Investigating the Applicability of Antibodies Generated within the

Article pubs.acs.org/jpr

Investigating the Applicability of Antibodies Generated within the Human Protein Atlas as Capture Agents in Immunoenrichment Coupled to Mass Spectrometry Tove Boström,† Henrik J. Johansson,‡ Janne Lehtiö,‡ Mathias Uhlén,§ and Sophia Hober*,† †

Department of Protein Technology, KTHRoyal Institute of Technology, SE-106 91 Stockholm, Sweden Science for Life Laboratory, Cancer Proteomics Mass Spectrometry, Department of Oncology−Pathology, Karolinska Institute, SE-171 21 Stockholm, Sweden § Science for Life Laboratory, Department of Proteomics, KTHRoyal Institute of Technology, SE-171 21 Stockholm, Sweden ‡

S Supporting Information *

ABSTRACT: For identification and characterization of proteins in complex samples, immunoenrichment coupled to mass spectrometry is a good alternative due to the sensitivity of the affinity enrichment and the specificity of mass spectrometry analysis. Antibodies are commonly used affinity agents; however, for high-throughput analysis, antibody availability is usually a bottleneck. Here we present a protocol for immunoenrichment coupled to mass spectrometry in a high-throughput setup, where all steps from bead coupling to mass spectrometry sample preparation are performed in parallel in a 96-well format. Antibodies generated within the Human Protein Atlas project were tested for applicability as capture agents. The antibodies were covalently attached to protein A beads, making it possible to reuse the coupled beads at least three times without destroying the antibody binding efficiency. Target proteins were captured from a U251 MG cell lysate, eluted, digested, and analyzed using mass spectrometry. Of 30 investigated antibodies, around 50% could successfully capture the corresponding native target protein, making the available library of more than 21 000 antibodies a valuable resource for immunoenrichment assays. Due to the diversity of different antibodies regarding affinity and specificity, analyzing antibodies in a high-throughput format is challenging. Even though protocol optimization for individual antibodies can be advantageous for future studies, our method enables a fast screening strategy to determine the usefulness of antibodies in immunoenrichment setups. In addition, we show that the specificity of the antibodies can be investigated by using label-free quantification. KEYWORDS: immunoenrichment, antibody enrichment, mass spectrometry, MS, label-free quantification, antibody validation, antibody specificity

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INTRODUCTION Many large-scale proteomics studies are currently ongoing with the aim to increase the understanding of the function of our proteins. This is done for example by studying protein interaction networks,1−12 investigating the connection of specific proteins to a certain disease,13−16 or investigating the tissue and subcellular protein localization.13,17−19 As our knowledge about protein function increases, focus is shifted toward developing targeted approaches for proteins of particular interest. This is, however, not always a straightforward process. For example, within the field of biomarker discovery, many potential biomarkers are identified in largescale proteomics studies, but it is difficult to set up robust assays for validation and clinical use.20 Analysis of proteins in complex samples such as cell lysates, tissue extracts, or plasma can generate important information regarding protein function. Detecting disease-related proteins in patient samples is also important in the area of clinical diagnostics. This protein characterization has traditionally been © 2014 American Chemical Society

performed using antibody-based methods, such as enzymelinked immunosorbent assay (ELISA) or antibody arrays, which both provide high sensitivity.21−26 However, the specificity of the assay is dependent on the antibody, and unspecific binding cannot always be distinguished from target−antibody interactions. In addition, no information regarding protein isoforms or interaction partners can be obtained. To obtain high enough specificity, a sandwich assay is preferred,27−29 where binding of two antibodies targeting separate epitopes on the protein of interest is required to obtain a signal output. Mass spectrometry (MS) is another technology widely used for studying proteins.30−32 In MS experiments, identification of peptides and proteins is based on analysis of the amino acid sequence, and hence, the specificity in the identification is very high. Compared to antibody-based methods, the detection limits in MS experiments are higher, although targeted approaches such Received: July 3, 2014 Published: September 18, 2014 4424

dx.doi.org/10.1021/pr500691a | J. Proteome Res. 2014, 13, 4424−4435

Journal of Proteome Research

Article

protein fragments (PrESTs) as internal standards.49 However, immunoenrichment of full-length proteins such as described in the present study also makes it possible to investigate the antibody specificity on a protein level, which is of great importance for a number of different antibody applications. When, for example, doing peptide enrichment, no information can be obtained regarding protein interactions for which enrichment of full-length proteins is necessary. Within the HPA project, more than 21 000 antibodies targeting around 16 500 human proteins have so far been successfully generated. Of these, a substantial number should be possible to use as capture agents for immunoenrichment coupled to mass spectrometry to characterize the corresponding proteins.

as multiple reaction monitoring (MRM or SRM) have been developed to increase sensitivity.33−36 MS furthermore enables the possibility to distinguish different protein isoforms from one another and also detect post-translational modifications. However, high sample complexity is a problem for MS-based proteomics, and a target enrichment step is often necessary in order to identify proteins of low abundance.37 An appealing solution is therefore to combine the specificity of MS with an antibody capture step to increase the sensitivity. This approach has been applied in different setups, where the antibody capture step is placed either before tryptic digestion to capture fulllength proteins38−45 or after digestion to enrich tryptic peptides46−49 or using both protein and peptide immunoenrichment in a two-step setup.50,51 For capture of full-length proteins, the following tryptic digestion can either be done directly on the beads, in solution after an elution step, or in-gel after electrophoretic separation. The latter option is convenient, as analyzing only a small part of the gel can decrease the degree of contaminating proteins. However, this option can potentially lower the sensitivity and reproducibility by introducing additional steps. It is also very time-consuming, and for highthroughput applications, one of the two first options would be preferable. If the aim is to study protein interactions, a good strategy is to use tagged proteins, where the gene of interest is first transfected into the target cell line and expressed as a tagprotein fusion by the machinery of the cell.52−54 This enables a streamlined protocol where the same antitag antibody or other affinity molecule can be used in all experiments. This is a great advantage, as the availability of antibodies suitable for immunoenrichment is usually a limiting factor. By using very mild washing conditions during the capture step, even weakly interacting proteins may be detected. Usually, a bead-based system is used, where affinity molecules are coupled to microspheres. This introduces the issue of background binding, as proteins tend to stick to the beads. However, nonspecific interactions with the beads can often be distinguished from specific ones by including relevant controls and adequate data analysis protocols.55−59 Different antibodies also tend to behave differently, which makes a high-throughput approach with primary antitarget antibodies more challenging than when using anti-tag antibodies. However, when using tagged proteins, there is always the issue of whether the bait protein will behave equally to the endogenous untagged protein. It is also impossible to apply the tagged protein strategy in patient sample analysis. It would therefore be desirable to develop robust, high-throughput systems for immunoenrichment coupled to MS using target-specific antibodies. However, due to the obstacles explained above, high-throughput studies using primary antitarget antibodies are rare, and earlier studies have used laborious gel-based methods for sample preparation prior to MS analysis.38,39 Here we present a 96-well format protocol for enrichment of target proteins using antitarget antibodies followed by in solution digestion and MS analysis. To test the developed protocol we used both antibodies generated within the Human Protein Atlas (HPA) project60 and monoclonal antibodies. In total, 30 monoclonal and polyclonal antibodies were used to enrich target proteins using lysates from the glioblastoma cell line U251 MG. We recently showed that HPA antibodies can be used for peptide immunoenrichment, where the immunoenrichment is performed after tryptic digestion. This enables absolute protein quantification using heavy-isotope-labeled

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EXPERIMENTAL SECTION

Antibody Generation and Selection

The rabbit polyclonal antibodies used were generated within the HPA project. Antigens of 50−150 amino acids (protein epitope signature tags; PrESTs) were used for immunization61 and sera containing the resulting polyclonal antibody mixture were purified on columns with immobilized antigen.62 Data from experiments using validated polyclonal antibodies is available on the HPA Web site (www.proteinatlas.org). Binding specificity of the polyclonal antibodies to the corresponding target proteins was determined using Western blot (WB), PrEST arrays, and immunohistochemistry (IHC).63−65 The polyclonal antibodies chosen for this study were selected randomly from the HPA antibody library. All included antibodies generated strong bands of correct molecular weight from a U251 MG cell lysate in WB; however, some of the antibodies also generated additional bands. mRNA transcript levels as determined by RNA sequencing varied with FPKM values of 0−168; however, this information was not taken into consideration in the selection process. This data is also available on the HPA Web site. Murine monoclonal antibodies were obtained from Atlas Antibodies (Atlas Antibodies, Stockholm, Sweden), of which half are currently commercially available (AMAb90655, AMAb90616, AMAb90663, AMAb90664, and AMAb90710). Monoclonal antibodies targeting the same protein were generated using the same PrEST antigen; however, these antibodies represent separate antibody clones with affinity to different epitopes. The criteria for these antibodies were the same as for the polyclonal antibodies and all chosen antibodies generated a band of correct molecular weight in WB analysis of a U251 MG cell lysate. In total, 10 monoclonal antibodies (mAb1−mAb10) and 20 polyclonal antibodies (pAb1−pAb20) were chosen. Cell Cultivation and Lysate Preparation

U251 MG cells were cultivated in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich, St. Louis, MO) with 10% fetal bovine serum (Sigma-Aldrich). A trypsin−EDTA solution (SigmaAldrich) was used to release the cells from the culture dish. Cells were counted and frozen at −80 °C. Tubes were thawed on ice and the cells were lyzed with 200 μL of lysis buffer (50 mM Tris, 150 mM NaCl, 2 mM EDTA, 1% Nonidet P40 pH 8) supplemented with protease inhibitor cocktail (Sigma-Aldrich) per 10 million cells. The sample was incubated at 4 °C for 30 min before cell debris was removed by centrifugation at 13 000 rpm for 15 min and subsequent filtration through a 0.45 μm filter. 4425



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Bead Coupling and Immunoenrichment

downloaded February 2014). The contaminant database available together with MaxQuant was also used in the search. Carbamidomethylation of cysteines was chosen as a fixed modification and methionine oxidation and N-terminal acetylation as variable modifications. A maximum of two missed cleavages was allowed, and identified peptides were required to consist of at least six amino acids. The false discovery rate was set to 0.01, and the MaxLFQ algorithm, implemented in the MaxQuant software, was used for label-free quantification (LFQ) with a LFQ minimum ratio count of 2. The match between runs setting was used with a retention time window of 1 min. The mass tolerance for precursors was set to 4.5 and 20 ppm for fragment ions. The output file proteingroups.txt was processed in the Perseus software (www.perseus-framework.org). Contaminants, reverse sequences, and proteins “only identified by site” were removed from the list of identified proteins. Replicates were grouped together, and only proteins identified in at least two out of the three replicates in at least one group were kept for further analysis. LFQ intensities were logarithmized (base 2), and data imputation was performed to replace missing values with intensity values in the background level. This was done with the “replace missing values by normal distribution” option in Perseus (width = 0.3, shift = 1.8).69 In order to distinguish enriched proteins from nonspecifically interacting proteins, independent two-sample t-tests were performed. Logarithmized LFQ intensities from each group of immunoenrichment replicates were compared to the corresponding LFQ intensities from a control value. The intensity values in the control consisted of median LFQ intensities from all samples. Separate control values were used for the monoclonal and polyclonal antibodies.

Wells in a filter plate (Millipore, Bedford, MA) were washed twice with 150 μL of phosphate-buffered saline (PBS; 10 mM NaP, 150 mM NaCl, pH 7.3) before 20 μL of protein A-coated sepharose bead slurry (GE Healthcare, Uppsala, Sweden) was added. The plate was positioned on a vacuum manifold (Millipore) during washing. All incubations were performed at room temperature on a plate shaker. The beads were washed twice with 150 μL of PBS and incubated with 10 μg of antibody in 50 μL of PBS for 1 h. Bound antibodies were cross-linked to the beads with 100 μL of 10 mM dimethyl pimelimidate (DMP) for 1 h. The cross-linking reaction was stopped with 100 μL of 50 mM ethanol amine, and antibodies not crosslinked were eluted with 2 × 100 μL of elution buffer [200 mM formic acid (FA), pH 2.2] with 3 min incubation. The beads were washed with 4 × 150 μL of wash buffer 1 (named WB1; 50 mM Tris, 150 mM NaCl, 1% Nonidet P40, 5% glycerol pH 8) before 200 μL of lysate (corresponding to 10 million cells) was added to each well. The plate was incubated for 2 h. The beads were washed with 150 μL of WB1 and 150 μL of wash buffer 2, (named WB2; 0.5 M NaCl). This was performed twice before the beads were washed with 2 × 150 μL of desalting buffer (20 mM NH4HCO3). Target proteins were eluted with 2 × 100 μL elution buffer with 3 min incubation. MS Sample Preparation

Eluted samples were dried in a vacuum centrifuge and proteins were resolved in denaturation buffer (6 M urea, 100 mM NH4HCO3). Proteins were reduced with 10 mM dithiothreitol for 45 min at room temperature and alkylated with 55 mM iodoacetamide for 30 min at room temperature. The samples were diluted with 100 mM NH4HCO3 to reach a urea concentration of 1.5 M. The samples were digested at room temperature overnight by the addition of 100 ng of trypsin (Sigma-Aldrich). The digestion reaction was stopped by addition of TFA to a concentration of 0.4% (v/v) in water. Peptide samples were desalted using C18 StageTips as described previously66 and dried in a vacuum centrifuge.

Western Blot Analysis

The U251 MG lysates before and after immunoenrichment and the samples eluted from the beads were run on SDS−PAGE gels (Invitrogen, Carlsbad, CA) and transferred to 0.45 μm PVDF membranes (Invitrogen). Transferred proteins were visualized with Ponceau staining (Sigma-Aldrich). Membranes were blocked in blocking buffer (5% dry milk, 0.5% Tween 20) for 1 h at room temperature. Membranes were incubated with primary antibodies (the same antibodies as in immunoenrichment) diluted 1:2500 in blocking buffer for 1 h at room temperature and washed 4 × 5 min with washing buffer (0.1 M Tris-HCl, 0.5 M NaCl, 0.1% Tween 20). A goat anti-rabbit antibody conjugated to horseradish peroxidase or a goat antimouse antibody conjugated to horseradish peroxidase (Dako, Glostrup, Denmark) was used as secondary reagent for the polyclonal and monoclonal antibodies, respectively. The antibody was diluted 1:2500 in blocking buffer and added to the membranes. After 1 h incubation at room temperature and subsequent washing, substrate (Millipore) was added to the membranes, and signals were detected with a CCD camera (Bio-Rad Laboratories, Hercules, CA).

MS Analysis

The samples were resuspended in 3% acetonitrile (ACN), 0.1% FA (v/v) in water before injection onto a HPLC column. The total sample volume was 8 μL, out of which 3 μL was injected. An Agilent 1200 nano-LC system was used for peptide separation, where peptides were first trapped on a Zorbax 300SB-C18 column (Agilent, Santa Clara, CA) and then separated on a NTCC-360/100-5-153 C18 stationary phase column (5 μm C18 beads, 100 μm internal diameter, 150 mm length, Nikkyo Technos. Co., Ltd., Tokyo, Japan). A gradient of 6−40% ACN in 15 min was used and the flow rate was 0.4 μL/ min. MS analysis was performed on a Q Exactive instrument (Thermo Fischer Scientific, San Jose, CA). The instrument was operated in a data-dependent manner, and five precursor ions were selected for fragmentation by higher energy collisional dissociation for each full MS scan. MS spectra were generated between 300 and 1700 m/z at 70,000 resolution for full scan MS spectra and 17,500 resolution for MS fragment ion spectra. Precursors were isolated with a width of 2 m/z and subsequently excluded for 50 s.

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RESULTS AND DISCUSSION The combination of immunoenrichment and subsequent MS analysis is very useful for identification of proteins in complex samples, as it combines the sensitivity obtained from the antibody-based target enrichment with the specificity of the MS analysis. Here we present a high-throughput method for identification of target proteins in cell lysates. First, antibodies covalently bound to protein A beads are used to capture the

Data Evaluation

Data analysis was performed using the MaxQuant software (version 1.4.1.2)67 with the built-in search engine Andromeda68 and the human database from Uniprot (www.uniprot.org, 4426



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Figure 1. Workflow for the immunoenrichment/mass spectrometry method. Antibodies are cross-linked to protein A beads. The beads are used for immunoenrichment of target proteins from a cell lysate. After elution, proteins are digested, desalted, and analyzed on a nano-LC−MS system. The MS data are evaluated, and significantly enriched proteins are identified.

corresponding target proteins in a lysate from U251 MG glioblastoma cells. After several washing steps, bound proteins are eluted and digested in solution with trypsin. The tryptic peptides are thereafter analyzed using MS, and peak intensities from samples and controls are compared to determine which proteins were specifically enriched by the different antibodies. The whole workflow of the process can be seen in Figure 1. Both monoclonal and polyclonal antibodies were tested with a dual aim: (1) to validate antibody specificity and (2) to investigate their potential as capture reagents in immunoenrichment experiments before MS detection. Immunoenrichment in a Filter-Plate Format

Antibodies were coupled to protein A-coated sepharose beads in a 96-well filter plate. The filter plate format enables fast and simple washing of the beads by using a vacuum manifold to efficiently remove buffer through the filters while leaving the beads in the wells. It also enables 96 immunoenrichment experiments to be performed in parallel. As the aim of this project was to identify antibody targets and by that determine specificity, extensive washing was performed to exclude as many proteins interacting nonspecifically with the beads or antibodies as possible. Washing was performed with salt (0.5 M NaCl), detergent (1% Nonidet P-40), and glycerol (5%) to minimize the unspecific binding. Since detergent was used during the washing steps, a buffer exchange step with 20 mM NH4HCO3 was included to hinder the detergent from interfering with the MS analysis. After the antibody capture step, the complexity of the eluted sample is significantly decreased (Figure 2A), enabling short HPLC gradients prior to MS analysis. The extensive washing reduces the amount of contaminating proteins further, and a 15 min nano-LC gradient is sufficient for peptide separation. This can be compared to protocols using milder washing conditions, where the larger sample complexity requires the use of longer gradients.1,2 In protocols where in-gel digestion of captured proteins is employed, several different gel slices are usually analyzed, which also increases the MS analysis time.38,39,70 The relatively short MS analysis time presented here is a requirement if a high-throughput method is desired. However, harsh washing conditions are not optimal in all applications. As mentioned above, the aim of this project was to identify specific interactions between antibody and target

Figure 2. Verification of immunoenrichment by two antibodies using Western blotting. (A) Unspecific protein staining of a PVDF membrane and (B) WB analysis with target-specific antibodies. pAb5 targets CHCHD3 and pAb9 targets G3BP2. Lane assignments: (1, 4, and 7) lysate before immunoenrichment; (2, 5, and 8) lysate after immunoenrichment; (3, 6, and 9) sample eluted from beads; (M) molecular weight marker. Theoretical molecular weights of the different splice variants of CHCHD3 are 6.8, 15.6, 26.2, 26.7, and 27.7 kDa. Molecular weights for G3BP2 are 2.4, 6.5, 6.7, 9, 11.1, 11.4, 12.5, 13.1, 14.1, 16, 22.5, 50.8, and 54.1 kDa.

protein. If, in addition, proteins that interact with the target protein are of interest, less-harsh washing buffers and fewer washing steps would be advisible. In total, 30 antibodies (10 monoclonal and 20 polyclonal) were used in the immunoenrichment experiments. All antibodies were generated by immunization using PrEST proteins. The target proteins are presented in Table 1, along with data from RNA sequencing experiments available on www.proteinatlas. org. In total, target proteins from seven of the 10 monoclonal antibodies (70%) were successfully identified in at least two out of the three replicate samples. For the 20 polyclonal antibodies, nine target proteins were detected in the MS analysis in at least two out of three replicates (45%). One additional target protein was detected in only one of the three replicates. 4427



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Table 1. Information about the Target Proteins for the 30 Antibodiesa gene

protein name

AMACR ANLN AQP4 ARL3 BTN3A3 CAAP1 CALD1 CES2 CHCHD3 CTCF DAZAP1 ETFB FOXK1 G3BP2 GCC1 GORAB MPHOSPH8 NSRP1 OXCT1 P4HA2 PPID RBM3 STX7 TFAP2A

α-methylacyl-CoA racemase anillin, actin binding protein aquaporin 4 ADP-ribosylation factor-like 3 butyrophilin, subfamily 3, member A3 caspase activity and apoptosis inhibitor 1 caldesmon 1 carboxylesterase 2 coiled-coil−helix−coiled-coil−helix domain containing 3 CCCTC-binding factor (zinc finger protein) DAZ associated protein 1 electron-transfer-flavoprotein, beta polypeptide Forkhead box K1 GTPase activating protein (SH3 domain) binding protein 2 GRIP and coiled-coil domain containing 1 golgin, RAB6-interacting M-phase phosphoprotein 8 nuclear speckle splicing regulatory protein 1 3-oxoacid CoA transferase 1 prolyl 4-hydroxylase, alpha polypeptide II peptidylprolyl isomerase D RNA binding motif (RNP1, RRM) protein 3 syntaxin 7 transcription factor AP-2 alpha (activating enhancer binding protein 2 alpha) znc finger, C3HC-type containing 1

ZC3HC1 a

protein class enzymes, potential transmembrane proteins − potential transmembrane proteins, transporters − potential transmembrane proteins − potential transmembrane proteins enzymes, potential transmembrane proteins potential transmembrane proteins transcription factors − − transcription factors − − − − − enzymes enzymes, potential transmembrane proteins enzymes potential transmembrane proteins potential transmembrane proteins potential transmembrane proteins, transcription factors −

U-251 MG (fpkm) 8.5 209 0 9.2 3.9 12 168 8.1 80 21 149 33 6.8 57 9.8 11 12 47 33 35 42 2256 27 17 21

The table contains gene name, protein name, protein class, and RNA levels based on RNA sequencing.

Figure 3. Intensity of target proteins in the three immunoenrichment replicates. Protein intensities are logged (base 2) for target proteins from each of the 30 antibodies.

Identification of Target Proteins

When using polyclonal antibodies, which are not renewable like monoclonal antibodies, it is especially important to decrease the required amount of antibody. Here, 10 μg of antibody is used for each immunoenrichment experiment; however, the coupled beads are reused for all replicates, which dramatically decreases the amount of antibody needed. As is demonstrated in Figure 3, there is not a decrease in the intensities of the target proteins across the three replicates, which shows that the coupled beads are stable for repetitive use.

The results from the immunoenrichment experiments are summarized in Table 2, including the number of replicates where the target proteins were detected. For the polyclonal antibodies, additional data from the HPA are also presented. In the HPA project the antibodies were used in IHC experiments on formalin-fixed, paraffin-embedded tissue sections (47 different tissue types and 20 different cancer forms). When comparing the results shown in Table 2, there is no correlation 4428



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Table 2. List of Investigated Antibodies and Corresponding Target Proteinsa antibody

antibody ID

gene

IHC validation scoreb

MS identification (no. of replicates)

no. of identified peptides in MS

mAb1 mAb2 mAb3 mAb4 mAb5 mAb6 mAb7 mAb8 mAb9 mAb10 pAb1 pAb2 pAb3 pAb4 pAb5 pAb6 pAb7 pAb8 pAb9 pAb10 pAb11 pAb12 pAb13 pAb14 pAb15 pAb16 pAb17 pAb18 pAb19 pAb20

AMAb90655 AMAb90657 AMAb90659 AMAb90661 AMAb90656 AMAb90616 AMAb90663 AMAb90664 AMAb90709 AMAb90710 HPA019369 HPA017330 HPA020404 HPA007904 HPA042935 HPA017998 HPA027208 HPA004201 HPA018304 HPA019527 HPA014784 HPA036292 HPA018897 HPA040035 HPA015593 HPA012047 HPA019692 HPA028850 HPA024023 HPA018921

RBM3 RBM3 ANLN ANLN RBM3 STX7 CTCF CTCF P4HA2 P4HA2 GCC1 CALD1 CAAP1 BTN3A3 CHCHD3 FOXK1 GORAB DAZAP1 G3BP2 AMACR AQP4 ARL3 CES2 MPHOSPH8 NSRP1 OXCT1 PPID TFAP2A ZC3HC1 ETFB

uncertain uncertain supportive supportive uncertain supportive supportive supportive supportive supportive supportive supportive uncertain uncertain uncertain supportive uncertain supportive supportive supportive supportive supportive supportive supportive supportive supportive supportive supportive supportive supportive

3/3 3/3 3/3 3/3 3/3 3/3 2/3 0/3 0/3 0/3 0/3 3/3 0/3 0/3 3/3 3/3 3/3 2/3 3/3 0/3 0/3 0/3 0/3 0/3 3/3 0/3 0/3 3/3 1/3 3/3

14 13 17 17 14 9 1 0 0 0 0 111 0 0 9 21 5 1 4 0 0 0 0 0 17 0 0 1 1 2

a IHC validation scores are presented for all antibodies. A supportive validation score indicates that results from the antibody validation correlate well with available gene data and/or that two antibodies targeting the same gene exist that show similar staining patterns. The number of replicates in which the target protein was detected in the mass spectrometry analysis is shown, as well as the number of identified unique peptides for each target protein. bData for polyclonal antibodies are available at www.proteinatlas.org.

would further decrease the leakage; however, since this agent is nonspecific, a larger antibody fraction would be inaccessible to the antigen, hence lowering the effective target binding capacity. The antibody pAb9 targets the protein G3BP2. Thirteen different molecular weights are reported for this protein, and the most intense band on the WB corresponds well to one of these isoforms. Several other bands are present in the lysate before and after immunoenrichment but not in the eluted sample. These bands possibly demonstrate cross reactivity of the antibody that only occurs when the antibody is exposed to proteins not in their native state. The context dependence of the performance of antibodies has earlier been discussed in Ä lgenäs et al.71 For the proteins that were not identified in the MS analysis there are, apart from the possibility that the antibody does not recognize the protein of interest, several other probable explanations: The target protein (1) is not abundant in the chosen cell line and below the limit of detection, (2) interacts weakly with the antibody and is therefore released in the extensive washing steps, or (3) is inaccessible to the antibody, for instance embedded in a cell membrane or involved in large protein complexes hiding the antibody epitope, or (4) the recognition sequence of the antibody, the epitope, is located in the interior of the protein, enabling binding of the antibody only to a denatured target protein. Table 1 shows that RNA levels of identified proteins in

between antibody IHC validation score and the result from immunoenrichment of native proteins. The lack of correlation between IHC and immunoenrichment could be due to the sample preparation, which for IHC includes harsh denaturing conditions,63 while the immunoenrichment is milder, potentially leaving the proteins in a more nativelike state. Western blotting (WB) verifies this difference and is exemplified by the results for two antibodies in Figure 2B. WB samples were collected from the lysate used in the immunoenrichment, the lysate after immunoenrichment, and the sample eluted from the beads. The same antibodies were used in immunoenrichment and WB. The antibody pAb5 targets the protein CHCHD3. The WB results show that this antibody gives rise to several bands, two of which are most intense. The molecular weights of these proteins correlate well with two of the isoforms of CHCHD3. However, in the lane containing the sample eluted from the beads, only one of these bands is present. These data support the notion that an antibody can have different recognition patterns when presented to a lysate containing proteins in their native or a denatured state. The 38 kDa band, in the sample eluted from the beads, may originate from leakage of antibody not covalently attached to protein A on the beads. The antibody immobilization protocol was optimized to minimize antibody leakage while antibody activity is still preserved. Applying a larger amount of cross-linking agent 4429



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Figure 4. Immunoenrichment−nano-LC−MS identification of target proteins. Volcano plots from immunoenrichment−mass spectrometry experiments using 14 different antibodies. Logarithmized LFQ intensity ratios from triplicate experiments are plotted against the negative logarithmic t-test p-values. The thresholds were set to log2(ratio) > 4 and p = 0.01 as indicated by the gray lines. Significantly enriched proteins appear in the upper right corner. Significantly enriched target proteins are dark blue, and significantly enriched proteins other than the target protein are light blue (probable interaction partners or proteins to which the antibody possibly shows cross-reactivity, CHCHD6 in pAb5, FOXK2 in pAb6, and TFG in pAb9) or light pink (specifically enriched proteins where the type of interaction is unknown). Significantly enriched proteins identified in the majority of the samples are dark pink. For pAb5 and pAb20, the target proteins were not significantly enriched; however, they can be identified just below the threshold values. These proteins (CHCHD3 and ETFB) are gray in the plots. See Table 3 and Supplementary Table 1 (Supporting Information) for more information.

alternative to acquire more efficient capture of the target protein is by increasing the amount of lysate for the potentially low abundant proteins, with the cost of identifying more proteins that bind to the matrix in a nonspecific manner. In these cases the washing procedure could be optimized for each specific case and thereby increase the success rate.

general are higher than for the undetected proteins, indicating that these proteins are more abundant in the U251 MG cell line. Increasing the antibody amount coupled to the beads would probably shift the equilibrium for the antibody antigen interaction, possibly leading to a larger amount of captured antigen and identification of the targeted protein. Another 4430



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However, different antibodies cannot be expected to behave equally; therefore, the threshold values were set to be strict. With lower threshold values, several proteins were identified as significant that were in fact captured by a majority of the antibodies/beads but to a different extent in the different samples. But even when using high threshold values, for most comparisons, additional proteins apart from the target proteins were identified as significantly enriched [log2(ratio) > 4 and p < 0.01]. These proteins correspond to either specific interaction partners of the target protein, proteins other than the target protein interacting specifically with the antibody, or proteins interacting nonspecifically with the antibody or the beads. Identified proteins that were significantly enriched in one sample but that were identified in a majority of the samples were considered false positives. Other proteins were negatively enriched [log2(ratio) < −4 and p < 0.01] in some samples. These proteins are probably common unspecific interaction partners that have been more efficiently removed in the washing steps in these samples compared to the majority of the samples. The probability of the significantly enriched proteins interacting specifically with the target protein was determined by looking at previously determined interaction networks. Sequence similarities between the identified proteins were also investigated to determine the possible cross-reactivity of the antibody. The monoclonal antibodies only enriched their corresponding target protein (Figure 4, mAb1−mAb6). A negative enrichment of HSPE1 [heat shock 10 kDa protein 1 (chaperonin 10)] was observed for mAb2; however, heat shock proteins are common contaminants found in immunoenrichment experiments,10 and HSPE1 was identified in the majority of the samples. For the polyclonal antibodies a larger number of proteins were identified as significantly enriched in the different samples. Since these antibodies recognize multiple epitopes, as compared to the monoclonal antibodies targeting a single epitope, this difference in specificity is not unexpected. Many of these were, like HSPE1, detected in the majority of the samples, but with different intensities, and can therefore be classified as unspecific interactors that can be excluded from the analysis (for example, RBM14, RPL35, SH3BGRL3, DYNLRB1, and DYNLRB2). However, some proteins could be distinguished as specific for a certain antibody. For example, pAb2 enriches the protein MAP7D1 (MAP 7 domain containing 1) apart from its target CALD1 (caldesmon 1). CALD1 is involved in smooth muscle and nonmuscle contraction, whereas not much is known about the function of MAP7D1, and therefore, it would be of interest to investigate if these proteins interact within the cell. The polyclonal antibody pAb5 targets CHCHD3, a mitochondrial protein involved in mitochondrial function and cristae integrity. CHCHD3 was, however, identified just below the threshold values (see Figure 4, pAb5), and the antibody only significantly enriched the protein CHCHD6. Studies of these proteins have shown that they interact with each other72,73 and therefore enrichment of both proteins is expected. However, when comparing the antigen sequence (75 amino acids) that was used for generation of this antibody against the CHCHD6 protein sequence, a stretch of 24 amino acids with 63% sequence identity was observed, and therefore, antibody cross reactivity cannot be ruled out. The polyclonal antibody pAb6 enriched nine different proteins, one of these being the target protein FOXK1 (Forkhead box K1). FOXK1 is a transcriptional regulator that has been found to be involved in

In total, 11 proteins (corresponding to 14 antibodies) are classified as potential transmembrane proteins (Table 1) and five of these (corresponding to six antibodies) were not identified in MS. Membrane proteins should be more difficult to enrich; however, the number of potential transmembrane proteins is not increased in the group of unidentified proteins compared to the group of identified proteins, and hence, firm conclusions are difficult to draw. Even though target-specific optimization of the immunoenrichment protocol would probably increase the success rate to some extent, it is likely that some of the antibodies are not suitable for immunoenrichment experiments due to inaccessible epitopes. The antigens within the HPA project (PrESTs) are chosen on the basis of low sequence similarity to other proteins. Special consideration is taken to avoid putative transmembrane regions; however, since many protein structures are still unsolved, there is no criteria for surface location and it is therefore unlikely that all HPA antibodies target epitopes at the protein surfaces. Determining Antibody Specificity

To determine the specificity of the different antibodies, labelfree quantification was performed. Proteins interacting specifically with the antibodies were separated from unspecific interactors by comparing protein signal intensities (LFQ intensities from MaxQuant) between samples. Median LFQ intensities from all samples (monoclonal and polyclonal antibodies separately) were used as control. This strategy was chosen instead of using a control sample with unconjugated beads or beads conjugated with a control antibody, as all contaminants would not likely be present in such a control sample. With this setup, the chance that a nonspecifically interacting protein would be identified as a contaminant is increased, since the median signal intensity from a large number of samples instead of the signals from one or a few control samples is used, resulting in a strategy resembling the CRAPome approach.59 Signal intensities were logarithmized and compared, and missing values were replaced by imputation. A two-sided, unpaired t-test was performed, where signal intensities from each immunoenrichment group (three replicates) were compared to those of the control group. The negative logarithms of the p-values from the t-test were plotted against the logarithmized ratios, and cutoff values of log2(ratio) = 4 (16-fold enrichment) and p = 0.01 were used to separate target proteins from proteins interacting in a nonspecific manner. Of the 17 identified target proteins, LFQ intensities were obtained for 14 (six monoclonal and eight polyclonal antibodies) in at least two out of three replicates (pAb8, pAb19, and mAb7 were excluded). Of the 14 target proteins, 12 were significantly enriched in the samples compared to the control intensities. Volcano plots with −log10(p-value) plotted against log2(ratio) for the 14 antibodies are shown in Figure 4 (target proteins marked with dark blue). The two remaining target proteins, CHCHD3 and ETFB, were not identified as significant with the chosen threshold values (proteins are marked with gray in Figure 4, pAb5 and pAb20). t-Test statistics of enriched proteins are presented in Supplementary Table 1 of the Supporting Information. Since unspecific interactions with the constant antibody regions should be equal or similar between the sample groups and the control, ratios for these proteins should theoretically appear in the background around log2(ratio) = 0. This should be the case even if the signal intensities of peptides originating from these proteins are significantly higher than the target peptides. 4431



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Table 3. Proteins Significantly Enriched by the Different Antibodies antibody mAb1 mAb2 mAb3 mAb4 mAb5 mAb6 pAb2 pAb5 pAb6

pAb7

pAb9

protein name

comment

RBM3 RBM3 ANLN ANLN RBM3 STX7 CALD1 MAP7D1 CHCHD6

RNA binding motif (RNP1, RRM) protein 3 RNA binding motif (RNP1, RRM) protein 3 anillin, actin binding protein anillin, actin binding protein RNA binding motif (RNP1, RRM) protein 3 syntaxin 7 caldesmon 1 MAP7 domain containing 1 coiled-coil−helix−coiled-coil−helix domain containing 6

SNRPD3 SS18 SRSF1 SS18L1 FOXK1 FOXK2

small nuclear ribonucleoprotein D3 polypeptide 18 kDa synovial sarcoma translocation, chromosome 18 serine/arginine-rich splicing factor 1 synovial sarcoma translocation gene on chromosome 18-like 1 Forkhead box K1 Forkhead box K2

UBAP2 LSM14A PKP4 TCEAL3; TCEAL5 ;TCEAL6 GORAB SS18 PAGR1 COA4 TFG

ubiquitin associated protein 2 LSM14A, SCD6 homologue A (S. cerevisiae) plakophilin 4 transcription elongation factor A (SII)-like 3, 5, and 6

pAb18

G3BP2 DYNLRB1; DYNLRB2 ATPIF1 NSRP1 CSRP1 CSRP2 TFAP2A

pAb20

RBM14

pAb15

a

enriched proteina

golgin, RAB6-interacting synovial sarcoma translocation, chromosome 18 PAXIP1 associated glutamate-rich protein 1 cytochrome c oxidase assembly factor 4 homologue (S. cerevisiae) TRK-fused gene GTPase activating protein (SH3 domain) binding protein 2 dynein, light chain, roadblock-type 1 and 2 ATPase inhibitory factor 1 nuclear speckle splicing regulatory protein 1 cysteine and glycine-rich protein 1 cysteine and glycine-rich protein 2 transcription factor AP-2 alpha (activating enhancer binding protein 2 alpha) RNA binding motif protein 14

mAb1 target mAb2 target mAb3 target mAb4 target mAb5 target mAb6 target pAb2 target specifically enriched by pAb2 specifically enriched by pAb5 - interaction partner to CHCHD3 identified in the majority of the samples identified in the majority of the samples identified in the majority of the samples identified in the majority of the samples pAb6 target specifically enriched by pAb6; possible antibody crossreactivity specifically enriched by pAb6 identified in the majority of the samples specifically enriched by pAb6 specifically enriched by pAb7 pAb7 target identified in the majority of the samples specifically enriched by pAb7 specifically enriched by pAb7 specifically enriched by pAb9; potential binding partner of G3BP2 pAb9 target identified in the majority of the samples identified in the majority of the samples pAb15 target identified in the majority of the samples identified in the majority of the samples pAb18 target identified in the majority of the samples

Target proteins are showed in bold and proteins identified in the majority of the samples are shown in italic.

promoting muscle progenitor cell proliferation and inhibiting myogenic differentiation.74 One of the additional identified proteins was FOXK2, a member of the same subgroup of Forkhead transcription factors as FOXK1. The antigen sequence (142 amino acids) used for antibody generation contains a stretch of 61 amino acids showing 48% sequence similarity with FOXK2. This indicates that the antibody is likely to be cross-reactive against FOXK2. Some of the other enriched proteins are involved in gene transcription (SS18, SS18L1) and pre-mRNA splicing (LSM14A, SNRPD3, SRSF1). However, these proteins were identified in the majority of the other samples as well, indicating that these interactions are unspecific. Two other identified proteins that seem to be specific for pAb6 are PKP4, a possible component of adhesion plaques, and UBAP2, which is involved in ubiquitination. Regarding the use of pAb6 in immunoenrichment experiments, it can be concluded that a more efficient washing protocol could potentially lead to higher reliability in the achieved data. Antibody pAb7 significantly enriched its target protein GORAB but also captured the proteins PAGR1, COA4, and TCEAL3, -5, and/or -6. GORAB is a member of the golgin family and may be involved in the secretory pathway. None of the

additionally enriched proteins are known to interact with GORAB or have any sequence similarities that would indicate cross-reactivity of the antibody. It is therefore not possible to verify the specificity of the antibody on the basis of this data. The polyclonal antibody pAb9 enriches TFG in addition to its target G3BP2. G3BP2 has been shown to interact with NF-κB complexes and be involved in the NF-κB signaling cascade.75 TFG also has been proposed to be involved in the NF-κB pathway,76 explaining the specific enrichment of this protein by the antibody targeting G3BP2. The polyclonal antibody pAb15 targets the RNA-binding protein NSRP1, which was significantly enriched in the samples. The antibody also enriched CSRP1 and CSRP2, two members of the cysteine-rich protein (CRP) family, as well as ATPIF1, mitochondrial ATPase inhibitor protein. CSRP1 and CSRP2 have a role in promoting protein assembly along the actin filaments of the cytoskeleton and are known to interact with one another.77 However, no interaction with NSRP1 has been reported, and CSRP1 and CSRP2 were identified in the majority of samples, indicating that they are unspecific interactors. Results from the label-free quantification evaluation for all 14 proteins are summarized in Table 3. 4432


Journal of Proteome Research Another strategy to analyze this type of data is to simply subtract all proteins identified in control samples (enrichment with a control antibody) from the enriched samples. This would, however, not be optimal here, as several of the target proteins were found in several of the other samples, although at a much lower level. For example, the protein CALD1 was found in many of the samples, but the intensity increased more than 1000-fold in the samples using the polyclonal antibody targeting CALD1 for enrichment. In the MS analysis, equal volumes of all samples were injected, regardless of the amount of target peptides present within each sample. This resulted in some observed carry-over, where peptides gave rise to signals in the next sample(s). The signals from the carry-overs could be clearly identified due to the systematic decrease in intensities across the samples. This was observed especially for the proteins CALD1 and RBM3, which also represent some of the highest RNA levels (see Table 1). Carry-over could increase ion suppression and generate misleading peptide intensities in following samples. However, since carry-over can be easily identified, the latter issue should not be very problematic to deal with. Injecting a smaller volume of these samples or including a more extensive wash of the column between the samples could easily solve the problem. However, optimizing sample injection and column washing for each sample is not convenient in a high-throughput setup but would of course be in focus when further optimizing individual antibodies for use in immunoenrichment experiments. If carry-over peptides can be easily identified, removal of this data is a straightforward process, which will not disturb the data evaluation.

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REFERENCES

(1) Paul, F. E.; Hosp, F.; Selbach, M. Analyzing protein−protein interactions by quantitative mass spectrometry. Methods 2011, 54, 387−395. (2) Hubner, N. C.; Bird, A. W.; Cox, J.; Splettstoesser, B.; Bandilla, P.; Poser, I.; Hyman, A.; Mann, M. Quantitative proteomics combined with BAC TransgeneOmics reveals in vivo protein interactions. J. Cell Biol. 2010, 189, 739−754. (3) Kaake, R. M.; Wang, X.; Huang, L. Profiling of protein interaction networks of protein complexes using affinity purification and quantitative mass spectrometry. Mol. Cell Proteomics 2010, 9, 1650− 1665. (4) Trinkle-Mulcahy, L. Resolving protein interactions and complexes by affinity purification followed by label-based quantitative mass spectrometry. Proteomics 2012, 12, 1623−1638. (5) Urbas, L.; Brne, P.; Gabor, B.; Barut, M.; Strlic, M.; Petric, T. C.; Strancar, A. Depletion of high-abundance proteins from human plasma using a combination of an affinity and pseudo-affinity column. J. Chromatogr. A 2009, 1216, 2689−2694. (6) Butland, G.; Peregrin-Alvarez, J. M.; Li, J.; Yang, W.; Yang, X.; Canadien, V.; Starostine, A.; Richards, D.; Beattie, B.; Krogan, N.; Davey, M.; Parkinson, J.; Greenblatt, J.; Emili, A. Interaction network containing conserved and essential protein complexes in Escherichia coli. Nature 2005, 433, 531−537. (7) Cai, B.; Wang, H.; Zheng, H.; Wang, H. Detection of protein complexes from affinity purification/mass spectrometry data. BMC Syst. Biol. 2012, 6 (Suppl 3), S4. (8) Ewing, R. M.; Chu, P.; Elisma, F.; Li, H.; Taylor, P.; Climie, S.; McBroom-Cerajewski, L.; Robinson, M. D.; O’Connor, L.; Li, M.; Taylor, R.; Dharsee, M.; Ho, Y.; Heilbut, A.; Moore, L.; Zhang, S.; Ornatsky, O.; Bukhman, Y. V.; Ethier, M.; Sheng, Y.; Vasilescu, J.; Abu-Farha, M.; Lambert, J. P.; Duewel, H. S.; Stewart, I. I.; Kuehl, B.; Hogue, K.; Colwill, K.; Gladwish, K.; Muskat, B.; Kinach, R.; Adams, S. L.; Moran, M. F.; Morin, G. B.; Topaloglou, T.; Figeys, D. Large-scale mapping of human protein−protein interactions by mass spectrometry. Mol. Syst. Biol. 2007, 3, 89. (9) Gavin, A. C.; Bosche, M.; Krause, R.; Grandi, P.; Marzioch, M.; Bauer, A.; Schultz, J.; Rick, J. M.; Michon, A. M.; Cruciat, C. M.; Remor, M.; Hofert, C.; Schelder, M.; Brajenovic, M.; Ruffner, H.; Merino, A.; Klein, K.; Hudak, M.; Dickson, D.; Rudi, T.; Gnau, V.; Bauch, A.; Bastuck, S.; Huhse, B.; Leutwein, C.; Heurtier, M. A.; Copley, R. R.; Edelmann, A.; Querfurth, E.; Rybin, V.; Drewes, G.; Raida, M.; Bouwmeester, T.; Bork, P.; Seraphin, B.; Kuster, B.; Neubauer, G.; Superti-Furga, G. Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature 2002, 415, 141−147. (10) Gingras, A. C.; Gstaiger, M.; Raught, B.; Aebersold, R. Analysis of protein complexes using mass spectrometry. Nat. Rev. Mol. Cell Biol. 2007, 8, 645−654. (11) Ho, Y.; Gruhler, A.; Heilbut, A.; Bader, G. D.; Moore, L.; Adams, S. L.; Millar, A.; Taylor, P.; Bennett, K.; Boutilier, K.; Yang, L.; Wolting, C.; Donaldson, I.; Schandorff, S.; Shewnarane, J.; Vo, M.; Taggart, J.; Goudreault, M.; Muskat, B.; Alfarano, C.; Dewar, D.; Lin, Z.; Michalickova, K.; Willems, A. R.; Sassi, H.; Nielsen, P. A.; Rasmussen, K. J.; Andersen, J. R.; Johansen, L. E.; Hansen, L. H.; Jespersen, H.; Podtelejnikov, A.; Nielsen, E.; Crawford, J.; Poulsen, V.; Sorensen, B. D.; Matthiesen, J.; Hendrickson, R. C.; Gleeson, F.; Pawson, T.; Moran, M. F.; Durocher, D.; Mann, M.; Hogue, C. W.; Figeys, D.; Tyers, M. Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry. Nature 2002, 415, 180−183. (12) Hirst, J.; Borner, G. H.; Edgar, J.; Hein, M. Y.; Mann, M.; Buchholz, F.; Antrobus, R.; Robinson, M. S. Interaction between AP-5

CONSCLUSIONS In summary, a high-throughput workflow for immunoenrichment coupled to mass spectrometry has been developed. In total, target proteins for 17 out of 30 antibodies (57%) were successfully detected in MS analysis after antibody capture. This means that it is likely that a large portion of the HPA antibodies, in fact, binds their native target, even though the antigen sequences were not selected on the basis of surface location. Many of the HPA antibodies should therefore be possible to use as capture agents in immunoenrichment experiments. In addition, the presented workflow can provide information regarding antibody specificity. The presented method could also be modified to enable analysis of protein interactions; however, protocol optimization would be needed where the washing steps are decreased in order to preserve weak but specific interactions. ASSOCIATED CONTENT

S Supporting Information *

Supplementary Table 1, which summarizes the t-test statistics of enriched proteins from the label-free quantification. This material is available free of charge via the Internet at http:// pubs.acs.org.

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ACKNOWLEDGMENTS

This work was founded by the Knut and Alice Wallenberg foundation.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +46-8- 790 87 94. Fax: +46 8-553 784 81. Notes

The authors declare no competing financial interest. 4433



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and the hereditary spastic paraplegia proteins SPG11 and SPG15. Mol. Biol. Cell 2013, 24, 2558−2569. (13) Asplund, A.; Edqvist, P. H.; Schwenk, J. M.; Ponten, F. Antibodies for profiling the human proteomeThe Human Protein Atlas as a resource for cancer research. Proteomics 2012, 12, 2067− 2077. (14) Eckhardt, B. L.; Francis, P. A.; Parker, B. S.; Anderson, R. L. Strategies for the discovery and development of therapies for metastatic breast cancer. Nat. Rev. Drug Discovery 2012, 11, 479−497. (15) Kumar, S.; Mohan, A.; Guleria, R. Biomarkers in cancer screening, research and detection: Present and future: A review. Biomarkers 2006, 11, 385−405. (16) Hudler, P.; Kocevar, N.; Komel, R. Proteomic approaches in biomarker discovery: New perspectives in cancer diagnostics. Sci. World J. 2014, 2014, 260348. (17) Uhlen, M.; Bjorling, E.; Agaton, C.; Szigyarto, C. A.; Amini, B.; Andersen, E.; Andersson, A. C.; Angelidou, P.; Asplund, A.; Asplund, C.; Berglund, L.; Bergstrom, K.; Brumer, H.; Cerjan, D.; Ekstrom, M.; Elobeid, A.; Eriksson, C.; Fagerberg, L.; Falk, R.; Fall, J.; Forsberg, M.; Bjorklund, M. G.; Gumbel, K.; Halimi, A.; Hallin, I.; Hamsten, C.; Hansson, M.; Hedhammar, M.; Hercules, G.; Kampf, C.; Larsson, K.; Lindskog, M.; Lodewyckx, W.; Lund, J.; Lundeberg, J.; Magnusson, K.; Malm, E.; Nilsson, P.; Odling, J.; Oksvold, P.; Olsson, I.; Oster, E.; Ottosson, J.; Paavilainen, L.; Persson, A.; Rimini, R.; Rockberg, J.; Runeson, M.; Sivertsson, A.; Skollermo, A.; Steen, J.; Stenvall, M.; Sterky, F.; Stromberg, S.; Sundberg, M.; Tegel, H.; Tourle, S.; Wahlund, E.; Walden, A.; Wan, J.; Wernerus, H.; Westberg, J.; Wester, K.; Wrethagen, U.; Xu, L. L.; Hober, S.; Ponten, F. A human protein atlas for normal and cancer tissues based on antibody proteomics. Mol. Cell Proteomics 2005, 4, 1920−1932. (18) Lundberg, E.; Uhlen, M. Creation of an antibody-based subcellular protein atlas. Proteomics 2010, 10, 3984−3996. (19) Geiger, T.; Velic, A.; Macek, B.; Lundberg, E.; Kampf, C.; Nagaraj, N.; Uhlen, M.; Cox, J.; Mann, M. Initial quantitative proteomic map of 28 mouse tissues using the SILAC mouse. Mol. Cell Proteomics 2013, 12, 1709−1722. (20) Anderson, N. L. The clinical plasma proteome: A survey of clinical assays for proteins in plasma and serum. Clin Chem. 2010, 56, 177−185. (21) Ayoglu, B.; Haggmark, A.; Neiman, M.; Igel, U.; Uhlen, M.; Schwenk, J. M.; Nilsson, P. Systematic antibody and antigen-based proteomic profiling with microarrays. Expert Rev. Mol. Diagn 2011, 11, 219−234. (22) Engvall, E.; Perlmann, P. Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immunochemistry 1971, 8, 871−874. (23) Fulton, R. J.; McDade, R. L.; Smith, P. L.; Kienker, L. J.; Kettman, J. R., Jr. Advanced multiplexed analysis with the FlowMetrix system. Clin. Chem. 1997, 43, 1749−1756. (24) Kingsmore, S. F. Multiplexed protein measurement: Technologies and applications of protein and antibody arrays. Nat. Rev. Drug Discovery 2006, 5, 310−320. (25) Schwenk, J. M.; Gry, M.; Rimini, R.; Uhlen, M.; Nilsson, P. Antibody suspension bead arrays within serum proteomics. J. Proteome Res. 2008, 7, 3168−3179. (26) Borrebaeck, C. A.; Wingren, C. Design of high-density antibody microarrays for disease proteomics: Key technological issues. J. Proteomics 2009, 72, 928−935. (27) Wiese, R.; Belosludtsev, Y.; Powdrill, T.; Thompson, P.; Hogan, M. Simultaneous multianalyte ELISA performed on a microarray platform. Clin Chem. 2001, 47, 1451−1457. (28) Fan, F.; Shen, H.; Zhang, G.; Jiang, X.; Kang, X. Chemiluminescence immunoassay based on microfluidic chips for alpha-fetoprotein. Clin. Chim. Acta 2014, 431C, 113−117. (29) Nielsen, U. B.; Geierstanger, B. H. Multiplexed sandwich assays in microarray format. J. Immunol Methods 2004, 290, 107−120. (30) Aebersold, R.; Mann, M. Mass spectrometry-based proteomics. Nature 2003, 422, 198−207.

(31) Domon, B.; Aebersold, R. Mass spectrometry and protein analysis. Science 2006, 312, 212−217. (32) Gillette, M. A.; Carr, S. A. Quantitative analysis of peptides and proteins in biomedicine by targeted mass spectrometry. Nat. Methods 2013, 10, 28−34. (33) Picotti, P.; Aebersold, R. Selected reaction monitoring-based proteomics: Workflows, potential, pitfalls and future directions. Nat. Methods 2012, 9, 555−566. (34) Anderson, L.; Hunter, C. L. Quantitative mass spectrometric multiple reaction monitoring assays for major plasma proteins. Mol. Cell Proteomics 2006, 5, 573−588. (35) Rafalko, A.; Dai, S.; Hancock, W. S.; Karger, B. L.; Hincapie, M. Development of a chip/chip/SRM platform using digital chip isoelectric focusing and LC−chip mass spectrometry for enrichment and quantitation of low abundance protein biomarkers in human plasma. J. Proteome Res. 2012, 11, 808−817. (36) Shi, T.; Fillmore, T. L.; Sun, X.; Zhao, R.; Schepmoes, A. A.; Hossain, M.; Xie, F.; Wu, S.; Kim, J. S.; Jones, N.; Moore, R. J.; PasaTolic, L.; Kagan, J.; Rodland, K. D.; Liu, T.; Tang, K.; Camp, D. G., 2nd; Smith, R. D.; Qian, W. J. Antibody-free, targeted massspectrometric approach for quantification of proteins at low picogram per milliliter levels in human plasma/serum. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 15395−15400. (37) Makawita, S.; Diamandis, E. P. The bottleneck in the cancer biomarker pipeline and protein quantification through mass spectrometry-based approaches: Current strategies for candidate verification. Clin. Chem. 2010, 56, 212−222. (38) Malovannaya, A.; Lanz, R. B.; Jung, S. Y.; Bulynko, Y.; Le, N. T.; Chan, D. W.; Ding, C.; Shi, Y.; Yucer, N.; Krenciute, G.; Kim, B. J.; Li, C.; Chen, R.; Li, W.; Wang, Y.; O’Malley, B. W.; Qin, J. Analysis of the human endogenous coregulator complexome. Cell 2011, 145, 787− 799. (39) Malovannaya, A.; Li, Y.; Bulynko, Y.; Jung, S. Y.; Wang, Y.; Lanz, R. B.; O’Malley, B. W.; Qin, J. Streamlined analysis schema for highthroughput identification of endogenous protein complexes. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 2431−2436. (40) Brockman, A. H.; Orlando, R. Probe-immobilized affinity chromatography/mass spectrometry. Anal. Chem. 1995, 67, 4581− 4585. (41) Kiernan, U. A.; Tubbs, K. A.; Gruber, K.; Nedelkov, D.; Niederkofler, E. E.; Williams, P.; Nelson, R. W. High-throughput protein characterization using mass spectrometric immunoassay. Anal. Biochem. 2002, 301, 49−56. (42) Berna, M. J.; Zhen, Y.; Watson, D. E.; Hale, J. E.; Ackermann, B. L. Strategic use of immunoprecipitation and LC/MS/MS for tracelevel protein quantification: Myosin light chain 1, a biomarker of cardiac necrosis. Anal. Chem. 2007, 79, 4199−4205. (43) Schwertman, P.; Bezstarosti, K.; Laffeber, C.; Vermeulen, W.; Demmers, J. A.; Marteijn, J. A. An immunoaffinity purification method for the proteomic analysis of ubiquitinated protein complexes. Anal. Biochem. 2013, 440, 227−236. (44) Neiman, M.; Fredolini, C.; Johansson, H.; Lehtio, J.; Nygren, P. A.; Uhlen, M.; Nilsson, P.; Schwenk, J. M. Selectivity analysis of single binder assays used in plasma protein profiling. Proteomics 2013, 13, 3406−3410. (45) Falk, R.; Ramstrom, M.; Eriksson, C.; Uhlen, M.; Wernerus, H.; Hober, S. Targeted protein pullout from human tissue samples using competitive elution. Biotechnol. J. 2011, 6, 28−37. (46) Anderson, N. L.; Anderson, N. G.; Haines, L. R.; Hardie, D. B.; Olafson, R. W.; Pearson, T. W. Mass spectrometric quantitation of peptides and proteins using stable isotope standards and capture by anti-peptide antibodies (SISCAPA). J. Proteome Res. 2004, 3, 235−244. (47) Warren, E. N.; Jiang, J.; Parker, C. E.; Borchers, C. H. Absolute quantitation of cancer-related proteins using an MS-based peptide chip. Biotechniques 2005, Suppl, 7−11. (48) Hoofnagle, A. N.; Becker, J. O.; Wener, M. H.; Heinecke, J. W. Quantification of thyroglobulin, a low-abundance serum protein, by immunoaffinity peptide enrichment and tandem mass spectrometry. Clin Chem. 2008, 54, 1796−1804. 4434



Article

(49) Edfors, F.; Bostrom, T.; Forsstrom, B.; Zeiler, M.; Johansson, H.; Lundberg, E.; Hober, S.; Lehtio, J.; Mann, M.; Uhlen, M. Immunoproteomics using polyclonal antibodies and stable isotope labeled affinity-purified recombinant proteins. Mol. Cell Proteomics 2014, 13, 1611−1624. (50) Neubert, H.; Muirhead, D.; Kabir, M.; Grace, C.; Cleton, A.; Arends, R. Sequential protein and peptide immunoaffinity capture for mass spectrometry-based quantification of total human beta-nerve growth factor. Anal. Chem. 2013, 85, 1719−1726. (51) Palandra, J.; Finelli, A.; Zhu, M.; Masferrer, J.; Neubert, H. Highly specific and sensitive measurements of human and monkey interleukin 21 using sequential protein and tryptic peptide immunoaffinity LC−MS/MS. Anal. Chem. 2013, 85, 5522−5529. (52) Chen, G. I.; Gingras, A. C. Affinity-purification mass spectrometry (AP−MS) of serine/threonine phosphatases. Methods 2007, 42, 298−305. (53) de Boer, E.; Rodriguez, P.; Bonte, E.; Krijgsveld, J.; Katsantoni, E.; Heck, A.; Grosveld, F.; Strouboulis, J. Efficient biotinylation and single-step purification of tagged transcription factors in mammalian cells and transgenic mice. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 7480−7485. (54) Rigaut, G.; Shevchenko, A.; Rutz, B.; Wilm, M.; Mann, M.; Seraphin, B. A generic protein purification method for protein complex characterization and proteome exploration. Nat. Biotechnol. 1999, 17, 1030−1032. (55) Boulon, S.; Ahmad, Y.; Trinkle-Mulcahy, L.; Verheggen, C.; Cobley, A.; Gregor, P.; Bertrand, E.; Whitehorn, M.; Lamond, A. I. Establishment of a protein frequency library and its application in the reliable identification of specific protein interaction partners. Mol. Cell Proteomics 2010, 9, 861−879. (56) Varjosalo, M.; Sacco, R.; Stukalov, A.; van Drogen, A.; Planyavsky, M.; Hauri, S.; Aebersold, R.; Bennett, K. L.; Colinge, J.; Gstaiger, M.; Superti-Furga, G. Interlaboratory reproducibility of largescale human protein-complex analysis by standardized AP−MS. Nat. Methods 2013, 10, 307−314. (57) Tackett, A. J.; DeGrasse, J. A.; Sekedat, M. D.; Oeffinger, M.; Rout, M. P.; Chait, B. T. I-DIRT, a general method for distinguishing between specific and nonspecific protein interactions. J. Proteome Res. 2005, 4, 1752−1756. (58) Armean, I. M.; Lilley, K. S.; Trotter, M. W. Popular computational methods to assess multiprotein complexes derived from label-free affinity purification and mass spectrometry (AP−MS) experiments. Mol. Cell Proteomics 2013, 12, 1−13. (59) Mellacheruvu, D.; Wright, Z.; Couzens, A. L.; Lambert, J. P.; StDenis, N. A.; Li, T.; Miteva, Y. V.; Hauri, S.; Sardiu, M. E.; Low, T. Y.; Halim, V. A.; Bagshaw, R. D.; Hubner, N. C.; Al-Hakim, A.; Bouchard, A.; Faubert, D.; Fermin, D.; Dunham, W. H.; Goudreault, M.; Lin, Z. Y.; Badillo, B. G.; Pawson, T.; Durocher, D.; Coulombe, B.; Aebersold, R.; Superti-Furga, G.; Colinge, J.; Heck, A. J.; Choi, H.; Gstaiger, M.; Mohammed, S.; Cristea, I. M.; Bennett, K. L.; Washburn, M. P.; Raught, B.; Ewing, R. M.; Gingras, A. C.; Nesvizhskii, A. I. The CRAPome: A contaminant repository for affinity purification−mass spectrometry data. Nat. Methods 2013, 10, 730−736. (60) Uhlen, M.; Oksvold, P.; Fagerberg, L.; Lundberg, E.; Jonasson, K.; Forsberg, M.; Zwahlen, M.; Kampf, C.; Wester, K.; Hober, S.; Wernerus, H.; Bjorling, L.; Ponten, F. Towards a knowledge-based human protein atlas. Nat. Biotechnol. 2010, 28, 1248−1250. (61) Berglund, L.; Bjorling, E.; Jonasson, K.; Rockberg, J.; Fagerberg, L.; Al-Khalili Szigyarto, C.; Sivertsson, A.; Uhlen, M. A whole-genome bioinformatics approach to selection of antigens for systematic antibody generation. Proteomics 2008, 8, 2832−2839. (62) Nilsson, P.; Paavilainen, L.; Larsson, K.; Odling, J.; Sundberg, M.; Andersson, A. C.; Kampf, C.; Persson, A.; Al-Khalili Szigyarto, C.; Ottosson, J.; Bjorling, E.; Hober, S.; Wernerus, H.; Wester, K.; Ponten, F.; Uhlen, M. Towards a Human Proteome Atlas: High-throughput generation of mono-specific antibodies for tissue profiling. Proteomics 2005, 5, 4327−4337. (63) Algenas, C.; Agaton, C.; Fagerberg, L.; Asplund, A.; Bjorling, L.; Bjorling, E.; Kampf, C.; Lundberg, E.; Nilsson, P.; Persson, A.; Wester,

K.; Ponten, F.; Wernerus, H.; Uhlen, M.; Ottosson Takanen, J.; Hober, S. Antibody performance in western blot applications is contextdependent. Biotechnol J. 2014, 9, 435−445. (64) Uhlen, M.; Hober, S. Generation and validation of affinity reagents on a proteome-wide level. J. Mol. Recognit. 2009, 22, 57−64. (65) Paavilainen, L.; Wernerus, H.; Nilsson, P.; Uhlen, M.; Hober, S.; Wester, K.; Ponten, F. Evaluation of monospecific antibodies: A comparison study with commercial analogs using immunohistochemistry on tissue microarrays. Appl. Immunohistochem. Mol. Morphol. 2008, 16, 493−502. (66) Rappsilber, J.; Mann, M.; Ishihama, Y. Protocol for micropurification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat. Protoc. 2007, 2, 1896−1906. (67) Cox, J.; Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 2008, 26, 1367−1372. (68) Cox, J.; Neuhauser, N.; Michalski, A.; Scheltema, R. A.; Olsen, J. V.; Mann, M. Andromeda: A peptide search engine integrated into the MaxQuant environment. J. Proteome Res. 2011, 10, 1794−1805. (69) Deeb, S. J.; D’Souza, R. C.; Cox, J.; Schmidt-Supprian, M.; Mann, M. Super-SILAC allows classification of diffuse large B-cell lymphoma subtypes by their protein expression profiles. Mol. Cell Proteomics 2012, 11, 77−89. (70) Mousson, F.; Kolkman, A.; Pijnappel, W. W.; Timmers, H. T.; Heck, A. J. Quantitative proteomics reveals regulation of dynamic components within TATA-binding protein (TBP) transcription complexes. Mol. Cell Proteomics 2008, 7, 845−852. (71) Algenas, C.; Agaton, C.; Fagerberg, L.; Asplund, A.; Bjorling, L.; Bjorling, E.; Kampf, C.; Lundberg, E.; Nilsson, P.; Persson, A.; Wester, K.; Ponten, F.; Wernerus, H.; Uhlen, M.; Ottosson Takanen, J.; Hober, S. Antibody performance in western blot applications is contextdependent. Biotechnol J. 2014, 9, 435−445. (72) Xie, J.; Marusich, M. F.; Souda, P.; Whitelegge, J.; Capaldi, R. A. The mitochondrial inner membrane protein mitofilin exists as a complex with SAM50, metaxins 1 and 2, coiled-coil−helix coiled-coil− helix domain-containing protein 3 and 6 and DnaJC11. FEBS Lett. 2007, 581, 3545−3549. (73) An, J.; Shi, J.; He, Q.; Lui, K.; Liu, Y.; Huang, Y.; Sheikh, M. S. CHCM1/CHCHD6, novel mitochondrial protein linked to regulation of mitofilin and mitochondrial cristae morphology. J. Biol. Chem. 2012, 287, 7411−7426. (74) Shi, X.; Wallis, A. M.; Gerard, R. D.; Voelker, K. A.; Grange, R. W.; DePinho, R. A.; Garry, M. G.; Garry, D. J. Foxk1 promotes cell proliferation and represses myogenic differentiation by regulating Foxo4 and Mef2. J. Cell Sci. 2012, 125, 5329−5337. (75) Prigent, M.; Barlat, I.; Langen, H.; Dargemont, C. IκBα and IκBα/NF-κB complexes are retained in the cytoplasm through interaction with a novel partner, RasGAP SH3-binding protein 2. J. Biol. Chem. 2000, 275, 36441−36449. (76) Matsuda, A.; Suzuki, Y.; Honda, G.; Muramatsu, S.; Matsuzaki, O.; Nagano, Y.; Doi, T.; Shimotohno, K.; Harada, T.; Nishida, E.; Hayashi, H.; Sugano, S. Large-scale identification and characterization of human genes that activate NF-κB and MAPK signaling pathways. Oncogene 2003, 22, 3307−3318. (77) Weiskirchen, R.; Gunther, K. The CRP/MLP/TLP family of LIM domain proteins: Acting by connecting. BioEssays 2003, 25, 152− 162.

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