Combining Filter-Aided Sample Preparation and Pseudoshotgun

Dec 6, 2012 - (S.N.W.) E-mail: [email protected]. ... The performance of two proteomic sample preparation methods, “pseudoshotgun” ...
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Technical Note pubs.acs.org/jpr

Combining Filter-Aided Sample Preparation and Pseudoshotgun Technology To Profile the Proteome of a Low Number of Early Passage Human Melanoma Cells Margarita Maurer,† André C. Müller,‡ Christine Wagner,† Marie L. Huber,†,‡ Elena L. Rudashevskaya,‡,# Stephan N. Wagner,*,† and Keiryn L. Bennett*,‡ †

Division of Immunology, Allergy and Infectious Diseases, Department of Dermatology, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria ‡ CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Lazarettgasse 14, AKH Building BT 25.3, A-1090 Vienna, Austria S Supporting Information *

ABSTRACT: The performance of two proteomic sample preparation methods, “pseudoshotgun” (PSG) and filter-aided sample preparation (FASP) were compared in terms of the number of identified proteins, representation of cellular component GO (gene ontology) categories in the obtained list of proteins, and the efficiency of both methods in the proteomic analysis of a very low number of cells. Both methods were combined to obtain a proteomic profile of a short-term culture (passage 3) of melanoma cells, established in our laboratory from a human metastatic melanoma lesion. The data revealed that with FASP, usually more proteins are identified than with PSG when analyzing a higher number of cells (≥5000/injection), whereas PSG is favorable when analyzing only a very small amount of cells (250−500/injection). PSG and FASP, however, are complementary techniques, as combining both methods further increases the number of identified proteins. Moreover, we show that it is feasible to identify a substantial number of proteins from only 250 cells/injection that is equivalent to 60 ng of protein. KEYWORDS: FASP, pseudoshotgun, in situ digest, SDS removal, orbitrap, proteomic profile, metastatic melanoma, ultrasensitive proteomics

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situ digestion5−7 or the recently developed filter-aided sample preparation (FASP).8−10 In general, precipitation, dialysis, and gel filtration require larger amounts of material and/or a certain concentration of protein. Thus, none of these methods are ideal for samples from a very small amount of cellular starting material with a low protein content. In this technical note, we chose to compare FASP and PSG. Both methods are very effective in removing high concentrations of SDS with minimal sample loss, while maintaining efficient peptide recovery following tryptic digestion.5−10 PSG is a modified version of the commonly practiced 1D-SDSPAGE (or 2D-SDS-PAGE) analysis followed by in situ digestion. Prior to digestion, proteins are usually separated by running a full-length gel, and each lane is excised into several slices. Gel slices are washed prior to digestion to remove SDS. After in situ digestion of the proteins in the gel pieces, the tryptic peptides from each slice are individually analyzed by LCMSMS. For the PSG approach, the proteins are only partially separated by running a short, 15 min gel that is cut into a few

INTRODUCTION Sample preparation preceding mass spectrometric analysis is critical for high quality results in proteomic research. In a classical “bottom-up” experiment, the sample preparation workflow includes the following: (i) extraction/solubilization of proteins; (ii) reduction of disulfide bonds; (iii) alkylation of free cysteine residues, and (iv) digestion of proteins into peptides. The membrane proteins could be underrepresented in the obtained proteome because of poor solubilization properties. For in-depth proteomic profiling of complex biological samples (e.g., cells, tissues), it is necessary to extract proteins from different cell compartments including membranes, membrane organelles, and stable, multicomponent protein complexes. This requires efficient solubilization of the proteins with the use of detergents, of which sodium dodecyl sulfate (SDS) is still the reagent of choice for successful protein extraction. Unfortunately, SDS and other detergents are detrimental for subsequent mass spectrometric analysis, and therefore a detergent removal step must be included in the sample preparation workflow. Several methods can be used to reduce SDS in samples prior to MS analysis, such as protein precipitation with organic solvents, dialysis, gel filtration,1−4 in © 2012 American Chemical Society

Received: October 25, 2012 Published: December 6, 2012 1040

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Technical Note

gel slices (e.g., four slices). After washing the gel slices and in situ digestion of the proteins, the peptides from all excised slices are pooled and analyzed as one injection by LC-MSMS. In contrast to PSG, FASP combines the removal of SDS with digestion of solubilized proteins in the same device, thus minimizing sample loss and improving peptide recovery. The primary focus of this study was to compare the performance of PSG and FASP rather than addressing extensive biological questions, mechanisms and function. Nonetheless, short-term cultured melanoma cells obtained from a human metastatic melanoma biopsy that was only in vitro passaged three times before proteomic analysis was chosen for the study. Although comparative proteomics of laser-microdissected melanoma cells, melanoma cell lines, and serum from melanoma patients have often been used for biomarker discovery using 2D-electrophoresis,11−16 we are not aware of any published detailed proteomic profile of a short-term cultured human melanoma sample. The aims of the present study were as follows: (i) compare the performance of FASP and PSG technology for the LCMSMS analysis of complex protein mixtures from small amounts of cell lysate; (ii) evaluate if the two methods differ with respect to identification of proteins from different cellular components (cytoplasm, nucleus, plasma membrane), and (iii) assess the feasibility of using a very small number of human melanoma cells and LC-MSMS analysis to obtain a proteomic profile of short-term cultured melanoma cells . The data revealed that with FASP, more proteins were identified than with PSG when analyzing a higher number of cells (≥5000/injection), whereas PSG was the favored technique when analyzing only a very small amount of cells (250−500/injection). The two methods, however, gave complementary results. In addition, we could show that as low as 250 cells/injection can be used to obtain a proteomic profile of (cancer) cells. To our knowledge, this is the first proteomic profile of short-term cultured melanoma cells obtained from a human metastatic melanoma biopsy.

Figure 1. General workflow for the proteomic analysis of short-term cultured melanoma cells from a human melanoma biopsy. Melanoma cells obtained from a fresh human metastatic melanoma lesion were short-term cultured until the third passage. For the proteomic analyses, the melanoma cells were detached, washed, and counted and separate aliquots of different numbers of melanoma cells were prepared. Each aliquot and replicate was lysed separately in 40 μL of lysis buffer containing 2% SDS. Half of each sample (20 μL) was used for the protein assay and the other half (20 μL) for either PSG or FASP. Cells were prepared in sufficient amounts to allow a maximum of three injections onto the LC-MSMS system (i.e., three technical replicates). Thus, the cell number of each injection was 1/6 of the original starting number of cells. For example, 1500 melanoma cells were lysed in 40 μL of lysis buffer. Twenty microliters (∼750 cells) were used for the protein assay, and the remaining 20 μL (∼750 cells) were further processed using PSG or FASP. Finally, the resultant tryptic peptides from these 750 cells correspond to three injections onto the LCMSMS system (i.e., 250 cells/injection). After purification and desalting, the samples were analyzed by nanoLC-MSMS on a LTQ Orbitrap Velos followed by bioinformatic analysis.

After dissociation, the cell suspension was passed through a 70 μm cell strainer (BD Falcon, Bedford, MA) to remove any large particles from the single cell suspension. Cells were seeded in tissue culture flasks (T25) containing RPMI, 10% fetal calf serum (FCS, Invitrogen) and penicillin/streptomycin (Gibco, Paisley, UK). Outgrowth of melanoma cells was confirmed by microscopy. Melanoma cells were frozen after passage 2 and stored in liquid nitrogen. For the proteomic experiments, cells were thawed and cultured until confluent (passage 3).

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MATERIAL AND METHODS The workflow of the experiments is shown in Figure 1. Isolation and Culture of Metastatic Melanoma Cells

Collection of Cells from Culture

The melanoma sample analyzed in this study was collected and prepared under a local ethics committee-approved protocol. The patient provided written, informed consent. Immediately after surgical resection, the metastatic melanoma tissue was dissociated into single cells using the tumor dissociation kit (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) in conjunction with the gentleMACS Dissociator (Miltenyi Biotec GmbH) enabling both mechanical and enzymatic dissociation of the tumor tissue. Briefly, the tissue was minced into 2−4 mm pieces and transferred into a gentleMACS C tube (Miltenyi Biotec GmbH) containing 4.7 mL RPMI (Invitrogen, Carlsbad, CA) and an enzyme mix consisting of 200 μL of solution I, 100 μL of solution II, and 25 μL of solution III (solutions I, II, and III are provided with the tumor dissociation kit). The C tube containing the tissue and the enzyme mix was attached to the sleeve of the gentleMACS Dissociator and sequentially run with the preinstalled programs h_tumor_1, h_tumor_2, and h_tumor_3. Between each program, the C tube containing the tumor tissue and the enzyme mix was incubated at 37 °C for 30 min under continuous rotation.

After the third in vitro passage, cells were detached using Versene (0.48 mM EDTA (Sigma-Aldrich, St. Louis, MO) in PBS without MgCl2 and CaCl2 (Gibco)), washed three times in PBS (Gibco), and counted using a hemocytometer. Aliquots containing different numbers of cells were pelleted by centrifugation using an Eppendorf centrifuge (5415R) at 400g for 4 min at RT. The supernatant was discarded, and cell pellets were snap frozen and stored at −80 °C until required. Flow Cytometric Analysis

An aliquot of the cell suspension was used to assess the purity of the seeded melanoma cells by flow cytometry. The cells were stained with an antibody against melanoma-associated chondroitin sulfate proteoglycan (MCSP), a well-established melanoma cell marker.17 Briefly, 105 cells were resuspended in 100 μL of PEB buffer (0.5% BSA, 2 mM EDTA) and incubated for 10 min at 4 °C in the dark with an APCconjugated MCSP antibody (Miltenyi Biotec GmbH) or with the corresponding isotype control (Miltenyi Biotec GmbH). Cells were washed by adding 1 mL of PBS and centrifuged at 1041

dx.doi.org/10.1021/pr301009u | J. Proteome Res. 2013, 12, 1040−1048

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300g for 10 min at 4 °C. The supernatant was discarded and the cells were resuspended in 100 μL of PBS before analysis on the FACS Calibur (Becton Dickinson, San Jose, CA). The percentage of MCSP positive cells was >97% (Figure S1, Supporting Information).

of 50 mM TEAB buffer (Sigma-Aldrich). Proteins were digested with trypsin overnight at 37 °C. Peptides were recovered using 40 μL of 50 mM TEAB buffer followed by 50 μL of 0.5 M NaCl (Sigma-Aldrich). Acidified tryptic peptides from the in situ digest PSG and FASP approaches were concentrated and desalted using C18 spin columns (The Nest Group, Southborough, MA).

Cell Lysis

After the melanoma cells were detached, washed, and counted and separate aliquots of different numbers of melanoma cells prepared (Figure 1), each aliquot and replicate was lysed separately in 40 μL of freshly prepared lysis buffer. Half of each sample (20 μL) was used for the protein assay (Figure 1). Lysis buffer contained 50 mM HEPES (pH 8.0), 2% SDS, 0.1 M DTT, 1 mM PMSF, and protease inhibitor cocktail (SigmaAldrich), and lysis took place at RT for 20 min. After being heated at 99 °C for 5 min and cooled to RT, the cell lysate was sonicated using a Covaris S2 high performance ultrasonicator. The lysate was centrifuged at 20 000g for 15 min at 20 °C, and the protein extract was collected from the supernatant. Total protein content of the whole cell lysates was determined using the BCA protein assay kit (Pierce Biotechnology, Rockford, IL) following the recommendations of the manufacturer. The limited size of the samples necessitated that the assay was performed in a 96-well plate using 10 μL of each lysate and standard protein. The samples were measured in duplicate. BSA (Pierce Biotechnology) was used as the standard protein.

Liquid Chromatography Mass Spectrometry

Mass spectrometry was performed on a hybrid LTQ-Orbitrap Velos mass spectrometer (ThermoFisher Scientific, Waltham, MA) using Xcalibur version 2.1.0 SP1.1160 coupled to an Agilent 1200 HPLC nanoflow system (dual pump with one precolumn and one analytical column) (Agilent Biotechnologies, Palo Alto, CA) via a nanoelectrospray ion source using liquid junction (Proxeon, Odense, Denmark). Solvents for HPLC separation of peptides were as follows: solvent A consisted of 0.4% formic acid (FA) in water, and solvent B consisted of 0.4% FA in 70% methanol and 20% 2-propanol. Note that in our hands, this composition of solvent B provides superior data compared to other more commonly used mobile phase combinations, e.g., acetonitrile (unpublished observations). It is known from the literature that the use of methanol in the mobile phase B augments and improves the limits of peptide detection compared to acetonitrile.18 From a thermostatted microautosampler, 8 μL of the tryptic peptide mixture were automatically loaded onto a trap column (Zorbax 300SBC18 5 μm, 5 × 0.3 mm, Agilent Biotechnologies) with a binary pump at a flow rate of 45 μL/min. 0.1% trifluoroacetic acid (TFA) was used for loading and washing the precolumn. After washing, the peptides were eluted by back-flushing onto a 16 cm fused silica analytical column with an inner diameter of 50 μm packed with C18 reversed phase material (ReproSil-Pur 120 C18-AQ, 3 μm, Dr. Maisch GmbH, AmmerbuchEntringen, Germany). The peptides were eluted from the analytical column with a 27 min gradient ranging from 3% to 30% solvent B, followed by a 25 min gradient from 30% to 70% solvent B, and finally a 7 min gradient from 70% to 100% solvent B at a constant flow rate of 100 nL/min.19 The analyses were performed in a data-dependent acquisition mode, and dynamic exclusion for selected ions was 60 s. A top 15 collisioninduced dissociation (CID) method was used, and a single lock mass at m/z 445.120024 (Si(CH3)2O)6)20 was employed. Maximal ion accumulation time allowed in CID mode was 50 ms for MSn in the LTQ and 500 ms in the C-trap. Automatic gain control was used to prevent overfilling of the ion traps and was set to 5000 in MSn mode for the LTQ and 106 ions for a full FTMS scan. Intact peptides were detected in the Orbitrap Velos at 60 000 resolution at m/z 400. All samples were analyzed as technical duplicates.

In Situ Protein Digest

For in situ digestion, 10.8 μL of 4 × NuPAGE LDS Sample Buffer (4×) (Invitrogen) was added to 20 μL of each cell lysate followed by heating the sample for 10 min at 70 °C. Each sample was alkylated using 4.3 μL of 719 mM/mL iodoacetamide (Sigma-Aldrich) for 20 min at RT in the dark. A total volume of 35.1 μL of each reduced, denatured, and alkylated protein extract was loaded onto a 4−12% NuPAGE NOVEX precast minigel (Invitrogen) and run at 120 mA, 200 V, for only 15 min. Gels were stained using Brilliant Blue Colloidal Coomassie (Sigma-Aldrich). Lanes containing proteins were excised from the gel and cut into four pieces using a scalpel blade. Each gel plug was transferred into an individual well of a 96-well microtiter plate with 1 mm diameter holes drilled in the bottom. Gel plugs were washed, reduced with 10 mM DTT (Sigma-Aldrich), and alkylated with 55 mM iodoacetamide (Sigma-Aldrich). The solutions in the wells of the plates were exchanged by centrifugation in a 5810 Eppendorf centrifuge at 370g for 2 min at RT. Peptides were digested with 12 ng/μL trypsin (Promega Corp., Madison, WI) overnight at 37 °C prior to extraction with 5% formic acid (HCOOH) (Merck, Darmstadt, Germany). Filter-Aided Sample Preparation (FASP)

Data Analysis

FASP was performed using a 30 kDa molecular weight cutoff filter (VIVACON 500; Sartorius Stedim Biotech GmbH, 37070 Goettingen, Germany) essentially according to the procedure described by Wisniewski et al.8 Briefly, each cell lysate was supplemented with lysis buffer to a final volume of 30 μL. Thirty microliters of each protein extract was mixed with 200 μL of 8 M urea in 100 mM Tris-HCl (pH 8.5) (UA) in the filter unit and centrifuged at 14 000g for 15 min at 20 °C to remove SDS. Any remaining SDS was exchanged by urea in a second washing step with 200 μL of UA. The proteins were alkylated with 100 μL of 50 mM iodoacetamide for 30 min at RT. Afterward, three washing steps with 100 μL of UA solution were performed, followed by three washing steps with 100 μL

The acquired raw MS data files were processed with msconvert (ProteoWizard Library v2.1.2708) and converted into MASCOT generic format (mgf) files. The resultant peak lists were searched against the human SwissProt database version v2012.05_20120529 (36 898 sequences, respectively, including isoforms, as obtained from varsplic.pl) with the search engines MASCOT (v2.3.02, MatrixScience, London, UK) and Phenyx (v2.5.14, GeneBio, Geneva, Switzerland).21 Submission to the search engines was via a Perl script that performs an initial search with relatively broad mass tolerances (MASCOT only) on both the precursor and fragment ions (±10 ppm and ±0.6 Da, respectively). High-confidence peptide identifications were 1042

dx.doi.org/10.1021/pr301009u | J. Proteome Res. 2013, 12, 1040−1048

Journal of Proteome Research

Technical Note

LC-MSMS analysis. Thus, we chose to perform a series of experiments to compare the performance of the FASP method with PSG (a modified version of standard gel-based approaches). A range of sample sizes were evaluated starting from 250 cells per injection onto an LC-MSMS system. The performance of PSG and FASP were compared based on the number of uniquely identified proteins. As both methods were conducted in parallel, the majority of the workflow steps were identical (Figure 1). Thus, a direct comparison of both methods was possible. We found that for samples with ≥5000 cells/injection, more proteins were identified with FASP than with PSG (Figures 2 and 3). At the same time, a limitation of

used to recalibrate all precursor and fragment ion masses prior to a second search with narrower mass tolerances (±4 ppm and ±0.3 Da). One missed tryptic cleavage site was allowed. Carbamidomethyl cysteine was set as a fixed modification, and oxidized methionine was set as a variable modification. To validate the proteins, MASCOT and Phenyx output files were processed by internally developed parsers. Proteins with ≥2 unique peptides above a score T1, or with a single peptide above a score T2, were selected as unambiguous identifications. Additional peptides for these validated proteins with score > T3 were also accepted. For MASCOT and Phenyx, T1, T2, and T3 peptide scores were equal to 16, 40, 10 and 5.5, 9.5, 3.5, respectively (P-value