Combination of FASP and StageTip-Based Fractionation Allows In

Combination of FASP and StageTip-Based Fractionation Allows In-Depth Analysis of the Hippocampal Membrane Proteome Jacek R. Wis´niewski,* Alexandre Zougman, and Matthias Mann* Department of Proteomics and Signal Transduction, Max-Planck Institute for Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany Received August 23, 2009

Abstract: Membrane proteomics is challenging because the desirable strong detergents are incompatible with downstream analysis. Recently, we demonstrated efficient removal of SDS by the filter aided sample preparation method (FASP). Here we combine FASP with our previously described small-scale membrane enrichment protocol. Analysis of a single mouse hippocampus enables identification of more than 1000 membrane proteins in a single LC-MS/MS run without protein or peptide prefractionation. To extend proteome coverage, we developed a simple anion exchange fractionation method in a StageTip format. When separating peptides into six fractions, a duplicate analysis resulted in identification of 4206 proteins of which 64% were membrane proteins. This data set covers 83% of glutamate and GABA receptor subunits identified in hippocampus in the Allen Brain Atlas and adds further isoforms. The combined method provides a streamlined protocol for rapid and sensitive membrane proteome mapping. We also provide a generic protocol for combining FASP with StageTip-based ion exchange fractionation, which is generally applicable to proteome analysis. Keywords: FASP • StageTip • SAX • Membrane Proteomics • Brain Proteome • High Resolution Mass Spectrometry

Introduction More than 30% of eukaryotic genes code for membrane proteins; however, these proteins are often underrepresented in proteomic studies. This is mainly caused by methods that are not efficient in extraction, separation, and digestion of membrane proteins, in particular those integral to membranes. Detergents are the reagents of choice for solubilization of membrane proteins and are regularly used in biochemical studies. Unfortunately, even in small concentrations, they can impair enzymatic digestion and dominate mass spectra due to their ready ionization ability and high-abundance compared to individual peptides. Therefore, removal of detergents is a prerequisite for successful mass spectrometric analysis. Since ‘in-solution’ removal of detergents has been thought to be difficult or impossible, various alternative approaches have * To whom correspondence should be addressed: E-mail: (J.R.W.) [email protected] or (M.M.) [email protected] Fax: +49 89 8578 2219.

5674 Journal of Proteome Research 2009, 8, 5674–5678 Published on Web 10/22/2009

been developed for the analysis of membrane proteomes. Early attempts involved membrane solubilization with acids and organic solvents.1,2 Digestion of the protein chains protruding from the membrane bilayer of nonsolubilized membranes yielded the first in-depth proteomic analyses of membrane proteomes.3-5 However, for general analysis of the membrane proteome, the use of detergents is unavoidable. Several novel detergents designed specifically for proteomic analysis (RapiGest and PPS Silent Surfactant) have been introduced. Their applicability to proteomic work has been tested,6,7 but this has not demonstrated unequivocal advantages over previously used gel-free approaches.5,8 Previously, we have shown that membrane proteins can be fully depleted from detergents by size exclusion chromatography in 8 M urea.8 A drawback of that method is the substantial dilution of proteins which accompanies the chromatographic separation of SDS and protein. This limitation was overcome in the filter aided sample preparation (FASP) method in which detergents can be fully removed in filter units and membrane proteins are digested with similar efficiency as soluble proteins.9 Here we report development of a rapid and sensitive method for membrane proteome analysis on the basis of a high yield enrichment protocol that is applicable to small amounts of tissue.8 Combination with FASP and single LC-MS/MS analysis resulted in identification of more than 1000 hippocampal membrane proteins. We also developed a pipet-based anion exchange fractionation for FASP generated peptides, capable of identifying 4000-5000 proteins from total cell lysate. Application to the hippocamal membrane proteome enabled identification of 4206 proteins including a nearly complete set of GABA and glutamate receptors as judged by comparison to the most in-depth atlas of localized brain gene expression.

Methods Isolation of Crude Membrane Fractions. Hippocampi were dissected from 10-14 week old female mice of the inbred strain C57BL/6 and flash frozen in liquid nitrogen followed by storage at - 80 °C. Crude membrane fractions were prepared using repeated tissue homogenization and extraction of nonmembrane proteins and solubilization with SDS as described.8 Briefly, single hippocampi (about 2 mg of total protein) were homogenized in 1 mL of 2 M NaCl, 10 mM HEPES/NaOH, pH 7.4, 1 mM EDTA containing protease inhibitor cocktail “Complete” (Roche) using a T-10 basic dispenser (IKA) equipped with S10N 5G dispersion tool at maximum speed (setting 6) for 20 s. 10.1021/pr900748n CCC: $40.75

 2009 American Chemical Society

technical notes

Combination of FASP and StageTip-Based Fractionation The homogenates were centrifuged in a benchtop centrifuge (Eppendorf) at 16 100g at 4 °C for 20 min. The pellets were rehomogenized in 1 mL of 0.1 M Na2CO3 and 1 mM EDTA, pH 11.3, incubated at 4 °C for 30 min, and collected by centrifugation. Subsequently, the pellets were extracted with 5 M urea, 100 mM NaCl, 10 mM HEPES, pH 7.4, and 1 mM EDTA and then washed twice with 0.1 M Tris/HCl, pH 7.6. The pellets were solubilized in 0.1 mL of 2% SDS, 50 mM DTT, and 0.1 M Tris/HCl, pH 7.6, at 90 °C for 1 min. Detergent Removal and Protein Digestion. Dissolved membranes were processed by the FASP procedure9 using 30k Microcon filtration devices (Millipore). Briefly, 25 µL aliquots were mixed with 0.2 mL of 8 M urea in 0.1 M Tris/HCl, pH 8.5 (UA solution), loaded into the filtration devices, and centrifuged at 14 000g for 15 min. The concentrates were diluted in the devices with 0.2 mL of UA solution and centrifuged again. After centrifugation, the concentrates were mixed with 0.1 mL of 50 mM iodoacetamide in UA solution and incubated in darkness at room temperature (RT) for 30 min followed by centrifugation for 15 min. Then, the concentrate was diluted with 0.2 mL of 8 M urea in 0.1 M Tris/HCl, pH 8.5 (UB solution), and concentrated again. This step was repeated twice. The resulting concentrate was diluted to 30 µL with UB solution and 2 µg of endoproteinase LysC was added. After overnight incubation at RT, the samples were diluted with 120 µL of 40 mM NaHCO3 containing 2 µg of trypsin. Following a 4 h digestion, peptides were collected by centrifugation of the filter units for 20 min. The concentration of peptides was determined by UV-spectrometry using an extinction coefficient of 1.1 for 0.1% (g/L) solution at 280 nm. Anion Exchange-Based Fractionation of Peptides. A total of 30 µg of peptides was separated on a pipet-based anion exchanger, which was assembled following the StageTip principle10,11 by stacking 6 layers of a 3M Empore Anion Exchange disk (Varian, 1214-5012) into a 200 µL micropipet tip. For column equilibration and elution of fractions, we used Britton & Robinson buffer composed of 20 mM acetic acid, 20 mM phosphoric acid, and 20 mM boric acid titrated with NaOH to the desired pH. Peptides were loaded at pH 11 and fractions were subsequently eluted with buffer solutions of pH 8, 6, 5, 4, and 3, respectively. The flow-through (peptides loaded at pH 11 but not bound to the exchanger material) was captured on a StageTip11 containing three layers of C18 membrane. The five pH eluted fractions were also captured on C18 StageTips. LC-MS/MS Analysis of Peptides. LC-MS/MS analysis was carried out essentially as described before.12 Briefly, samples were separated on an in-house made 15 cm reversed phase capillary emitter column (inner diameter 75 µm, 3 µm ReproSilPur C18-AQ media (Dr. Maisch GmbH, Ammerbuch-Entringen, Germany)) using 240 min gradients and analyzed on an LTQOrbitrap instrument (Thermo Fisher Scientific). Survey MS scans were acquired in the orbitrap with 60 000 resolution. For accurate mass measurements, the lock-mass option was employed.13 Up to the 10 most intense ions in each full MS scan were fragmented and analyzed in the linear ion trap part of the instrument. Raw MS files were processed with MaxQuant, a freely available software suite.14 Peak list files were searched by the MASCOT search engine15 against the IPI-mouse or human database (version 3.46) containing both forward and reversed protein sequences. Initial maximum precursor and fragment mass deviations were set to 7 ppm and 0.5 Da, respectively, but MaxQuant achieved sub-parts per million (sub-ppm) mass accuracy for the majority of peptide precur-

Figure 1. (A) Workflow for streamlined and sensitive membrane proteome analysis. Direct analysis (1) and analysis with peptide fractionation (2) are shown. (B) Assembly of anion-exchange fractionation and StageTip desalting columns fixed in the lid of a centrifugal tube.

sors. The search included variable modifications for oxidation of methionine and protein N-terminal acetylation. Peptides with at least six amino acids were considered for identification. The false discovery rate for both peptides and proteins were set at 0.01. All peptides and proteins identified in this study are listed in Supplementary Tables 1-3.

Results and Discussion Brain function is mainly mediated by membrane proteins and their dysfunction is involved in many diseases of the nervous system. The brain is composed of several functionally and anatomically distinct regions, of which the hippocampus has a central role in memory and learning. Mouse hippocampus is a small brain region with a total volume of 20-30 µL, which contains about 2 mg of total protein. Thus, in-depth analysis of the hippocampal membrane proteome of one single mice is a considerable analytical challenge. To analyze the membrane proteome of mouse hippocampus, we prepared crude membrane fraction from individual animals by a three-step extraction method,8 which yielded about 100-200 µg of protein material. This was sufficient for multiple downstream analyses, with or without peptide fractionation. The membranes were solubilized in a buffer containing 4% SDS and were processed by the FASP method using Microcon filters with nominal 30k cutoff. However, even small proteins are retained because they are unfolded. The presence of the strong detergent SDS ensured practically complete solubilization of membrane proteins. After FASP, peptides are detergentfree and essentially pure9 and were analyzed by high resolution LC-MS/MS with 240 min gradients on an LTQ-Orbitrap either directly or after prefractionation using anion exchange chromatography (Figure 1). Single Run Analysis of FASP Generated Peptide Mixtures of the Membrane Proteome. We generated hippocampal membrane peptide samples by FASP from four different mice and analyzed each in triplicate by single LC-MS/MS runs. These individual runs consumed 5 µg aliquots of the digests as Journal of Proteome Research • Vol. 8, No. 12, 2009 5675

technical notes

Figure 2. Protein identified across 12 LC-MS/MS runs. The plot shows the number of proteins identified in all 12 runs, in exactly 11 runs, and so on down to the proteins identified in only a single run. (Inset) Identification of proteins with Gene Ontology (GO) annotation terms ‘membrane’,‘integral to membrane’, and ‘cytoplasm’. Membrane proteins are identified more consistently across fractions than nonmembrane proteins.

determined by UV absorbance of FASP-generated peptides, representing 3-5% of the total from a single mouse. MaxQuant processing of the 12 individual experiments yielded 1531 ( 52 protein identifications per run. Triplicate analyses of the same sample increased the number of identified proteins to 1818 ( 24 per single hippocampus. In total, the 12 LC-MS/MS runs identified 2078 proteins. More than 1000 of these proteins were identified in common in each separate experiment and more than 1400 were present in at least six of the 12 experiments (Figure 2). Interestingly, the proteins identified in all or most runs have a higher proportion of membrane and integral membrane proteins compared to the proteins that are only identified in some experiments (Figure 2 inset). In contrast, the proportion of proteins with the GO annotation term “cytoplasm” remained unchanged between proteins identified in all runs and in single runs. Therefore, the run-to-run variation in the identity of proteins is not only due to variation caused by undersampling of the mass spectrometer. Instead, membrane proteins are consistently enriched by the protocol, whereas background proteins (such as cytoplasmic ones) only associate with the membrane enriched fraction in an inconsistent manner. It follows that repetitions tend to yield identifications with a larger proportion of contaminating proteins and do not lead to substantial improvement in coverage of the membrane proteome. Of the 2078 proteins identified in the 12 single runs, 1354 were annotated as ‘membrane’ in the GO ontology (72% of those with a GO compartment annotation). A total of 955 and 534 protein had annotations ‘integral to membrane’ and ‘plasma membrane’, respectively (Figure 3). Low percentage of nuclear and cytosolic proteins (less than 10%) additionally supports efficient enrichment of membranes in our preparations. More than 20% of the found proteins appear to be involved in signal transduction or cell communication (Figure 3). In summary, single run analysis of the hippocampal membrane proteome by membrane enrichment and FASP demonstrated robust, rapid and very sensitive analysis of a large number of membrane and membrane associated proteins. 5676

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Figure 3. GO ontology annotations of proteins identified in membrane-FASP preparations from mouse hippocampus in 12 LC runs (red bars) or in two combined membrane-FASP-SAX experiments (green bars).The numbers of proteins matching each annotation are indicated. Lengths of the bars are proportionate to the percentage value.

Analysis of Anion Exchange Fractionated Protein Digests. To achieve even greater depth in the characterization of the membrane proteome and to identify very low-abundance proteins, we decided to perform an additional step of peptide fractionation of the FASP-generated peptides prior to LC-MS/ MS analysis. For this purpose, we adopted and further developed the StageTip concept of peptide fractionation.10,11 We used ion-exchanger filter-plugs in pipet tips to provide peptide separation orthogonal to reverse phase material. The fractionation column was assembled from 6 layers of ion exchange membrane. Eluted peptides were directly bound on standard, C18 type StageTips (Figure 1B). To develop and to test the ion exchange separation, we used FASP-prepared digests of HeLa cells. Both anion (‘SAX’) and cation (‘SCX’) exchanger membranes were tested. A single experiment comprising LC- MS/MS analysis of six SAX fractions allowed identification of 4699 proteins (one day of measurement time). Similar experiments using SCX membranes identified 4158 proteins (Supplementary Table 2). Because of the 13% higher number of identified proteins in the SAX-based approach, we used anion exchanger for fractionation of peptides in this investigation. However, it is possible that SCX-based fractionation would perform similarly or better than SAX-based fractionation in other applications. The protocol for downstream fractionation of FASP-generated peptides is provided as Supporting Information. For analysis of hippocampal membranes, we generated six fractions by sequential elution with buffers of decreasing pHvalues, chosen to optimally fractionate tryptic peptides. Triplicate analysis of each fraction indeed resulted in the identification of a similar number of peptides per fraction (Figure 4A). Half of the peptides were identified in only one fraction, and on average, peptides were identified in two fractions (Figure 4B).

technical notes

Combination of FASP and StageTip-Based Fractionation

Figure 4. Fractionation of hippocampal peptides by pipet-based SAX. (A) Number of identified peptides per single fraction. Error bars are calculated from three runs. (B) Redundancy of peptide identification shows in how many fractions the same peptide was repeatedly identified.

We repeated the analysis of the SAX-fractionated sample of the same mouse, which resulted in the identification of 4206 proteins containing 2305 proteins with ‘membrane’ annotation (64% of the 3623 identified proteins with GO annotation) (Supplementary Table 3). Interestingly, there were 1604 proteins with GO term ‘integral to membrane’ and 906 proteins with GO term ‘plasma membrane’ (Figure 3) (Supplementary Table 3). These numbers are encouraging considering that previous studies using 2D gel electrophoresis typically found just a few integral membrane proteins at most and considering that the plasma membrane only constitutes a few percent of cellular volume. Nevertheless, this did not indicate how comprehensive the coverage of the membrane proteome was. To answer this question, we turned to the Allan Mouse Brain Atlas (http://mouse.brain-map.org/), which is an anatomically comprehensive digital atlas containing the expression patterns of approximately 20 000 genes in the adult mouse brain.16 To estimate the percentage of the membrane proteome that we had identified, we checked GABA and glutamate receptor subunits reported in the hippocampus section of the Brain Atlas (Figure 5). As shown in Figure 5, our data set identified 83% of the subunits of these two major neurotransmitters. The missing proteomic identifications reflect transcripts that were observed at very low signal intensities in the in situ hybridization (see for example Grik4 and Gabra6 in Figure 5), and thus are close to the limit of detection in the Atlas data. The only exception was kainate receptor 1 (Grik1), which was observed in the Allen Brain Atlas at moderate intensity but was not identified in our experiments. Conversely, the proteomic approach allowed identification of several isoforms of AMPA and NMDA glutamate

Figure 5. Identification of GABA and glutamate receptors. (A) Sequence coverage of the proteins identified in 12 single runs and two SAX-fractionated samples. (B) Intensity of in situ hybridization with transcript specific probes in the Allen Brain Atlas. Asterisk (*) indicates absence of transcript intensity information. Protein isoforms of the same gene are in ‘Italics’ and ‘underlined’. The unique sequence coverage (and the number of unique peptides) of the isoforms is indicated on the right to the bars. These values refer to the SAX-LC experiment.

and GABA receptors, which were absent in the Allen Brain Atlas (Figure 5). Furthermore, we found 68 subunits of K+, Na+, and Ca2+ channels and 182 transmembrane transporter proteins of Slc and Abc gene families (Supplementary Table 3). Together, our results provide the most comprehensive list of proteins identified in any neural cell membrane and clearly demonstrate that the combined method presented here can probe the membrane proteome in considerable depth.

Conclusion Previously, multiple runs were necessary to cover a substantial number of brain-specific membrane proteins. Here we demonstrate that such a characterization is attainable in a single analysis, due to effective membrane enrichment and efficient solubilization by SDS and digestion of membrane proteins. Additional separation of peptides into six fractions by anion exchange chromatography allows an in-depth analysis of the membrane subproteome providing an almost complete map of the two major neurotransmitter receptors. Journal of Proteome Research • Vol. 8, No. 12, 2009 5677

technical notes Our “off-line” SAX-based fractionation together with the ‘online’ C18-LC separation resembles the well-known ‘multidimensional protein identification technology’ (MuDPIT)1 but does not require any special equipment. The disposable SAX tip-columns provide an uncomplicated way for peptide fractionation. The SAX column size can be scaled up or scaled down to fit the sample amount. In our group, this fractionation method has already been successfully applied to analysis of various types of samples. In our hands, compared to OFFGEL fractionation,17 it is more robust, much faster and simpler to use. Furthermore, peptide fractions do not contain potentially interfering ampholytes. Whereas pipet-based fractionation does not provide the separation power of an optimal OFFGEL run, we typically identify 4000-5000 proteins by FASP-SAX in a one day analysis, sufficient for many applications.

Acknowledgment. This work was supported by the Max-Planck Society for the Advancement of Science and the Munich Center for Integrated Protein Science (CIPSM). Supporting Information Available: Table 1, 12 single runs of lysates from nonfractionated hippocampal membranes. Table 2, SAX and SCX-based separations of whole cell lysates of HeLa cells. Table 3, SAX-based separation and identification of hippocampal proteins. Protocol: Anion Exchanger Separation. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Washburn, M. P.; Wolters, D.; Yates, J. R., III. Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat. Biotechnol. 2001, 19 (3), 242–7. (2) Blonder, J.; Conrads, T. P.; Veenstra, T. D. Characterization and quantitation of membrane proteomes using multidimensional MSbased proteomic technologies. Expert Rev. Proteomics 2004, 1 (2), 153–63. (3) Wu, C. C.; MacCoss, M. J.; Howell, K. E.; Yates, J. R., III. A method for the comprehensive proteomic analysis of membrane proteins. Nat. Biotechnol. 2003, 21 (5), 532–8. (4) Le Bihan, T.; Goh, T.; Stewart, I. I.; Salter, A. M.; Bukhman, Y. V.; Dharsee, M.; Ewing, R.; Wis´niewski, J. R. Differential analysis of membrane proteins in mouse fore- and hindbrain using a labelfree approach. J. Proteome Res. 2006, 5 (10), 2701–10. (5) Nielsen, P. A.; Olsen, J. V.; Podtelejnikov, A. V.; Andersen, J. R.; Mann, M.; Wis´niewski, J. R. Proteomic mapping of brain plasma membrane proteins. Mol. Cell. Proteomics 2005, 4 (4), 402–8. (6) Chen, E. I.; McClatchy, D.; Park, S. K.; Yates, J. R., III. Comparisons of mass spectrometry compatible surfactants for global analysis of the mammalian brain proteome. Anal. Chem. 2008, 80 (22), 8694–701. (7) Chen, E. I.; Cociorva, D.; Norris, J. L.; Yates, J. R., III. Optimization of mass spectrometry-compatible surfactants for shotgun proteomics. J. Proteome Res. 2007, 6 (7), 2529–38.

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