Modular Stop and Go Extraction Tips with Stacked Disks for Parallel and Multidimensional Peptide Fractionation in Proteomics Yasushi Ishihama,*,†,| Juri Rappsilber,‡,| and Matthias Mann*,§ Center for Experimental BioInformatics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark Received November 7, 2005
Abstract: Proteome complexity necessitates protein or peptide separation prior to analysis. We previously described a pipet-tip based peptide micropurification system named StageTips (STop and Go Extraction Tips), which consists of a very small disk of membrane-embedded separation material. Here, we extend this approach in several dimensions by stacking disks containing reversed phase (C18) and strong cation exchange (SCX) materials. Multidimensional fractionation as well as desalting, filtration, and concentration prior to mass spectrometry in single or tandem columns is described. C18-SCX-C18 stacked disks significantly improved protein identification by LC-MS/ MS for an E. coli protein digest and by MALDI-MS for a 12 standard protein digest. Sequential fractionation based on C18- followed by SCX material was also developed. This multidimensional fractionation approach was expanded to parallel sample preparation by incorporating C18-SCX-StageTips into a 96-well plate (StagePlate). Fractions were collected into other C18-StagePlates and desalted and eluted in parallel to sample well plates or MALDI targets. This approach is suitable for high throughput protein identification for moderately complex, low abundance samples using automated nanoelectrosprayMS/MS or MALDI-MS. Keywords: peptide multidimensional fractionation • StageTip • microtip • SCX • C18 • proteomics • LC-MS/MS • MALDI-MS • nanoelectrospray-MS
Introduction Proteomics seeks a comprehensive understanding of the cellular functions of proteins and therefore requires tools to analyze protein samples with high complexity and a wide dynamic range. Modern mass spectrometry (MS) with the ionization techniques electrospray ionization (ESI) and matrixassisted laser desorption ionization (MALDI) is currently a * To whom correspondence should be addressed. Y.I. Tel: +81 29 847 7192. Fax: +81 29 847 7614. E-mail:
[email protected]. M.M. Tel: +49 89 8578 2557. Fax: +49 89 8578 3209. E-mail:
[email protected]. † Current address: Laboratory of Seeds Finding Technology, Eisai Co., Ltd., Ibaraki, Japan. ‡ Current address: The FIRC Institute for Molecular Oncology, Milan, Italy. § Current address: Department of Proteomics and Signal Transduction, Max Planck Institute for Biochemistry, Martinsried, Germany. | Both authors contributed equally.
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Journal of Proteome Research 2006, 5, 988-994
Published on Web 03/07/2006
cornerstone of proteomics.1 Separation tools at the front end of MS are required to reduce the complexity and dynamic range of samples, and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) is one of the most powerful tools available for this purpose.2 Even LC-MS/MS, however, cannot be used to analyze complex samples such as cell lysates in a single run with the current instrumentation. Several forms of fractionation, at either the protein or peptide level, have therefore been developed. For example, on-line multidimensional LC is popular for large-scale proteomic studies.3-6 Likewise, one-dimensional gel electrophoresis followed by LCMS (‘GeLC-MS’) is a robust method for expanding the dynamic range of proteome analysis, especially for samples containing particular proteins with very high abundance.7,8 Isoelectric focusing in peptide separation prior to LC-MS/MS is also applied to fractionate complex mixtures.9 These approaches, however, were developed for samples with typically large amounts (milligrams) of total protein and are not necessarily suitable for medium-scale experiments when only a few micrograms of sample are available. For these situations, a flexible off-line system might achieve the best fractionation and analysis conditions. Off-line microscale solid-phase extraction (SPE) tools are often employed for MALDI and nanoelectrospray (nanoES) analysis to increase the sensitivity as well as the system stability by combining desalting, concentration, and filtration.10-14 Commonly, loosely packed SPE microtips such as ZipTip are used with repeated aspirate-eject cycles of a pipet. The loose structure, however, leads to a relatively low capacity and large elution volume, requires time for diffusion, and might result in low peptide recovery. In addition, it is difficult to remove contaminant particles or precipitates because the sample is loaded and eluted from the same side. Recently, we reported the concept of “stop and go extraction tips (StageTips)” in which Teflon embedded chromatographic beads are immobilized in a very small volume ( 3)
no. of unique peaks
flow-through fr. 1 fr. 2 fr. 3 fr. 4 total without fractionation
12 47 41 35 38 173 39
12 46 32 23 27 140 39
a The fractionation procedure is described in the Supporting Information (procedure 2).
tips with single disks was selected, and this configuration is now routinely used in our laboratories. In addition, because the chromatographic particles are trapped in the Teflon mesh structure, different kinds of particles are isolated at the interface between disks as shown in Figures 1 and 2A, whereas packed particles could mix at the interface as shown in Figure 2C. We were, however, not able to construct a pipet tip column with a sandwich of three materials using loose beads. Because it is sufficient that one side of the interface is fixed, a triple StageTip can also be prepared in sandwich fashion, with Teflon disks on the top and bottom and a loose bead ‘filling’ in the middle (Figure 2B). In this way, SCX particles, which are not available in a Teflon-embedded membrane, can also be employed. This particular setup is also advantageous for phospho-affinity separations with titania particles.2 If StageTips are used to prepare samples for LC-MS, the presence of salt is not a problem and a C18-SCX combination would normally be sufficient. Complete elution from SCX material, however, often requires relatively large elution volumes (e.g., 30 µL in our experience). The bottom C18 disk was therefore necessary to reduce the loading volume. To quantify the qualitative observations of stacked StageTips, we analyzed two model systems with LC-MS, nanoES or MALDI. First, we fractionated the soluble portion of E. coli cell lysate with C18-SCX-C18-StageTips and analyzed the fractions by LC-MS. Compared to the nonfractionation approach, both triple-StageTips using an SCX disk or loose SCX beads between two C18 disks significantly improved both peptide and protein identification (see statistics in Figure 3A and B). The peptides
were well distributed into fractions (Figure 4), although the salt concentration was not exhaustively optimized. Regarding the C18-SCXdisk-C18-StageTip, when 5% acetonitrile with different concentrations of ammonium acetate was used for salt elution from SCX, few peptides were observed because of the hydrophobic nature of this SCX disk. Therefore, we increased the acetonitrile concentration to 15% and added different ion pair reagents to increase the hydrophobicity of peptides for the final trapping step in the bottom C18 disk. We found that 0.1% perfluoropentanoic acid (PFPA) provided the best performance, retaining most of the peptides with 15% acetonitrile without changing the retention times in the subsequent nanoLC-MS analysis. When shorter length heptafluorobutyric acid was used as the ion pairing reagent, some hydrophilic peptides were lost (see Supporting Information C) and longer-chain perfluorinated acids such as perfluoroheptanoic acid and perfluorooctanoic acid20 resulted in shifted retention times of peptides in the subsequent nanoLC-MS analysis (approximately 15 min shift in 60 min gradient up to 30% acetonitrile for perfluoroheptanoic acid and no elution for perfluorooctanoic acid). Unlike the SCX disk, polysulfoethylaspartamide beads as the SCX material are sufficiently hydrophilic to allow peptide elution in 5% acetonitrile, although the production of sandwiched StageTips was not as robust as that of pure disk tips. Note that the flow-through fraction of the C18-SCXdisk-C18-StageTip contained only 17 proteins with 17 unique peptides. Next, we tested our approach with MALDI analysis using P10 type StageTips. Sample fractionation should be especially beneficial for MALDI, because there is a greater influence of sample complexity on MALDI. For MALDI and nanoES, reversed phase (RP) separation under acidic conditions is effective to reduce the complexity.11 The direct coupling of RP with MALDI or nanoES, however, is problematic in that the fractionated sample solutions have different organic solvent compositions, which impacts crystallization of the matrix or the spray, respectively; a critical parameter for sensitivity. This problem was solved by the use of SCX followed by RP for desalting (Figure 5 and Table 1). Using a mixture of 12 standard proteins, we observed 39 peptides without fractionation, while 140 peptides were observed with StageTip fractionation into four fractions. These results encouraged us to develop further fractionation steps adding a reversed phase mode prior to SCX. The elution conditions from low pH RP separation are fully compatible with
Table 2. C18 and SCX Fractionation Using C18-SCX-StageTips Analyzed by C18-StageTips Followed by NanoES-MS/MSa C18-SCX fractionation Swiss-Prot ID
TRFE•BOVIN CATA•BOVIN ALBU•BOVIN PHS2•RABIT PERL•BOVIN DHE3•BOVIN AMY•BACAM TRY1•BOVIN KCRM•RABIT ALFA•RABIT LDHA•RABIT KPY1•RABIT TPIS•RABIT Total a
992
SCX fractionation
without fractionation
protein amount (fmol)
protein score
no. of peptides
protein score
no. of peptides
protein score
no. of peptides
100 100 100 100 100 100 100 unknown 20 30 10 20 20
355 289 271 116 176 184 132 100 106 79 55 31 25 1919
14 10 10 10 9 8 6 5 4 3 2 1 1 83
165 238 138 86 29 84 76 60 33 44 0 26 17 996
8 8 6 5 1 5 2 2 1 2 0 1 1 42
106 72 0 65 66 81 0 0 0 0 0 0 0 390
4 2 0 3 2 3 0 0 0 0 0 0 0 14
The fractionation procedure is described in the Supporting Information (procedure 3).
Journal of Proteome Research • Vol. 5, No. 4, 2006
technical notes
Ishihama et al.
Figure 6. StagePlate with vacuum deposit device for MALDI in parallel fractionation mode. Table 3. Automated NanoES-MS/MS for 13 Proteins Fractionated by C18-SCX StagePlate-C18 StagePlatea fractionation, n)4 combined
without fractionation
Swiss Prot ID
protein score
no. of unique peptides
protein score
no. of unique peptides
CATA•BOVIN TRFE•BOVIN ALBU•BOVIN AMY•BACAM DHE3•BOVIN PHS2•RABIT TRY1•BOVIN ALFA•RABIT PERL•BOVIN KCRM•RABIT KPY•RABIT LDHA•RABIT TPIS•RABIT Total
363 349 280 223 230 183 102 135 46 61 44 40 32 2088
9 11 9 5 8 6 3 6 1 2 1 1 1 63
44 107 0 0 52 71 0 0 107 0 0 0 0 381
2 4 0 0 2 3 0 0 4 0 0 0 0 15
a The fractionation procedure is described in the Supporting Information (procedure 4).
the trapping conditions for SCX, while the elution conditions for SCX do not affect the peptides still bound to RP, which is well-known as it is the basis of the MudPit approach in LCMS.3,6 We fractionated the standard protein sample using 3 conditions in RP mode in combination with 4 different conditions in SCX mode, resulting in a total of 12 fractions, which were then manually analyzed by nanoES-MS/MS. As listed in Table 2, the number of identified peptides as well as proteins was drastically improved by this multidimensional fractionation in comparison with those by SCX fractionation and nonfractionation analyses. Finally, the use of multi-StageTips was extended to parallel handling. One advantage of using these off-line disposable tips is that they allow parallel sample treatment, reducing total preparation time prior to MS. In addition, because the disks in the StageTips are prepared from one uniform large disk, these tips are all identical and therefore suitable to perform parallel analysis. Using a vacuum-assisted deposit system with a 96-well format originally designed for 96-well filter plates, 96 samples were simultaneously fractionated (Figure 6). A 12
standard protein digest sample (100-1000 fmol each) for nanoES-MS/MS was also prepared using 96-well plate with StageTips (StagePlate), centrifuged, and analyzed by automated nanoES-MS/MS using LC-MS equipment, but without employing a column. Only 5 proteins were identified without fractionation. With fractionation, on the other hand, 13 proteins, including trypsin, were identified with more than twice the number of identified peptides on average (4 samples × 12 fractions), as listed in Table 3. This high-throughput sample preparation for MALDI-MS and nanoES-MS/MS is helpful for the analysis of moderately complex samples. In contrast to on-line separation systems, our approach separates the sample into discrete portions that can be analyzed by MALDI-MS/MS or nanoES robots. The latter systems are becoming increasingly fast and powerful, due to developments such as high frequency lasers and chip-based, low-flow nanoES. On the other hand, pipet-based fractionation is also readily performed in nonspecialized proteomic laboratories as it involves little setup and no extra equipment. Multi-StageTips therefore should be an ideal addition to the rapidly evolving proteome analysis pipeline.
Acknowledgment. We thank the group of Prof. Peter Roepstoff, especially Dr. Shabaz Mohammed, for measurements using their AP-MALDI instrument. Y.I. thanks Eisai Co., Ltd. for support during the duration of his sabbatical. The work in M.M.’s laboratory in Denmark is supported by a generous fund from the Danish National Research Foundation to the Center of Experimental BioInformatics (CEBI). Supporting Information Available: Detailed procedures of StageTip fractionation for LC9MS, MALDI, and nanoES. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Aebersold, R.; Mann, M. Nature 2003, 422, 198-207. (2) Ishihama, Y. J. Chromatogr. A 2005, 1067, 73-83. (3) Washburn, M. P.; Wolters, D.; Yates, J. R., 3rd Nat. Biotechnol. 2001, 19, 242-247. (4) Peng, J.; Elias, J. E.; Thoreen, C. C.; Licklider, L. J.; Gygi, S. P. J. Proteome Res. 2003, 2, 43-50.
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technical notes
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