11.73 10.72 10.49 10.44 10.56 10.56 11.01 9.65 11.04 11.50 11.03 11.48 9.21 10.50 11.26 12.01 11.58 10.54 11.32 11.38 10.91 10.44 12.55 5.72 4.26 4.42
** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** * * * *# *# * *# *# * * * * * ** * *
ribosomal protein S2 ribosomal protein S3 ribosomal protein S3a ribosomal protein S4, X-linked ribosomal protein S6 ribosomal protein S7 ribosomal protein S9 ribosomal protein S13 ribosomal protein S14 ribosomal protein S15a ribosomal protein S16 ribosomal protein S18 ribosomal protein S19 ribosomal protein S5 ribosomal protein S8 ribosomal protein S11 ribosomal protein S15 ribosomal protein S23 ribosomal protein S24 ribosomal protein S25 ribosomal protein S26 ribosomal protein S30
Small Ribosomal Subunit Proteins 1 710 756 31 324.6 417 719 26 688.5 4 506 723 29 945.1 4 506 725 29 598.0 4 506 731 28 696.7 337 518 21 848.6 4 506 745 22 571.6 4 506 685 17 222.4 5 032 051 16 272.8 4 506 689 14 853.6 4 506 691 16 445.4 133 840 17 718.8 4 506 695 16 060.6 15 929 961 22 876.6 4 506 743 24 205.4 4 506 681 18 430.9 4 506 687 17 040.3 4 506 701 15 807.7 4 506 703 15 423.3 4 506 707 13 742.2 4 506 709 12 930.3 548 855 6 647.9
10.25 9.68 9.75 10.16 10.90 10.09 10.74 10.53 10.08 10.14 10.21 10.99 10.31 9.73 10.32 10.31 10.39 10.50 10.79 10.12 10.86 12.15
** ** ** ** ** ** ** ** ** ** ** ** ** * *# * *# *# * *# * *
a **, identified by both analyses; *, identified only by DNLC-MS/MS analyses. #, proteins identified by single peptide hits are assigned by #. Identified proteins shown here are the sums of three-repeated DNLC-MS/MS analytical runs.
(1000-500 nL/min) and then automatically bring down the flow rate for separation and analysis. Thus, DNLC has versatility and is most practical for use. 4732
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CONCLUSION Our new analytical platform for interaction proteomics, DNLCMS/MS, provides a fully integrated approach to non-gel-based
protein identification. High resolution of the system is achieved using a fritless ESI column packed with 1-µm RP beads combined with high-resolution mass spectrometry. Reproducibility is maintained with the ReNCon gradient device and automation of the total system. Because the system is most sensitive and requires only 0.2∼0.5 µg of total protein to identify ∼100 protein components, it could be used for high-throughput protein analysis of interaction proteomics, in particular, for mass identification of protein components in a biological complex such as functional cellular domains and a multi-protein cell signaling complex available in limited amounts. Thus, we propose that the DNLC-MS/MS system reported here is an excellent analytical platform for non-gel-based technologies of interaction proteomics.
ABBREVIATIONS LC, liquid chromatography; MS, mass spectrometry; DNLC, direct nano flow liquid chromatography; ESI, electrospray ionization; RP, reversed phase; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; ReNCon, revolving nanoconnection; NCL, nucleolin. ACKNOWLEDGMENT We thank Dr. F. W. Putnam for valuable comments on the manuscript, K. Nishimura for expert technical contributions, and M. Ohara for critical reading. This work was supported by MEXT and partly by NEDO. Received for review January 8, 2002. Accepted June 14, 2002. AC020018N
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