Anal. Chem. 2006, 78, 5109-5118
Automated Ultra-High-Pressure Multidimensional Protein Identification Technology (UHP-MudPIT) for Improved Peptide Identification of Proteomic Samples Akira Motoyama, John D. Venable, Cristian I. Ruse, and John R. Yates, III*
Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037
Multidimensional separation is one of the most successful approaches for proteomics studies that deal with complex samples. We have developed an automated ultra-highpressure multidimensional liquid chromatography system that operates up to ∼20 kpsi to improve separations and increase protein coverage from limited amount of samples. The reversed-phase gradient is operated in the constantflow mode opposed to the constant-pressure mode, which is typical of previous ultra-high-pressure systems. In contrast to constant-pressure systems, the gradient shape is fully controllable and can be optimized for the type of samples to be run. The system also features fast sample loading/desalting using a vented column approach to improve sample throughput. This approach was validated on a soluble fraction from yeast lysate where we achieved ∼30% more protein identifications using a 60-cm-long triphasic capillary column than with our traditional approach. Advantages of the use of a relatively long reversedphase column (∼50 cm) for MudPIT-type experiments are also discussed. Analytical demands in the proteomics field have been continuously increasing over recent years. Especially for so-called “shotgun” approaches, where large numbers of peptides produced by protease digestion are analyzed to identify thousands of proteins, improved separation is key for further improvement. One of the most widely accepted methods for improving separation power is achieved by adding one or more orthogonal separation dimension(s).1 Our group has previously developed an online twodimensional peptide separation strategy known as multidimensional protein identification technology (MudPIT) by coupling strong cation-exchange (SCX) and reversed-phase (RP) separations followed by tandem mass spectrometric analysis for enzymatically digested peptide mixtures.2,3 It has been demonstrated that MudPIT can identify thousands of proteins by an automated single experiment,4 which typically consists of ∼12 salt steps and * To whom correspondence should be addressed. E-mail: jyates@ scripps.edu. Fax: +1-858-784-8883. Telephone: +1-858-784-8862. (1) Issaq, H. J.; Chan, K. C.; Janini, G. M.; Conrads, T. P.; Veenstra, T. D. J. Chromatogr., B: Anal. Technol. Biomed. Life Sci. 2005, 817, 35-47. (2) Wolters, D. A.; Washburn, M. P.; Yates, J. R., 3rd. Anal. Chem. 2001, 73, 5683-5690. (3) Link, A. J.; Eng, J.; Schieltz, D. M.; Carmack, E.; Mize, G. J.; Morris, D. R.; Garvik, B. M.; Yates, J. R., 3rd. Nat. Biotechnol. 1999, 17, 676-682. 10.1021/ac060354u CCC: $33.50 Published on Web 05/26/2006
© 2006 American Chemical Society
completes within 24 h. However, the human proteome, for example, is known to consist of more than 40 000 proteins with a difference of 10 orders of magnitude in expression levels.5 Depletion of abundant proteins and prefractionation methodologies have been shown to be effective for identification of some important low-abundant species.6,7 However, because of the need for dynamic range and sensitivity in these analyses, it is crucial to improve separation performance. Most of the current LC-based proteomics techniques rely on RP-LC for the final peptide separation. This is primarily due to its high separation power compared to other separation modes including SCX and also due to good compatibility of mobile phases with mass spectrometric analysis. The eluent is usually directly electrosprayed without flow splitting to maximize sensitivity. Because electrospray ionization demonstrates a concentrationdependent response8 and the ionization efficiency increases with decreasing flow rate, capillary columns having inner diameters of 10 kpsi is referred to as ultrahigh pressure in this report. Despite the fact that the advantages of ultra-high-pressure RP separations have been well documented by Jorgenson and other pioneering works,9-11 only a few research groups have been able to implement such formats thus far. The lack of commercially available instrumentation and technical difficulties inherent to the handling of capillary separation formats are likely to blame. Probably for the same reasons, an online multidimensional separation at ultrahigh pressure (>10 kpsi) has not yet been reported, whereas Smith’s group reported an offline approach resulting in improved performance compared to a single-phase counterpart.15 In this article, we describe a novel analytical format for fully automated, ultra-high-pressure, multidimensional online LC analysis for proteomic samples. Using an increased operating pressure to drive a relatively long column (∼60 cm) packed with small packing materials (3 µm), we have increased proteome coverage compared to a conventional pressure system. The UHP system was constructed with all commercially available components except for an in-house LabVIEW program used to control the actions of high-pressure valves and pumps. This system allows rapid online sample loading/desalting, as well as the generation of flexible gradients for ultra-high-pressure analysis. This approach was validated on a soluble fraction from a whole-cell yeast lysate and compared to a conventional pressure MudPIT system. EXPERIMENTAL PROCEDURES Materials. Urea, Tris(2-carboxyethyl)phosphine (TCEP), iodoacetoamide (IAM), Tris-HCl, ammonium acetate, formic acid, and dithiothreitol were obtained from Sigma-Aldrich (St. Louis, MO). HPLC grade acetonitrile was purchased from Fisher Scientific (Fair Lawn, NJ). Purified water was generated by a (10) Lippert, J. A.; Xin, B.; Wu, N.; Lee, M. L. J. Microcolumn Sep. 1997, 11, 631-643. (11) Shen, Y.; Zhao, R.; Belov, M. E.; Conrads, T. P.; Anderson, G. A.; Tang, K.; Pasa-Tolic, L.; Veenstra, T. D.; Lipton, M. S.; Udseth, H. R.; Smith, R. D. Anal. Chem. 2001, 73, 1766-1775. (12) Shen, Y.; Zhao, R.; Berger, S. J.; Anderson, G. A.; Rodriguez, N.; Smith, R. D. Anal. Chem. 2002, 74, 4235-4249. (13) Shen, Y.; Zhang, R.; Moore, R. J.; Kim, J.; Metz, T. O.; Hixson, K. K.; Zhao, R.; Livesay, E. A.; Udseth, H. R.; Smith, R. D. Anal. Chem. 2005, 77, 30903100. (14) Shen, Y.; Moore, R. J.; Zhao, R.; Blonder, J.; Auberry, D. L.; Masselon, C.; Pasa-Tolic, L.; Hixson, K. K.; Auberry, K. J.; Smith, R. D. Anal. Chem. 2003, 75, 3596-3605. (15) Shen, Y.; Jacobs, J. M.; Camp, D. G., 2nd; Fang, R.; Moore, R. J.; Smith, R. D.; Xiao, W.; Davis, R. W.; Tompkins, R. G. Anal. Chem. 2004, 76, 11341144.
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NANOpure water purifier system (Barnstead International, Dubuque, IO). Sequence-grade modified trypsin was purchased from Promega Corp. (Madison, WI). Endoproteinase Lys-C was obtained from Roche Diagnostics (Indianapolis, IN). A proteasedeficient Saccharomyces cerevisiae strain BJ546016 was purchased from American Type Culture Collection (Manassas, VA). Growth and Lysis of S. cerevisiae. Strain BJ5460 was grown to mid log phase (OD 0.6) in YPD, and cells collected by centrifugation were lysed as described previously.4 The lysed cells were separated in three fractions4 (soluble and lightly and heavily washed), and the soluble fraction was used in this study. Digestion of Soluble Fraction. The soluble fraction of cells was digested by a method slightly modified from the one described previously.4 Urea was added to the soluble fraction of cell lysate to denature proteins. The proteins were then reduced with TCEP, alkylated using IAM, and subsequently digested with endoproteinase Lys-C and trypsin. The digestion reaction was stopped by adding formic acid at the final concentration of 1%. The peptide mixture was aliquoted and stored at -80 °C prior to use. Preparation of Packed Capillary Columns. Fused-silica capillary analytical columns (50-µm i.d.) were prepared by slurry packing using a high-pressure syringe pump (Teledyne Isco, Lincoln, NE). PEEK tubing of 0.40-mm i.d. was cut to 1 in. (2.54 cm) and used as a sleeve to plumb capillaries to conventional 1/16in. HPLC fittings. Bulk C18 resins were first dispersed in acetone with an aid of vortex mixing. Then, the suspension was placed in a sonication bath for ∼1 min. The slurry was immediately transferred to the chamber of a gradient mixer (Jasco Inc., Easton, MD) and kept agitated by stirring in the chamber during packing. Capillaries were previously pulled by a laser puller (Sutter Instrument Co., Novato, CA) to form a fritless capillary of 2-3µm openings.17 The tip end was gently and carefully trimmed with a ceramic capillary cutter right after the tip was packed with resins. This process consistently gave ∼5-µm tip openings while still keeping the 3-µm packing materials inside the capillary. Analytical capillary columns were packed to ∼60 cm, conditioned with buffer A (described below in the Buffer System section) at ∼16 kpsi over 1 h, and then cut to length. For the packing of the biphasic C18/ SCX trapping column, an in-house-made packing bomb driven by high-purity He gas was used. The biphasic column was conditioned by running buffers with the following orders, B, A, C, and A, each for more than 10 column volumes prior to use (buffer compositions are described below in the Buffer System section). Automated Ultra-High-Pressure Multidimensional Protein Identification Technology. System Components. The system consists of two 20 kpsi syringe pumps for the UHP reversed-phase gradient LC, a standard HPLC quaternary pump for salt pulse generation, and four 20 kpsi valves to achieve automated MudPIT analysis (Figure 1). The 20 kpsi pumps (model 65D) were purchased from Teledyne Isco. Four 20 kpsi passivefeedback valves (model C2XU, V1-V4 in Figure 1) were obtained from Valco Instrument Co., Inc. (Houston, TX). Inline check valves (P/N 250-2000UHP) were purchased from Analytical Scientific Instruments, Inc. (El Sobrante, CA). A stainless steel column packed with silica-based C8 materials (1 mm i.d. × 50 mm) was (16) Jones, E. W. Methods Enzymol. 1991, 194, 428-453. (17) Gatlin, C. L.; Kleemann, G. R.; Hays, L. G.; Link, A. J.; Yates, J. R., 3rd. Anal. Biochem. 1998, 263, 93-101.
Figure 1. Schematic of the automated UHP-MudPIT system. Solid line connections were made by 1/16-in. stainless steel tubes. The dashed line connections were of 100-µm-i.d. fused-silica capillaries. V0 was a standard six-port valve equipped with the mass spectrometer and used for the introduction of sample or salt buffer solutions to the sample loop on V1. V1-V4 were 20 kpsi passive feedback valves from Valco Instrument. V1 was for the injection of sample/salt buffer solution to the triphasic capillary column array. V2 served as a vent/split position valve to allow rapid on-line sample loading/desalting. V3 and V4 were used to refill the 20 kpsi syringe pumps between MudPIT salt steps. The V0 status and the composition of salt buffer were controlled by Xcalibur software from Thermo Electron. V1-V4 are controlled by an in-house program run on LabVIEW.
used as a static mixer. Fused-silica capillaries (0.36-mm o.d.) are from Polymicro Technologies, LLC (Phoenix, AZ). Stainless steel tees, unions, and tubes are from Valco Instrument Co., Inc. PEEK tubing of 0.40-mm i.d. for sleeves (P/N 1565) was purchased from Upchurch Scientific Inc. (Oak Harbor, WA). A photodiode array (PDA) detector used to monitor the gradient profile was from Thermo Electron Corp. (San Jose, CA). An in-house program written in LabVIEW (v7.0, National Instrument Corp., Austin, TX) was developed to control the two 20 kpsi pumps and four 20 kpsi valves. Buffer System. The buffer solutions used were as follows: water/acetonitrile/formic acid (95:5:0.1, v/v/v) as buffer A, water/ acetonitrile/formic acid (20:80:0.1, v/v/v) as buffer B, and buffer A including 500 mM ammonium acetate as buffer C. Digested proteins were analyzed using a modified 4- or 15-step MudPITtype separation as described previously.4 Gradient Conditions. For the comparison experiments of 4and 15-step MudPIT, two gradient profiles having different slopes were used, which made the total run time nearly identical (∼24 h, see Table 2). The gradient profile of step 1, which was common for both 4- and 15-step MudPIT, was as follows: a 7-min gradient from 0 to 5% buffer B, a 3-min gradient from 5 to 15% buffer B, a 60-min gradient from 15 to 45% buffer B, a 10-min gradient from 45 to 75% buffer B, and 10 min of 75% buffer B (70-min gradient). For a 15-step MudPIT, a 2-min salt buffer step was programmed to be conducted during the first shallow gradient (a 7-min gradient from 0 to 5% buffer B). After this step, the same gradient was repeated for the 15-step MudPIT. The percentages of buffer C for the 15-step MudPIT were 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, and 100%, 95 + 5% buffer B, and 90 + 10% buffer B,
respectively, for the 15-step analysis (the ammonium acetate concentration was 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 375, and 500 mM, 475 mM + 5% buffer B, and 450 mM + 10% buffer B, respectively). For the 4-step UHP-MudPIT, the percentages of buffer C were 10%, 25%, and 90% + 10% buffer B (50 mM, 125 mM, and 450 mM + 10% buffer B as ammonium acetate concentration). The gradient profile of steps 2-4 for the 4-step UHP-MudPIT run was as follows; a 7-min gradient from 0 to 5% buffer B, a 3-min gradient from 5 to 15% buffer B, a 300-min gradient from 15 to 45% buffer B, a 50-min gradient from 45 to 75% buffer B, and 10 min of 75% buffer B (350-min gradient). Automated MudPIT Operations. Detailed operation events are summarized in Table 1. After the introduction of a sample solution to the loop on V1 (through V0, Figure 1), 4- or 15-step UHP-MudPIT analysis was conducted in an unattended manner. Briefly, the sample solution loaded into the loop was transferred to a trapping/desalting biphasic capillary column by the position change of V1 (A to B). The biphasic trapping/desalting column was composed of 5 cm of 5-µm Aqua C18 (Phenomenex, Torrance, CA) and 5 cm of 5-µm Partisphere SCX (Whatman, Clifton, NJ) resins packed in a 100-µm-i.d fused-silica capillary (C18 upstream). The 2-µm mesh stainless steel screens (P/N 2SR1-10, Valco) were placed at both ends of the biphasic column (inside the stainless tees, Figure 1). After the sample loading to the trapping/desalting column, the column was washed with buffer A at 5-10 kpsi constant pressure (typical flow rate, 7-15 µL/min). More than five sample volumes of buffer A were passed through to wash out nonvolatile buffers included in a sample. After desalting, V2 (Figure 1, Valco) was flipped to the position B and the 20-kpsi Analytical Chemistry, Vol. 78, No. 14, July 15, 2006
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Table 1. Timed Events of Automated UHP-MudPITa First Step (No Salt Pulse Gradient) valve status time (min) 0 13b 15b
flow rate (µL/min)
event
V0c
V1d
V2d
V3d
V4d
P1d
P2d
P3c
load sample to the loop sample injection and desalting prepressurize P1, P2 and equilibration start gradient
B (sample) B (sample) B (sample)
A (load) B (injection) A (load)
A (vent) A (vent) B (Split)
A (close) A (close) A (close)
A (close) A (close) A (close)
(7-15)e (7-15)e (∼125)f
0 0 (∼0)f
0 0 0
B (sample)
A (load)
B (split)
A (close)
A (close)
125g
0g
0
d
d
Salt Steps valve status time (min)
0 2 4 10
c
event
V0
refilling pumps and salt soln to the loop prepressurize P1, P2 and equilibration salt soln injection equilibration and salt wash start gradienth
A (salt soln) A (salt soln) A (salt soln) A (salt soln) A (salt soln)
d
d
V1
flow rate (µL/min) P1d
P2d
P3c
V2
V3
V4
A (load)
B (split)
B (open)
B (open)
A (load)
B (split)
A (close)
A (close)
(∼125)f
(∼0)f
2
A (close) A (close) A (close)
125g
0g
125g 125g
0g 0g
0 0 0
B (injection) A (load) A (load)
B (split) B (split) B (split)
A (close) A (close) A (close)
200
a V , regular 6-port valve equipped with a mass spectrometer; V , V , V , and V , 20 kpsi passive-feedback Valco valves; P and P , 20 kpsi 0 1 2 3 4 1 2 syringe pumps from Teledyne Isco; P3, Agilent 1100 quarternary pump. b Adjusted depending on sample volume. c Controlled by the Xcalibur d e f software from Thermo Electron. Controlled by an in-house LabVIEW program. Operated at a constant-pressure mode (5∼10 kpsi). Operated at a constant-pressure mode (∼15 kpsi). g Operated at a constant-flow mode. h Shallow slope gradient was started just before the salt introduction. See text for details.
syringe pumps (Teledyne Isco) were prepressurized to ∼15 kpsi. The prepressurization procedure was programmed to first pressurize P1 (aqueous buffer) and then P2 (organic). Flow from P2 was blocked by the check valve while the same pressures were maintained at P1, P2, and the line downstream of the check valves (Figure 1 and Table 1). The prepressurization pressure was previously determined by running a short blank gradient with the flow rate set (0% B f 50% B f 0% B for 10 min). The system pressure at the end of the survey gradient was considered at equilibrium, and the prepressurization pressure was set to ∼200 psi above equilibrium. The addition of extra pressure was found necessary to achieve reproducible gradients due to a slight (∼100 psi) but rapid pressure drop caused by the change of the operation mode of the pumps (from constant pressure to constant flow). The magnitude of additional pressure was experimentally determined by observing the starting shape of gradients. Too much additional pressure resulted in a delay of gradient and too small caused a surge of buffer B. In the final optimized procedure, the constant-flow gradient was started 2 min after both pumps were equilibrated. During this 2-min period (operated in the constantpressure mode), the flow from P1 dominated and that of P2 was negligible. The flow from P2 was initiated after the pressure of P2 exceeded that of the downstream check valve. The gradient delay related to the system volume was found to be insignificant since the estimated volume between the check valve and the middle tee was relatively small (∼50 µL) compared to the pump flow rate (125 or 250 µL/min). For the MudPIT salt steps, V1 was flipped 2 min after the prepressurization and equilibration to introduce salt buffer solution to the binary gradient liquid stream (to elute peptides from the SCX phase). The salt buffer solutions were prepared by an Agilent 1100 quaternary HPLC pump to the set compositions (25-500 mM (12 steps), 475 mM + 5% buffer B, and 450 mM + 10% buffer 5112 Analytical Chemistry, Vol. 78, No. 14, July 15, 2006
B) and delivered to the sample loop during refilling of the 20 kpsi pumps. The main part of the ultra-high-pressure gradient was programmed to start after 8 min of salt introduction leaving time for the salt to be washed through the column. The analytical column was a 50-µm-i.d capillary packed with 50 cm, 3-µm Aqua C18 particles (Phenomenex, Ventura, CA). The flow rate of the column was measured by carefully connecting a measured glass pipet to the tip under a magnifier for observation. For the “triphasic” column setup (C18/SCX+C18) described as above, ∼15 kpsi system pressure consistently produced a column-flow rate of ∼0.16 µL/min (with 100% buffer A). The typical pump flow rate used was 125 and 250 µL/min, and ∼15 kpsi of system pressure was maintained by adjusting the resistance of the splitting capillary. To maintain a constant split ratio throughout the gradient, a stainless steel column packed with silica-based C18 material identical to that used in the analytical column was connected between V2 and the split capillary (Figure 1). Mass Spectrometric Conditions. As peptides eluted from the capillary column, they were electrosprayed directly into an LCQ-Deca 3D ion trap or an LTQ linear ion trap mass spectrometer (Thermo Electron, Palo Alto, CA). A distal 2.4-kV spray voltage was applied to the tee in the back (Figure 1) with the combination of a fritless integrated electrospray tip column.17 A cycle of one full-scan mass spectrum (400-1400 m/z for LCQDeca, 400-1600 for LTQ) followed by three data-dependent MS/ MS spectra at a 35% normalized collision energy was repeated continuously throughout each step of the multidimensional separation. Application of mass spectrometer scan functions and salt solution preparation with an Agilent quaternary pump were controlled by the Xcalibur data system (Thermo Electron).
Data Analysis. Collected MS/MS spectra were processed using RawExtract18 and then filtered with the PARC algorithm.19 The filtered MS/MS spectra were searched (without enzyme specificity) against a yeast database that includes both regular and reversed protein sequences to estimate a false positive rate.20 The SEQUEST search algorithm was used to interpret MS/MS spectra as described previously.2,4,21,22 SEQUEST results were then filtered with DTASelect v2.0 using XCorr and ∆Cn values determined by a 5% false positive rate.23 Peptides were accepted regardless of their tryptic nature. The minimum number of peptides to identify proteins was set at two. Additional database search criteria are as follows; mass tolerance of (3 mass units for the precursor peptide was used in the SEQUEST database search, an average mass was used for the predicted mass in the search, and monoisotopic masses were used for the predicted fragment ions. Cysteine residues were considered to have a static modification of +57 mass units. Oxidized methionine was searched as a dynamic modification of +16 mass units. Safety Considerations. The operators should wear protective glasses and gloves for all the ultra-high-pressure operations including capillary column packing. The electric grounding must be secured since leakage of mobile phase can cause a short of the high voltage used for electrospray generation. RESULTS AND DISCUSSION Design and Performance of Automated UHP-MudPIT System. Constant-Flow Flexible Gradient UHP System. The objective of this study was to develop an automated UHP online multidimensional LC/MS/MS system to improve proteome coverage from limited amount of samples. Increased operating pressure was a prerequisite to drive a long column packed with small packing materials, which gives better chromatographic resolution for complex peptide mixtures. As shown in Figure 1, our system was relatively simple compared to the similar 20 kpsi system recently reported by Shen et al.13 In our system, the UHP binary gradient was simply achieved by a pressure balance between two inline check valves. The mobile phases combined at the tee were then mixed in the static mixer of ∼30-µL inner volume. V0 was the standard six-port valve on the mass spectrometer and served as a flow selector to load sample to the system or to supply salt buffer solution for MudPIT salt steps. V1 and the attached sample loop were used to introduce sample or salt buffer solution into the ultra-high-pressure liquid stream. V2 worked as a vent valve for quick sample loading and buffer exchange at the reversedphase trapping column placed upstream of the SCX resin. V3 and V4 were used to refill pumps between MudPIT steps. The system was tested to withstand at least 16 kpsi after plumbing and operated at ∼15 kpsi under the constant-flow mode. The typical pump flow rate used was 125 and 250 µL/min and was split to (18) McDonald, W. H.; Tabb, D. L.; Sadygov, R. G.; MacCoss, M. J.; Venable, J.; Graumann, J.; Johnson, J. R.; Cociorva, D.; Yates, J. R., 3rd. Rapid Commun. Mass Spectrom. 2004, 18, 2162-2168. (19) Bern, M.; Goldberg, D.; McDonald, W. H.; Yates, J. R., 3rd. Bioinformatics 2004, 20 (Suppl 1), I49-I54. (20) Peng, J.; Elias, J. E.; Thoreen, C. C.; Licklider, L. J.; Gygi, S. P. J. Proteome Res. 2003, 2, 43-50. (21) Eng, J. K.; McCormack, A. L.; Yates, J. R., 3rd. J. Am. Soc. Mass Spectrom. 1994, 5, 976-989. (22) Yates, J. R., 3rd; Morgan, S. F.; Gatlin, C. L.; Griffin, P. R.; Eng, J. K. Anal. Chem. 1998, 70, 3557-3565. (23) Cociorva, D.; Yates, J. R., 3rd (manuscript in preparation).
Figure 2. Gradient profiles of the constant-flow automated UHPMudPIT system. (A) A stair-shape gradient profile obtained by changing the ratio of buffers A and B stepwise (10% steps from 100% A to 100% B and then back to 100% A, 2-min holds for each step). A PDA detector was connected to the split line and used to monitor UV absorption at 265 nm for acetone. Buffer A is purified water, and buffer B is purified water containing 0.1% acetone. (B) Linear gradient profiles of various slopes ranging from 0.06 to 3.00% buffer B/min. In this case, buffer A is purified water/acetonitrile/formic acid (95:5: 0.1), and buffer B is purified water/acetonitrile/formic acid/acetone (20:80:0.1:0.1). The system was operated at 250 µL/min constant flow (system pressure ∼10 kpsi).
produce ∼0.16 µL/min liquid flow in the column. Importantly, the system was composed of all commercially available components except for a control program running on LabVIEW (National Instrument). The program is capable of controlling most basic pump functions such as binary gradient generation, prepressurization, refilling, etc. We added several necessary functions such as valve control and an external start using a contact closure to make the system entirely automated. Gradient Generation. To date, most published UHP methods employed a constant-pressure mode for gradient generation, which resulted in gradients with exponential profiles (Supporting Information, Figure S-1).9-13,15,24 Since most peptides elute at ∼1530% of organic modifier, they are resolved in the steepest region of the gradient. On the contrary, hydrophobic peptides (which account for a relatively small fraction of the total number of peptides typically identified) are separated in the shallowest region of the gradient at the expense of time. Although there is some room for optimization, depending on the objectives, compromise is often required to best balance resolution and run time. In addition, the down time between gradients can be significant in the constant-pressure format, due to the fact that remaining Analytical Chemistry, Vol. 78, No. 14, July 15, 2006
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Figure 3. Typical base peak chromatograms of automated 15-step UHP-MudPIT. A 1-µg sample of yeast Lys-C + tryptic digest was injected in the system. A triphasic column composed of 5-cm C18 trap (5 µm)/5-cm SCX/50-cm C18 analytical (3 µm) was operated at 125 µL/min constant flow (system pressure, ∼15 kpsi; column flow rate, ∼0.16 µL/min). The gradient profile was as follows: a 10-min gradient from 0 to 5% buffer B, a 5-min gradient from 5 to 15% buffer B, a 90-min gradient from 15 to 45% buffer B, a 20-min gradient from 45 to 75% buffer B, and 10 min of 75% buffer B (120-min gradient). For the second to 15th salt steps, 2 min of salt buffer introduction was conducted during the first shallow gradient (0-7 min). The salt (ammonium acetate) concentration of each salt step is provided in the figure.
organic modifier needs to be completely purged from the gradient chamber after every run. As a result, the constant-pressure format 5114
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cannot achieve rapid cycle times and is, therefore, not ideal for multidimensional type approaches.
Figure 4. High-resolution separation demonstrated by the UHP-MudPIT system. (A) A base peak chromatogram of tryptic peptides from yeast lysate separated by a 60-cm triphasic column with a 350-min gradient. (B) A mass chromatogram of 6 typical peptides used to estimate a peak capacity. (C) A gradient profile monitored by UV 265 nm for acetone in buffer B. (D) Representative MS/MS spectra and their assignments. The triphasic column had the same dimensions as those of Figure 3. The pump flow rate was 125 µL/min, resulting in the operating pressure of ∼15 kpsi. A 10-µg sample of yeast Lys-C + tryptic digest was injected in the system. Peptides were eluted by a two-step UHP-MudPIT (i.e., the chromatogram shown in (A) is of the second step eluted with 500 mM ammonium acetate (see Results and Discussion for detail)). In (B), six mid-intensity peaks distributed nearly evenly across the chromatogram were picked. wb stands for a peak width at the baseline (in minutes).
Clearly, a constant-flow approach is more flexible and versatile than its constant-pressure counterpart as evidenced by the fact that the constant-flow mode is dominant in the majority of conventional RP-LC gradient applications. By definition, the gradient generated in a constant-flow mode is not limited to an exponential profile and can be adjusted and optimized as desired to suit the application. This feature is also extremely important in fields such as metabolomics, where high-resolution separations of complex mixtures of more diverse hydrophobicity are required.
To address limitations of constant-pressure operation in UHP separation systems, we developed a system that is capable of automated UHP online multidimensional capillary LC/MS/MS with full control over the shape of the gradient. Figure 2 demonstrates the precise gradient control realized by the system. For this experiment, acetone was added to buffer B at the concentration of 0.1% to monitor the shape of the gradient. A PDA detector was connected to the outlet of a restriction capillary for the monitoring of UV absorption at 265 nm. A stair-shaped step Analytical Chemistry, Vol. 78, No. 14, July 15, 2006
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Table 2. Number of Proteins Identified by UHP-MudPIT. Comparison with “Standard” MudPIT RP column lengtha (cm)
operating pressure (kpsi)
no. of MudPIT steps
gradient length (min)
gradient vol (gradient length/ column length)
UHP
50
∼15
standard
10
∼3
4b 15c 15c 4b
350d 70 70 70
7 1.4 7 7
MudPIT type
total run time (h)
no. of proteins identifiedg
fold increaseh of the no. of proteins identified
24.3e 27.5f 27.5f 7.3f
1090 742 830 397
1.31 0.89 1 0.48
a Length of an analytical C18 column used for the final separation. A 10-cm biphasic column (RP + SCX, 5 cm each) was connected upstream. Ammonium acetate concentrations for the 4 steps; 0, 50, 150, 450 mM + 10% buffer B. c Ammonium acetate concentrations for the 15 steps; 0, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 375, 500, 475 mM + 5% buffer B, 450 mM + 10% buffer B. d 70-min gradient for the first step. e 45 min for pump refilling between steps. f 20 min for pump refilling between steps. g False positive rate 5%, minimum 2 peptides/protein, subset of others removed. h Compared to standard 15-step MudPIT.
b
gradient profile in Figure 2A clearly shows an accurate control of the gradient, good mixing of the buffers, and reasonable delay of the system. We confirmed that once both inline check valves reached equilibrium after pressurization of both pumps, the flow from both pumps were precisely controlled regardless of the direction of the gradient (upward or downward). We also demonstrated the ability to generate accurate shallow slope gradients that are typically used for peptide separations (Figure 2B). The gradient slopes shown are proportional in the range between 0.5 and 0.06% buffer B/min (0.4-0.048% acetonitrile/ min). Thus, we conclude that flexible and reproducible ultra-highpressure constant-flow gradients that are suitable for proteomic separations can be generated using our system. Vented Column Configuration. To maximize sample throughput, we incorporated a “vented column” feature to our system. The vented column setup was originally proposed by Licklider et al.,25 and it is ideal for automated analysis by enabling quick sample loading to a narrow capillary column and online desalting of samples.25,26 It was challenging, however, to implement it in an ultra-high-pressure system simply because of the increased number of capillary connections. We tested several ways of plumbing methods to connect capillaries to conventional 1/16-in. ports, and concluded that the method reported by Shen et al.13 worked best for our setup. Automated 15-Salt Step, Ultra-High-Pressure Separations. To evaluate the performance of the UHP-MudPIT system, we analyzed a 1-µg sample of the soluble fraction of a yeast wholecell lysate using a 15-salt step procedure (Figure 3). The system pressure was ∼15 kpsi to drive the mobile phases at near the optimum linear velocity for the analytical column described in the Experimental Section (column flow rate, ∼0.16 µL/min (0.13 cm/ s); pump flow rate, 125 µL/min). It is clear from these chromatograms that yeast peptides were nicely distributed in 15 SCX fractions by the salt steps employed. Retention time was also found to be reproducible by investigating several peptides that eluted across adjacent salt steps. Retention time variations for the peptides were ranging from 0.1 to 0.8 min. The only manual operation required for this 15-step analysis was sample loading
to the loop and starting the mass spectrometer that triggered all other components. Separation Efficiency (Peak Capacity) of UHP-MudPIT System. Peak capacity is a good index to estimate the separation efficiency of multiple components.27 To demonstrate the peak capacity obtained with the UHP-MudPIT system, a two-step MudPIT was conducted that consisted of a short RP gradient for desalting followed by a salt introduction of 500 mM ammonium acetate and subsequent separation of eluted peptides with a shallow 350-min RP gradient (Figure 4). A UV-monitored gradient profile and typical tandem mass spectra were also presented to show the validity of the system. A triphasic column of the same dimension as that used for Figure 3 was used for this experiment (10-cm C18 (5 µm)/SCX (5 µm) + 50-cm C18 (3 µm)). As shown in previous work, the gradient length is an important parameter when comparing different chromatography approaches.28 To maintain consistency, we used a 350-min gradient for a 50-cmlong column compared to our typical low-pressure MudPIT system that uses a 70-min gradient and 10-cm column. In other words, the gradient slope for a long column was set to 1/5 of that used for a 10-cm column, and the gradient volume was constant. For the estimation of peak capacity, the following equation29,30 was used in this study:
(24) MacNair, J. E.; Patel, K. D.; Jorgenson, J. W. Anal. Chem. 1999, 71, 700708. (25) Licklider, L. J.; Thoreen, C. C.; Peng, J.; Gygi, S. P. Anal. Chem. 2002, 74, 3076-3083. (26) Yi, E. C.; Lee, H.; Aebersold, R.; Goodlett, D. R. Rapid Commun. Mass Spectrom. 2003, 17, 2093-2098.
(27) Giddings, J. C. United Separation Science; John Wiley & Sons Inc.: New York, 1991. (28) Gilar, M.; Daly, A. E.; Kele, M.; Neue, U. D.; Gebler, J. C. J. Chromatogr., A 2004, 1061, 183-192. (29) Wren, S. A. J. Pharm. Biomed. Anal. 2005, 38, 337-343. (30) Neue, U. D. J, Chromatogr., A 2005, 1079, 153-161.
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n ) 1 + tg/wb where n is the peak capacity, tg the gradient time, and wb the peak width at the baseline. Taking a peak width at half-height instead of at the baseline is generally accepted because of a known issue for the estimation of peak capacity from complex chromatograms. wb values in this study were therefore estimated by doubling a peak width at half-height. The other concern is how to pick peak widths from millions of candidate peaks. The peak width greatly depends on the nature of analyte and can vary across the chromatogram. To have a conservative and practical estimation of peak capacity,30 we picked six mid-intensity typical peaks distributed evenly across the chromatogram (Figure 4B). The estimated peak capacity was ∼400, which is comparable to the
value reported by Jorgenson et al. with their 130 kpsi system using 1.0-µm nonporous particles.24 Shen et al., who employed a system similar to ours, have reported peak capacities of more than 1000 by extending column length and greatly extending gradient time.12,13 It should be mentioned that, the peak capacity obtained using a constant-pressure gradient can be overestimated due to its exponential profile, as is obvious from Figure S-1 (Supporting Information). In the chromatogram shown in Figure 4A, peptides are well distributed across the chromatogram and the gradient time frame was fully and evenly used for the separation. Unfortunately, it is difficult or even impossible to directly compare peak capacities determined in different studies since there is no known consensus to report peak capacities. The use of a calculation algorithm31 seems ideal for a head-to-head comparison but may not be suitable for mass spectrometry data that tend to be noisier than that of UV. The shape of the gradient in the above experiment, which was measured from the split eluent, was reasonably smooth even for such a long run and a shallow slope (Figure 4C). Note that the slight distortion apparent in the steeper gradient regions (340400 min) was due to the change of the UV spectrum, which is solvent composition dependent, and not due to nonlinear delivery of the binary solvents. In addition, one of the notable benefits of our triphasic vented-column format is that peptides released from the SCX phase at each MudPIT salt step are refocused onto the top of the C18 analytical column for the final separation. As a result, the inner dead volume of the vent tee has little effect on the quality of the final separation. This is a clear contrast to the system proposed by Shen et al.13,14 or others,26 where the dead volume between a C18 trap column and an analytical column should be carefully minimized. Protein Identifications by UHP-MudPIT System. To demonstrate the performance of our UHP-MudPIT system regarding protein identifications, we compared the UHP system with a traditional MudPIT analysis (Table 2). The standard MudPIT here is referred to as a 15-salt step multidimensional LC analysis with a 10-cm column operated at a standard pressure (∼3 kpsi). Practically speaking, the analytical RP column was simply replaced on the UHP system with a 10-cm column of the same i.d., and a short 75-min gradient was run at the lower required pressure of ∼3 kpsi by adjusting the splitter resistance. Since throughput is also an important factor in proteomics studies, the number of salt steps for UHP-MudPIT was modified so as to keep the total cycle time approximately constant (∼24 h). Because the time required for a standard MudPIT took ∼28 h (i.e., 70-min gradient × 15 salt steps + between run equilibration and pump refill time), and the gradient volume was kept constant (i.e., 350-min gradient length), the number of steps for UHP-MudPIT was set to four including one 70-min gradient for the first step. To minimize potential biases associated with detection scan rate, a fast linear ion trap mass spectrometer (LTQ) was used for this evaluation. Table 2 also includes results of (1) a short gradients15-step UHPMudPIT with a 50-cm-long column. and (2) a short gradients4step standard MudPIT with a 10-cm column for a comparison (rows 2 and 4, respectively). As shown in Table 2, the 4-step UHP-MudPIT gave rise to the largest number (i.e., 1090 unique IDs) of protein identification (31) Lan, K.; Jorgenson, J. W. Anal. Chem. 1999, 71, 709-714.
Figure 5. Number of peptides (A)/proteins (B) uniquely identified in each UHP-MudPIT step (bar graphs), and their accumulation plots (line plots). gray bar, 350 min, 4 steps; white bar, 70 min, 15 steps; 2, 350 min, 4 steps; 0, 70 min, 15 steps. (C) Percentages of carryover peptides that were identified in previous salt steps. 2, 350 min, 4 steps; 0, 70 min, 15 steps.
from 20 µg of a yeast soluble fraction digested by Lys-C and trypsin. This was ∼1.3-fold the number of IDs obtained from the 15-step standard MudPIT benchmark taking the same amount of analysis time (a list of identified proteins is provided as Table S-1). The comparison between the number of IDs shown in rows 2 and 3 in Table 2 shows the importance of the gradient volume.28 The difference between the two experiments was only the column length and resulting operational pressure. In this case, the gradient volume was not optimized for the long column. As a result, both chromatograms showed similar peak profiles and peak capacities (data not shown) resulting in similar identification numbers for both. In addition, the power of multidimensional separations on protein identification can be seen by comparison of rows 1 and 3, Analytical Chemistry, Vol. 78, No. 14, July 15, 2006
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and 3 and 4 in Table 2. If the second dimension has sufficient separation power, the number of salt steps is less important. However, if the second-dimension separation has insufficient resolving power for the sample analyzed, less salt steps result in less protein identifications. We concluded from this comparison that the combination of less salt steps and high-resolution RP separation has the potential to provide an increased number of identifications with the same total run time. Figure 5A and B shows a graphical comparison of the 4- and 15-step UHP MudPIT results. The only differences between the two experiments were the number of salt steps (4 and 15) and the length of gradient (350 and 70 min) as the same 50-cm-long column was used to simplify the comparison (both operated at ∼15 kpsi). It is apparent from these plots that better separation in the final dimension can result in improved protein identification with fewer first-dimension steps over the same total analysis time. This is presumably due to the fact that the resolution of the SCX separation is not as high as that of the RP separation and the total peak capacity of a multidimensional separation is a function of the peak capacity of all separation dimensions.30 Another potential benefit of our approach is a reduction in “carryover” peptides. One of the biggest criticisms of the online multidimensional LC approach is the carryover of peptides between salt steps. This is believed to be caused by hydrophobic interactions between peptides and the SCX resin that results in broader peaks with mobile-phase compositions that are typical in online multidimensional column setups.32 Some argue that this is a benefit because the low-resolution, first-dimension separation increases the chance of precursor selection from the same peptides using data-dependent sampling. However, a potential tradeoff is a decreased ability to identify low-abundant peptides because they are split into two or more fractions. One offline approach was reported to ease this problem by allowing independent optimization for each dimension of separation.20 However, this approach suffers from potential sample loss during fraction collection and sample handling and can result in a very large number of fractions further increasing the time required for the analysis. (32) Burke, T. W.; Mant, C. T.; Black, J. A.; Hodges, R. S. J. Chromatogr. 1989, 476, 377-389.
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Figure 5C shows a comparison of the percentage of carryover peptides for 4-step, 350-min and 15-step, 70-min gradients. The carryover percentage was calculated by dividing the number of peptides detected in two adjacent salt steps by the total number of peptides detected in each salt step. As expected, the 4-step gradient exhibited less carryover (
