Using Guanidine-Hydrochloride for Fast and Efficient Protein

Nov 28, 2012 - SBiN Lab, Department of Biology, University of Copenhagen, Faculty of Science, ... overnight incubation prior to mass spectrometry anal...
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Technical Note pubs.acs.org/jpr

Using Guanidine-Hydrochloride for Fast and Efficient Protein Digestion and Single-step Affinity-purification Mass Spectrometry Jon W. Poulsen,† Christian T. Madsen,† Clifford Young,† Flemming M. Poulsen,‡,§ and Michael L. Nielsen*,† †

Department of Proteomics, The Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Faculty of Health Sciences, DK-2200 Copenhagen ‡ SBiN Lab, Department of Biology, University of Copenhagen, Faculty of Science, DK-2200 Copenhagen S Supporting Information *

ABSTRACT: Protein digestion is an integral part of the “shotgun” proteomics approach and commonly requires overnight incubation prior to mass spectrometry analysis. Quadruplicate “shotgun” proteomic analysis of whole yeast lysate demonstrated that Guanidine-Hydrochloride (GndHCl) protein digestion can be optimally completed within 30 min with endoprotease Lys-C. No chemical artifacts were introduced when samples were incubated in Gnd-HCl at 95 °C, making Gnd-HCl an appropriate digestion buffer for shotgun proteomics. Current methodologies for investigating protein− protein interactions (PPIs) often require several preparation steps, which prolongs any parallel operation and high-throughput interaction analysis. Gnd-HCl allow the efficient elution and subsequent fast digestion of PPIs to provide a convenient highthroughput methodology for affinity-purification mass spectrometry (AP-MS) experiments. To validate the Gnd-HCl approach, label-free PPI analysis of several GFP-tagged yeast deubiquitinating enzymes was performed. The identification of known interaction partners demonstrates the utility of the optimized Gnd-HCl protocol that is also scalable to the 96 well-plate format. KEYWORDS: protein digestion, shotgun proteomics, guanidine hydrochloride, urea, endoproteinase Lys-C, protein−protein interactions, deubiquitinating enzymes, Saccharomyces cerevisiae

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INTRODUCTION Mass spectrometry (MS)-based proteomics has emerged as a powerful tool for identifying the protein content of complex biological samples.1 The “shotgun” proteomics approach, where all proteins in the sample are proteolytically digested into peptides prior to mass spectrometric detection, has widely become the method of choice for the large-scale identification of proteins, post-translational modifications and protein− protein interactions (PPIs).2,3 For protein digestion, trypsin is the protease of choice as it cleaves C-terminal to arginine and lysine residues with very high specificity.4 Typically the procedure requires long durations (>16 h) to ensure appropriate protein digestion and since large-scale proteomics experiments require a high number of samples to be analyzed,5,6 any reduction in the time spent on sample digestion would increase throughput. Protocols have emerged for quicker protein digestion procedures, but these often require supplementary equipment to perform microwave assisted and high-pressure aided enzymatic digestion.7,8 Other methods using surfactant-aided digestion have demonstrated improved digestion procedures as well,9−11 but these surfactants are generally expensive causing a major concern for large scale proteome analysis. Furthermore, the reported approaches are aimed at improving protein digestion only and are not easily applicable to other areas of proteomics sample © 2012 American Chemical Society

preparation. It would therefore be beneficial to have an efficient sample preparation procedure that allows for rapid protein digestion and for easy implementation in other proteomics areas such as MS-based interaction experiments. Many important cellular processes are carried out by a large number of protein components influenced by their PPIs. In the postgenomic era, PPI analysis has played a key role in untangling protein functions.12 In recent years, identification of PPIs by affinity purification (AP) coupled with mass spectrometry for protein identification (AP-MS) has become the favored method. It has been the basis of comprehensive interaction mapping in various organisms such as Saccharomyces cerevisiae13,14 and Drosophila melanogaster,6 as well as elaborate network studies on ER-associated degradation15 and autophagy.16 However, the AP-MS approach suffers from the presence of unspecific background proteins due to interactions with the affinity matrix.17 Quantitative MS allows the true biological PPIs to be distinguished from background proteins, either with labeling techniques such as Stable Isotope Labeling of Amino Acids in Cell Culture (SILAC)18 or label-free approaches.19 Generally, quantitative AP experiments are performed in parallel, with one Received: September 19, 2012 Published: November 28, 2012 1020

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experiment containing the ‘bait’ protein of interest against another experiment with a negative control. Quantitative assessment of the peptide abundances is then used to determine the identified proteins as true PPIs or unspecific background binders.20 AP-MS experiments have frequently been performed by subjecting AP eluates to electrophoresis on 1D SDS PAGE gels and excising the gel lane into several bands prior to in-gel digestion.3,21 This procedure has ensured that any surfactants used for the efficient elution of both “bait” and “prey” proteins in AP experiments are removed prior to MS sample analysis, as surfactants are often MS incompatible. Although the gel protocol generates MS compatible samples, the entire procedure requires several days of sample preparation22 and the sensitivity of gel-based AP-MS can be limited as the extraction efficiency can be very low (∼20%).23 Approaches that are more sensitive in solution have been previously described, although these methods do not markedly improve throughput as they require overnight digestion and employ sample cleanup steps prior to MS analysis.24,25 In order to minimize sample handling steps in AP-MS, onbead digestion procedure has recently been demonstrated for interaction network analysis.26 Although this study entails an elegant and gel-free AP-MS approach for the analysis of PPIs, the described method required four hours of on-bead predigestion, which was followed by overnight digestion to achieve sufficient proteolysis prior to mass spectrometry analysis. This demonstrates that current AP-MS protocols still require time-consuming sample handling steps, primarily due to the lengthy digestion procedure (>20 h). In comparison, MS analysis of a single AP-MS sample often requires less than two hours of instrument time.20 Any time reduction in AP-MS sample preparation would be particularly useful and increase analytical throughput, considering that comprehensive interaction studies often require several hundreds (or thousands) of AP-MS experiments.6 Here we describe a novel approach for the fast and efficient digestion of proteins, which can also be combined into a rapid method for AP-MS based interaction proteomics. The method entails a single-buffer approach based upon GuanidineHydrochloride (Gnd-HCl) as a combined AP elution and digestion buffer. Although Gnd-HCl is one of the most efficient chaotropic reagents,27 it is not commonly used in proteomics experiments because it hampers trypsin activity at low molar concentrations and precipitates in the presence of SDS.28 Earlier comparisons of different chaotropic reagents have shown that tryptic digestion in Gnd-HCl decreases the number of proteins identified, making Gnd-HCl in combination with trypsin undesirable for protein digestion.29,30 A thorough investigation into whether Gnd-HCl hampers other proteases, such as Endoproteinase Lys-C, has to our knowledge not been previously conducted. We therefore decided to examine whether Gnd-HCl could be used with Lys-C for efficient protein digestion. We find that Gnd-HCl in combination with Lys-C allows for efficient proteome digestion in just 30 min incubation time. The presented results were validated with quadruplicate MS analysis of a Lys-C digested yeast lysate and led to an investigation on whether Gnd-HCl could be employed as a fast and efficient AP-MS protocol. The elution efficiency of Gnd-HCl was compared against commonly employed AP-MS elution buffers and an optimized AP-MS protocol involving Gnd-HCl was tested by conducting interaction proteomics analyses on several yeast Deubiquitinat-

ing enzymes (DUBs). The Gnd-HCl approach allowed AP experiments and the subsequent MS analysis to be conducted in just a few hours, with the protocol easily scalable to a 96 well-plate format.

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EXPERIMENTAL PROCEDURES

Sample Preparation

Wild type yeast and yeast cells with endogenously GFP tagged DUBs were grown in YPD until OD600=0.7, with the pellet resuspended in lysis buffer (75 mM NaCl, 75 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM N-ethylmaleamide (NEM)) and complete protease inhibitor cocktail (Roche, Penzberg, Germany). Protein measurements were conducted using the Bradford Quick-step dye (Bio-Rad, Berkeley, CA) at OD595. For the Gnd-HCl comparison, all yeast lysates were acetone precipitated and redissolved in either 8 M Gnd-HCl or 8 M urea. For assessment of in vitro artifacts, yeast lysates where heated for one hour at 25, 37, 50, 60, 70 and 90 °C respectively prior to reduction with DTT and alkylation with chloroacetamide. 31 Samples were diluted in 25 mM ammonium bicarbonate to a final chaotrope concentration of 2 M. Insolution digestion was conducted overnight at 25 °C with endopeptidase Lys-C before the samples were loaded onto C18 StageTips. For the assessment of digestion efficiency, all investigated yeast lysates were digested with endopeptidase Lys-C in a time course experiment starting from 30 min to overnight digestion at various temperatures. The samples were then loaded onto C18 StageTips and analyzed by MS as described below. Pull-down Assay

The GFP-multitrap 96 well plate (Chromotek, Martinsried, Germany) was initially washed with 200 μL wash buffer (100 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 mM NEM and complete protease inhibitor cocktail). A second wash step was used while shaking for 10 min at 4 °C before the wash buffer was removed. GFP-tagged DUB protein lysates (2.5 mg) were loaded into each well of the plate and the samples were incubated for two hours at 4 °C. The samples were then washed on ice three times with fresh wash buffer. After the final wash step, the supernatant was removed and 50 μL of 8 M Gnd-HCl was added to the washed wells and the plate was heated at 95 °C for 10 min. The plate was spun down and the Gnd-HCl eluate was transferred to a normal 96-well plate. The GFP multitrap was washed with 20 μL of 25 mM ammonium bicarbonate and subsequently pooled with the earlier eluted proteins prior to preparation for MS. Elution Efficiency Assay

A direct comparison was performed to compare the efficiency of Gnd-HCl against other agents used for elution. GFP-trap beads were loaded with 2 mg of yeast lysate and subsequently eluted with Gnd-HCl (95 °C), urea (25 °C) or 2× LDS (95 °C) for 10 min. The efficiency of these elutions were compared by reboiling all the GFP-beads with 2× LDS (95 °C) for another 10 min. The second elution steps were loaded onto a 1D SDS-PAGE gel. After electrophoresis, the gel was subjected to Western blot analysis and the elution efficiency of the reboiling step was used to comparatively measure the initial elution efficiency. For Western blot analysis, mouse anti-GFP (Santa Cruz Biotechnology, Santa Cruz, CA) and mouse anti-βactin (Abcam, Cambridge, UK) antibodies were used. 1021

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Figure 1. (A) Comparison of digestion properties for Gnd-HCl and urea at 25 °C. Digestion in Gnd-HCl at room temperature (RT) for 30 min produces complete digestion as illustrated by the low occurrence of miscleaved peptides. (B) Similar digestion comparison of Gnd-HCl and urea at 37 °C, (C) 50 °C and (D) 60 °C. An increased number of miscleaved peptides confirms that digestion at RT is the most efficient.

Mass Spectrometric Analysis

fragment ion masses. Carbamidomethylation was specified as a fixed modification, with protein N-terminal acetylation and methionine oxidation set as variable modifications. For the analysis of in vitro artifacts, data were additionally searched with either carbamylation or guanidinylation as variable modifications. A maximum of two miscleavages was allowed while strict Lys-C specificity was specified. Peptide assignments were statistically evaluated in a Bayesian model on the basis of sequence length and Andromeda score. Peptides and proteins were only accepted with a false discovery rate (FDR) of less than 1%, which was estimated on the number of accepted reverse hits.

All MS experiments were performed on a nanoflow EASY-nLC system (Proxeon Biosystems, Odense, Denmark) connected to an Orbitrap Q-Exactive equipped with a nanoelectrospray source (Thermo Fisher Scientific, Bremen, Germany). Each peptide sample was loaded by an autosampler and separated on a 15 cm analytical column (75 um inner diameter) packed with 3 um C18 beads (Reprosil Pur-AQ, Dr. Maisch, Germany) with a one hour gradient ranging from 5 to 40% acetonitrile in 0.5% acetic acid. The eluate from the column was subjected to electrospray ionization before the ions were directly introduced into the mass spectrometer. The Q Exactive mass spectrometer was operated in data-dependent acquisition mode and all samples were analyzed using the previously described “fast” acquisition method.26

Label-free Quantification

Label-free quantification was performed with MaxQuant as previously described.20,35 Briefly, signals that were originally zero were imputed with random numbers from a normal distribution, whose mean and standard deviation were chosen to best simulate low abundance values below the noise level (Replace missing values by normal distribution − Width = 0.3; Shift = 1.8). Significant interactors were determined by a volcano plot-based strategy, which combined t test p-values with ratio information. Significance lines in the volcano plot to represent a given FDR (p < 0.01) were determined by a permutation-based method and SO values (line curvature) between 0.5 and 1.0 were used. Each analyzed “bait” protein (Ubp1p, Ubp2p, Ubp3p, Ubp12p, Ubp15p and Bre5p) was quantified against a control pull-down experiment from wildtype yeast.

Identification of Peptides and Proteins by MaxQuant

All raw data analysis was performed with the MaxQuant software suite (www.maxquant.org)32 version 1.2.0.34 supported by the Andromeda search engine (www.andromedasearch.org).33 The data were searched against a concatenated target/decoy34 (forward and reversed) version of the Yeast ORF database containing 6717 protein entries. Protein sequences of common contaminants (such as human keratins and proteases used) were added to the database. We followed the step-by-step protocol of the MaxQuant software suite to generate MS/MS peak lists that were filtered to contain at most six peaks (per 100 Da interval) prior to the Andromeda database search. The mass tolerances for searches were set to a maximum of 7 ppm for peptide masses and 20 ppm for HCD 1022

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Figure 2. (A) Set-up for testing chemical inertness. Quadruplicates of lysates were incubated for one hour at 25, 37, 50, 60, 70 and 90 °C prior to standard proteolytic digest and MS analysis. (B) Comparison of chemical inertness of Gnd-HCl and urea. The number of MS/MS scans did not change despite one hour incubation at different temperatures in either Gnd-HCl (blue bars) or urea (green bars). At elevated temperatures (>25 °C), urea decomposes into isocyanic acid and hampers peptide detection (MS/MS ID rate decreases for urea, red line). In contrast, the MS/MS ID rate increases for Gnd-HCl (purple line), demonstrating that Gnd-HCl does not produce any chemical in vitro artifacts that hampers MS analysis. (C) The chemical inertness of Gnd-HCl does not hamper protein identification (blue bars), while carbamylation of peptides by urea significantly reduces the number of identified proteins (green bars). (D) Number of urea derived modifications (carbamylation) on Lys, Met, Arg and N-termini after incubation at different temperatures. The percentage of modified peptides increase with incubations up to 60 °C, whereas the number of modified peptides decreases at higher temperatures. (E) Number of Gnd-HCl derived modifications (guanidinylation) on Lys and N-termini is at every temperature lower than 0.1% and at all times lower than the 1% FDR. (F) Comparison of elution efficiency of Gnd-HCl, urea and Laemmli (LDS) buffer. GFP-trap beads were first eluted with Gnd-HCl, urea or LDS buffer (not shown due to sample precipitation occurring with LDS and Gnd-HCl). A second elution of the same beads in LDS show Gnd-HCl is a highly efficient elution buffer compared to urea and LDS buffer. 1023

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Technical Note

RESULTS AND DISCUSSION

spanning from the addition of chemical groups (e.g., methylation) to the loss of water leading to structural changes in a protein/peptide. Importantly, in vitro artifacts can mimic true in vivo modifications by having the same mass and chemical composition, which leads to the incorrect identification of artifacts as post-translational modifications.31 To avoid such artifacts, improved digestion and fractionation methods have been established, with the in-solution digest containing chaotropic agents currently the method of choice for preparing samples.41,42 To test the chemical robustness of Gnd-HCl, its performance was compared against urea by incubating yeast lysates for one hour at various temperatures prior to digestion (Figure 2A). Although urea is a denaturant and often used as an elution buffer in immunoprecipitation experiments and for protein digestion,43 it quickly decomposes into isocyanic acid at elevated temperatures, resulting in the carbamylation of peptide amino groups.44 These in vitro peptide modifications increase the overall sample complexity and hamper the dynamic range of proteomic experiments.40 The proteomic analysis demonstrates Gnd-HCl did not cause any obvious chemical artifacts in the analyzed lysates, as shown by the high MS/MS identification rate throughout the temperature experiment (Figure 2B (purple line), and Supporting Information Table 2). In contrast, the MS/MS identification rate for the urea experiments decreased considerably at elevated temperatures, most probably due to urea decomposition and increased peptide carbamylation (Figure 2B (red line), and Supporting Information Tables 5 and 6). A lower MS/MS identification rate in the urea experiments also led to fewer proteins identified at increased temperatures, whereas incubating samples in Gnd-HCl for one hour at 95 °C generally improved the overall number of protein identifications (Figure 2C and Supporting Information Figure 2). To determine whether the decreased number of MS/MS identifications from the urea experiment was caused by in vitro artifacts, the data were searched again with carbamylation as a variable modification on various amino acids (Lys, Met, Arg) and peptide N-termini. It was discovered that the identification rate of carbamylated peptide species increased at elevated temperatures, with 80% of all peptides being modified already at 60 °C (Figure 2D). Interestingly, the percentage of modified peptides decreased at temperatures above 60 °C, while the overall MS/MS identification rate was reduced to below 10% (Figure 2B). This strongly indicates that some peptides may have become multiply carbamylated at higher temperatures, so the data were searched again to allow up to six single carbamylations. At temperatures above 60 °C, a significant drop in peptides harboring one or two carbamylations was observed (Figure 2D). This indicates that multiple carbamylations could be the cause for the lower number of modified peptides identified. Nevertheless, the decreased number of identified peptides at above 60 °C demonstrates that urea hampers proteomic analysis at elevated temperatures. In contrast to the large number of in vitro modified peptides caused by urea, no artifacts were detected with Gnd-HCl use. To validate this observation, the Gnd-HCl data were resubmitted to MaxQuant with the guanidinylation of lysines and peptide N-termini specified as variable modifications as described in Unimod (www.unimod.org45). The results from this search revealed that less than 0.1% of all the identified peptides possessed a guanidinyl modification. Considering that the percentage of guanidinylated peptides is an order of

Digestion Efficiency of Gnd-HCl

Gnd-HCl is a widely used denaturant involved in RNA isolation, globular protein denaturation and protein refolding studies.36,37 Although Gnd-HCl is one of the strongest chaotropes available, it has not been extensively used in proteomics studies because it precipitates in SDS and significantly reduces trypsin activity at concentrations higher than 1 M.28 To investigate whether Gnd-HCl hampers other proteases than trypsin, we decided to determine the digestion efficiency of Lys-C in a 2 M Gnd-HCl buffer. The investigation involved the LC−MS analysis of whole yeast lysates at various digestion time-points (30, 60, 120, 240, 360, 480, and 720 min) and temperatures (25, 37, 50, and 60 °C). Digested yeast lysate samples were analyzed in quadruplicate on a Q Exactive Orbitrap mass spectrometer,38 with control experiments similarly conducted with urea buffer. All experiments were analyzed using a 60 min LC gradient and the comparison required 192 yeast LC−MS experiments for evaluating the Gnd-HCl digestion efficiency in comparison to urea (Supporting Information Table 1). The results revealed that efficient protein digestion was already achieved after 30 min of Lys-C digestion at 25 °C, which was validated by the total number of proteins and peptides identified, coupled with the low occurrence of singly and doubly miscleaved peptides (Figure 1A, Supporting Information Tables 3 and 4). It should be noted that the occurrence of singly and doubly miscleaved peptides after 30 min of Lys-C digestion in Gnd-HCl was significantly lower than any of the urea based digestion steps, demonstrating the high digestion efficiency of the Lys-C/Gnd-HCl approach (Figure 1A,B and Supporting Information Figure 1). In comparison, digestion at elevated temperatures (50 and 60 °C) resulted in more than twice the number of both singly and doubly miscleaved peptides identified throughout the timecourse experiment (Figure 1C,D). Digestion in both Gnd-HCl and urea at 50 and 60 degrees for 720 min leads to a dramatic decrease in identified peptides. This is due to a significantly lower ID rate on the MS/MS scans (data not shown) which most probably could be caused by reduced specificity of Lys-C at elevated temperatures (>37 °C) combined with prolonged digestion. Overall, this results in a larger pool of nonspecific Lys-C peptides, and as a consequence longer digestion times at higher temperatures should be avoided when using either urea or Gnd-HCl. Although comparable efficient digestion was observed at 37 °C, it was decided to conduct all experiments at room temperature for simplicity. Moreover, the use of GndHCl required no additional considerations for optimal digestion efficiency, while the overall performance was on par with previously reported digestion protocols that utilized microwave-assisted and high-pressure-aided protein digestion.7,8 Thus, our experiments demonstrate that the activity of Lys-C is higher in Gnd-HCl than in urea, making Gnd-HCl a suitable reagent for rapid (30 min) protein digestion. These results are in stark contrast to using trypsin in Gnd-HCl, where the activity is reduced.29,30 Chemical Inertness of Gnd-HCl

In proteomics workflows, in vitro artifacts represents a major challenge, with several artifacts previously reported on gel separated proteins.39 Such artifacts compromise the in-gel digestion procedure and impair the sensitivity of proteomics experiments.40 In vitro artifacts can appear in various forms 1024

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Figure 3. Gnd-HCl as single-step component for rapid AP-MS. (A) Experimental setup for rapid AP-MS experiments using a GFP-coated 96-well plate and Gnd-HCl. (B) Investigation of the required amount of lysate needed for proper identification of either GFP-tagged Ubp1p (cyan) or Ubp3p (blue) using the GFP-coated well plate approach. (C) Pearson correlations of the summed peptide intensities between replicate pull-down analyses of Ubp3p using the GFP-coated well plate approach. The strong correlation (Pearson correlation above 0.95) signifies the high reproducibility of the demonstrated Gnd-HCl AP-MS method.

magnitude lower than the peptide 1% FDR rate of the data set, these identifications are most likely to be false-positives. We therefore conclude that Gnd-HCl does not cause in vitro artifacts on proteins and peptides at elevated temperatures.

considerably more efficient in eluting the GFP-bound target proteins and thus more appropriate for the investigation of PPIs (Figure 2F).

Elution Efficiency of Gnd-HCl

Gnd-HCl as a Single-step Component for Rapid AP-MS Experiments

An important aspect of AP-MS is the efficient elution of bound protein-complexes. The elution efficiency of Gnd-HCl was compared to the commonly used denaturing agents urea and SDS/LDS. Because proteins become randomly coiled in 6 M Gnd-HCl,46 it makes it an ideal component for the efficient elution of targeted protein complexes in AP-MS experiments. We performed parallel AP experiments of a GFP-tagged protein and assessed the elution performance of different chaotropic agents. The target protein was bound to GFP-beads (Chromotek, Germany) and subsequently eluted with 8 M Gnd-HCl, 8 M urea or LDS buffer, with the individual elution from each chaotropic agent compared by Western blot. Because Gnd-HCl precipitates in the presence of SDS, it was not possible to make a direct Western blot comparison. Instead, the efficiency of the initial elution was assessed by reboiling the beads with LDS buffer. In this comparison, Gnd-HCl was

Collectively our experiments demonstrate that Gnd-HCl is a suitable sample buffer for both the efficient elution of affinitypurified protein complexes and the subsequent protein digestion stage in a single step approach. To demonstrate that Gnd-HCl allows for an optimized AP-MS sample preparation, the protocol was applied to a biological interaction study of yeast DUBs in a 96 well-plate format (Figure 3A). Yeast was primarily chosen due to the availability of excellent genome-wide resources, such as proteome-wide GFP-tagged gene libraries.47 The GFP-tag has been established as a robust tag for AP-MS experiments and using GFP as the affinity tag permits a direct combination with sophisticated imaging possibilities.48 Interaction partners for the yeast DUBs were expected from the validation experiments as this protein family contains many well-characterized interactors. It is known that Ubp3p requires 1025

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Figure 4. Interaction partners of selected GFP-tagged DUBs. (A) Volcano plot representing results of the label-free pull-down of GFP-tagged Ubp3p. The logarithmic ratio of protein intensities in the Ubp3p/WT pull-downs were plotted against negative logarithmic p-values of the t test performed from quadruplicates. A hyperbolic curve separates specific Ubp3p-interacting proteins from background (red dotted line). The known interactor Bre5p is identified with high confidence and reproducibility. (B) Similar Volcano plot representing results of the label-free pull-down of GFP-tagged Bre5p. Green lines represent known Bre5p and Ubp3p interactors identified from STRING analysis (http://string.embl.de). Combined, these results confirm the reliability and reproducibility of the Gnd-HCl approach. (C) String analysis of the significant interactors for Ubp3p and Bre5p confirms the robustness of the method. (D) Gene Ontology analysis confirms the involvement of Ubp3p in ER-to-Golgi transport, ribophagy and protein deubiquitination.

which are typically present in the yeast cell with 8970 and 2210 protein copies, respectively.50 For each DUB, eight different concentrations were analyzed by GFP pull-downs (50 to 2000 μg) and the required amount of protein was established by comparing the number of MS identified peptides belonging to each target protein as a function of the lysate concentration. A total of 48 parallel GFP pull-down experiments in one GFPcoated well-plate was performed, with the AP-MS analysis revealing that approximately 2000 μg of whole yeast lysate would be required for the lower expressed DUB (Figure 3B). The results also uncovered an important limitation of the labelfree approach with regard to the expression levels of the investigated target candidate. Since the experiments were performed in a limited volume on the GFP-plate (250 μL maximum), the highest amount of whole yeast lysate that could be analyzed was restricted to 2000 ug. Using a higher amount increases the chances of protein aggregation and precipitation, which would hamper the results of any AP-MS experiment. Despite the fact that more than 60% of all expressed yeast genes

the cofactor Bre5p to deubiquitinate substrates within the COPII complex,49 so an effective AP-MS approach should detect Bre5p as a significant PPI for GFP-Ubp3p. As the GndHCl approach allows parallel AP-MS analysis, we decided to investigate the interactome for a number of DUBs. All interaction experiments were performed using label-free quantification, as the available GFP-library does not contain a knockout of the LYS2 gene that typically makes SILAC labeling tenable. Nonetheless, label-free analysis allows for the determination of unspecific background binding proteins20 but requires multiple target and control experiments for strong statistical significance. Lysates of yeast strains expressing endogenously GFP-tagged DUBs47 were individually incubated in a 96 well-plate coated with an anti-GFP antibody. A concentration assay was initially performed to determine the minimum amount of whole yeast lysate required for optimal protein pull-down efficiency. Triplicate pull-down experiments for two differentially expressed yeast DUBs (Ubp1p and Ubp3p) were conducted, 1026

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Figure 5. Interaction partners of selected GFP-tagged DUBs. Identified significant interactors for selected DUBs using the described Gnd-HCl methodology and GFP-coated 96 well-plate. Label-free analysis and Volcano plots for these DUBs can be found in Supporting Information Figure 3. Yeast protein names are given within circles. (A) Direct interactors for Ubp1p, (B) direct interactors for Ubp2p, (C) direct interactors for Ubp12p, (D) direct interactors for Ubp15p, (E) direct interactors for Ubp3p and (F) direct interactors for Bre5p.

are present in the cell with at least 2000 copies,51 the minimum expression level of target candidates should preferentially exceed 1500 copies per cell to produce meaningful PPIs for some yeast DUBs. After establishing the suitable lysate concentration, interaction profiles for several yeast DUBs (Ubp1p, Ubp2p, Ubp3p, Ubp12p, Ubp15p) and the Ubp3p-associated protein Bre5p were performed. A Pearson correlation above 0.95 was observed for the replicates of the GFP-Ubp3p AP-MS experiments, signifying strong reproducibility between experiments (Figure 3C). All pull-down experiments were performed in parallel and were ready for MS analysis within six hours of

laboratory time using the described Gnd-HCl approach, allowing interaction studies to be conducted more rapidly than traditional Western blot procedures.52 The Gnd-HCl pull-down experiments found Bre5p as a strong interaction partner of Ubp3p (Figures 4A, 5E,F and Supporting Information Table 7) and the reversed pull-down experiment (using GFP-Bre5p as the target protein) confirmed Ubp3p as a strong interaction partner of Bre5p (Figure 4B). This is in agreement with the literature describing Bre5p as an important cofactor for Ubp3p. A functional evaluation of identified PPIs from the label-free analysis was performed using the STRING software tool, which reports known and predicted 1027

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protein interactors based upon direct interactions.53 STRING extracts experimental data from databases such as BIND, DIP, GRID, HPRD, IntAct, MINT and PID, as well as curated data from Biocarta, BioCyc, GO, KEGG and Reactome to create a large number of previously reported interactions. We took the identified PPIs for Ubp3p and Bre5p (p < 0.01) and plotted these into STRING to extract known interactions. All the reported interactions between Ubp3p PPIs were plotted on Volcano plots (Figure 4A and B, green lines), which demonstrates the Gnd-HCl protocol allows the identification of known interaction partners. For a more detailed analysis, the entire STRING data set reported for the identified Ubp3p PPIs (Figure 4C) was extracted to perform a Gene Ontology analysis. The results show that the identified PPIs belong to biological processes known to involve Ubp3p, such as ER-toGolgi transportation,49 ribophagy54,55 and protein deubiquitination56 (Figure 4D). In summary, these experiments confirm that Gnd-HCl, in combination with the GFP-tagging strategy, is a reproducible and reliable method for high-throughput interaction proteomics. The Ubp1p pull-down experiment revealed the known protein interactor Ubi4p57 (Supporting Information Figure 3), along with several ribosomal proteins and the suppressor protein Stm1p (Figure 5A). Because ribosomal proteins are highly abundant and formed by tight macromolecular interactions, it is possible that some of the identified proteins are contaminants or secondary interactors. However, interactions between Ubp1p/Ubi4p and the identified ribosomal proteins and Stm1p have previously been reported.58,59 For the Ubp2p pull-down experiment, several rRNA-related proteins were identified as PPIs (Figure 5B). Rsp5p is a HECT E3 ligase known to regulate the nuclear export of rRNA60 and has previously been demonstrated to be antagonized by Ubp2p.61 Our data suggests a novel role for Ubp2p in nuclear rRNA export, which is strengthened by the observation that many significant Ubp2p interactors are involved in the translational machinery or in other closely related cellular functions (ribosome biogenesis, rRNA processing, ribosomal LSU assembly and rRNA maturation). The data also indicate novel roles for Ubp12 in the biosynthesis of secondary metabolites (Figure 5C), as the majority of the identified PPIs are involved in related processes (amino acid metabolism, glycolysis and the biosynthesis of secondary metabolites). Likewise, for Ubp15p several novel interaction partners were identified (Figure 5D). Although confirmation of all observed PPIs by complementary methods would be highly desirable, such biological validation studies are beyond the scope of this report. It is worth noting that the significantly identified PPIs appear in a reproducible manner across the technical replicates, while not being observed across the investigated DUBs. This clearly demonstrates that the identified PPIs are specific to individual DUBs and highly unlikely to result from commonly occurring contaminants.

properties are desirable for the MS analysis of peptides and favorably compare to commonly used denaturing reagents. We have demonstrated that Gnd-HCl is a suitable component for conducting rapid and single-step AP-MS experiments. The described method is not solely limited to AP-MS experiments with GFP-tagged targets since Gnd-HCl allows for an efficient elution of a large variety of available tags (data not shown), and the protocol can be adjusted to encompass other protein interaction approaches such as DNA/ RNA-protein and post-translationally modified protein studies. We examined the capabilities of Gnd-HCl on a number of characterized and uncharacterized DUBs, showing that the approach is able to identify known and novel interactors for the investigated target genes.

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ASSOCIATED CONTENT

S Supporting Information *

Supplementary tables and figures. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest. § Deceased November 9, 2011.

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ACKNOWLEDGMENTS We thank members of the NNF-CPR for fruitful discussions and the careful reading of the manuscript. The work carried out in this study was in part supported by the Novo Nordisk Foundation Center for Protein Research, the Lundbeck Foundation, and by the European Union 7th Framework Programs PRIME-XS, grant agreement number 262067, and EURATRANS, grant agreement number HEALTH-F4-2010241504.

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REFERENCES

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CONCLUSION In conclusion, the use of Gnd-HCl as a chaotropic reagent for proteomic experiments allows the rapid and efficient Lys-C digestion of proteins. The chaotropic property of Gnd-HCl ensures efficient denaturing of protein samples, hereby abolishes the need for adding denaturing surfactants to the sample. The chemical inertness of Gnd-HCl does not seem to introduce any in vitro artifacts during sample preparation. These 1028

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