Advertisement

Revealing Novel Telomere Proteins Using in Vivo Cross-linking, Tandem Affinity Purification, and Label-free Quantitative LC-FTICR-MS*

  • Thalia Nittis
    Affiliations
    Departments of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110-1093
    Search for articles by this author
  • Lionel Guittat
    Footnotes
    Affiliations
    Departments of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110-1093
    Search for articles by this author
  • Richard D. LeDuc
    Affiliations
    Departments of Medicine, and Washington University School of Medicine, St. Louis, Missouri 63110-1093
    Search for articles by this author
  • Ben Dao
    Affiliations
    Departments of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110-1093
    Search for articles by this author
  • Julien P. Duxin
    Affiliations
    Departments of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110-1093
    Search for articles by this author
  • Henry Rohrs
    Affiliations
    Departments of Chemistry, Washington University School of Medicine, St. Louis, Missouri 63110-1093
    Search for articles by this author
  • R. Reid Townsend
    Correspondence
    To whom correspondence may be addressed: Dept. of Cell Biology and Physiology, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110-1093. Tel.: 314-362-7709; Fax: 314-362-9136;
    Affiliations
    Departments of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110-1093

    Departments of Medicine, and Washington University School of Medicine, St. Louis, Missouri 63110-1093
    Search for articles by this author
  • Sheila A. Stewart
    Correspondence
    A Sidney Kimmel Scholar. To whom correspondence may be addressed: Dept. of Cell Biology and Physiology, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110-1093. Tel.: 314-362-7437; Fax: 314-362-7463;
    Affiliations
    Departments of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110-1093

    Departments of Medicine, and Washington University School of Medicine, St. Louis, Missouri 63110-1093
    Search for articles by this author
  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grant R21 AG02532 from the NIA (to S. A. S.) and Grants P41RR000954 and UL1RR024992 from the National Center for Research Resources and from the NCI, P30 CA91842. This work was also supported by the Sidney Kimmel Foundation for Cancer Research, the Edward Mallinckrodt, Jr. Foundation, the W. M. Keck Foundation, and the Siteman Cancer Center and by institutional resources to the Proteomics Center at Washington University (to R. R. T.).
    This article contains supplemental Table 1, which reports the masses (observed and theoretical) and MASCOT scores of the peptide sequences for all identified proteins, including common contaminants such as keratins, trypsin, immunoglobulins, and BSA.
    § Present address: INSERM U978 Adaptateurs de Signalisation en Hematologie UFR-SMBH Universite Paris 13 74 rue Marcel Cachin 93017 Bobigny cedex, France.
Open AccessPublished:January 22, 2010DOI:https://doi.org/10.1074/mcp.M900490-MCP200
      Telomeres are DNA-protein structures that protect chromosome ends from the actions of the DNA repair machinery. When telomeric integrity is compromised, genomic instability ensues. Considerable effort has focused on identification of telomere-binding proteins and elucidation of their functions. To date, protein identification has relied on classical immunoprecipitation and mass spectrometric approaches, primarily under conditions that favor isolation of proteins with strong or long lived interactions that are present at sufficient quantities to visualize by SDS-PAGE. To facilitate identification of low abundance and transiently associated telomere-binding proteins, we developed a novel approach that combines in vivo protein-protein cross-linking, tandem affinity purification, and stringent sequential endoprotease digestion. Peptides were identified by label-free comparative nano-LC-FTICR-MS. Here, we expressed an epitope-tagged telomere-binding protein and utilized a modified chromatin immunoprecipitation approach to cross-link associated proteins. The resulting immunoprecipitant contained telomeric DNA, establishing that this approach captures bona fide telomere binding complexes. To identify proteins present in the immunocaptured complexes, samples were reduced, alkylated, and digested with sequential endoprotease treatment. The resulting peptides were purified using a microscale porous graphite stationary phase and analyzed using nano-LC-FTICR-MS. Proteins enriched in cells expressing HA-FLAG-TIN2 were identified by label-free quantitative analysis of the FTICR mass spectra from different samples and ion trap tandem mass spectrometry followed by database searching. We identified all of the proteins that constitute the telomeric shelterin complex, thus validating the robustness of this approach. We also identified 62 novel telomere-binding proteins. These results demonstrate that DNA-bound protein complexes, including those present at low molar ratios, can be identified by this approach. The success of this approach will allow us to create a more complete understanding of telomere maintenance and have broad applicability.
      Numerous redundant systems exist to maintain the genome and ensure proper segregation of genetic material upon cellular division. Elucidation of the molecular mechanisms that constitute these systems is an area of intense inquiry. In model systems, elegant genetic approaches have been used extensively to identify proteins and interrogate their role in these mechanisms. Unfortunately, mammalian systems are refractory to similar approaches, and thus protein identification has relied heavily on homology searches and mass spectrometry. For this reason, the development of isolation procedures and refined mass spectrometric approaches capable of identifying proteins within large protein complexes, including those present as transient interactors and in substoichiometric quantities, is an important area of research. Previous studies have successfully utilized quantitative proteomics with stable isotopic peptide labeling to identify specific components of cellular macromolecular complexes by affinity purification (
      • Ranish J.A.
      • Yi E.C.
      • Leslie D.M.
      • Purvine S.O.
      • Goodlett D.R.
      • Eng J.
      • Aebersold R.
      The study of macromolecular complexes by quantitative proteomics.
      ,
      • Blagoev B.
      • Kratchmarova I.
      • Ong S.E.
      • Nielsen M.
      • Foster L.J.
      • Mann M.
      A proteomics strategy to elucidate functional protein-protein interactions applied to EGF signaling.
      ,
      • Ross P.L.
      • Huang Y.N.
      • Marchese J.N.
      • Williamson B.
      • Parker K.
      • Hattan S.
      • Khainovski N.
      • Pillai S.
      • Dey S.
      • Daniels S.
      • Purkayastha S.
      • Juhasz P.
      • Martin S.
      • Bartlet-Jones M.
      • He F.
      • Jacobson A.
      • Pappin D.J.
      Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents.
      ,
      • Schmidt A.
      • Kellermann J.
      • Lottspeich F.
      A novel strategy for quantitative proteomics using isotope-coded protein labels.
      ,
      • Ong S.E.
      • Blagoev B.
      • Kratchmarova I.
      • Kristensen D.B.
      • Steen H.
      • Pandey A.
      • Mann M.
      Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.
      ,
      • Himeda C.L.
      • Ranish J.A.
      • Angello J.C.
      • Maire P.
      • Aebersold R.
      • Hauschka S.D.
      Quantitative proteomic identification of six4 as the trex-binding factor in the muscle creatine kinase enhancer.
      ). More recently, high resolution mass spectrometry with label-free quantification has been shown to improve and extend quantitative proteomics toward comprehensive analysis of protein complexes (
      • Rinner O.
      • Mueller L.N.
      • Hubálek M.
      • Müller M.
      • Gstaiger M.
      • Aebersold R.
      An integrated mass spectrometric and computational framework for the analysis of protein interaction networks.
      ).
      Telomeres are DNA-protein structures located at the ends of linear eukaryotic chromosomes (see Fig. 1). The DNA portion of telomeres consists of a double-stranded region and a single-stranded 3′ overhang, both composed of repetitive non-coding G-rich sequences (TTAGGG). In addition to the DNA component, proteins bind the telomere and contribute to its stability. Six core proteins (TRF1, TRF2, POT1, TIN2, RAP1, and ACD/TPP1), collectively known as the shelterin (or telosome) complex, are constitutively present at the telomere (for reviews, see Refs.
      • de Lange T.
      Shelterin: the protein complex that shapes and safeguards human telomeres.
      and
      • Liu D.
      • O'Connor M.S.
      • Qin J.
      • Songyang Z.
      Telosome, a mammalian telomere-associated complex formed by multiple telomeric proteins.
      ). Together, the telomeric DNA and shelterin complex maintain a “capped” or functional telomere that protects the end of the chromosome by distinguishing it from a bona fide double strand DNA break (
      • Blackburn E.H.
      Switching and signaling at the telomere.
      ). When telomeres become uncapped or “dysfunctional,” they no longer carry out this protective function, rendering the chromosome ends susceptible to DNA repair enzymes. In the absence of functional checkpoints, uncapped telomeres can lead to end-to-end fusions that drive genomic instability, a hallmark of human cancer (
      • Ferreira M.G.
      • Miller K.M.
      • Cooper J.P.
      Indecent exposure: when telomeres become uncapped.
      ).
      Figure thumbnail gr1
      Fig. 1Fluorescent in situ hybridization reveals presence of telomeres at termini of human chromosomes. Top panel, representative metaphase spread from human cells. FISH analysis reveals the presence of telomeres (red) and centromeres (green), and chromosomal DNA (blue) was detected by DAPI staining. Bottom panel, schematic drawing of a telomere loop (T-Loop) showing the shelterin core complex (TRF1, TRF2, POT1, TIN2, RAP1, and TPP1) as well as a subset of known telomere-binding proteins (in gray). Question marks indicate that more telomere-binding proteins remain to be identified. WRN, Werner, BLM, Bloom, and XPF, xeroderma pigmentosum type F.
      Recent work has revealed that in addition to the shelterin complex a growing list of proteins associate with the telomere and play essential roles in telomere maintenance (a subset of these proteins, colored in gray, is depicted in Fig. 1). Paradoxically, many of these proteins play roles in DNA repair and recombination. These proteins include the MRE11-Rad50-Nbs1 complex involved in recombinational repair (
      • Takai H.
      • Smogorzewska A.
      • de Lange T.
      DNA damage foci at dysfunctional telomeres.
      ); Ku70 and Ku80, which are members of the non-homologous end joining complex (
      • Hsu H.L.
      • Gilley D.
      • Blackburn E.H.
      • Chen D.J.
      Ku is associated with the telomere in mammals.
      ); the ERCC1/XPF nucleotide excision repair endonuclease (
      • Zhu X.D.
      • Niedernhofer L.
      • Kuster B.
      • Mann M.
      • Hoeijmakers J.H.
      • de Lange T.
      ERCC1/XPF removes the 3′ overhang from uncapped telomeres and represses formation of telomeric DNA-containing double minute chromosomes.
      ); and the ataxia telagiectasia mutated (ATM) kinase (
      • Takai H.
      • Smogorzewska A.
      • de Lange T.
      DNA damage foci at dysfunctional telomeres.
      ,
      • Karlseder J.
      • Hoke K.
      • Mirzoeva O.K.
      • Bakkenist C.
      • Kastan M.B.
      • Petrini J.H.
      • de Lange T.
      The telomeric protein TRF2 binds the ATM kinase and can inhibit the ATM-dependent DNA damage response.
      ). Additional proteins have been found at the telomere in low stoichiometric ratios, including telomerase, which binds the telomere during S phase and adds telomeric repeats to the ends of the chromosomes (
      • Wright W.E.
      • Tesmer V.M.
      • Liao M.L.
      • Shay J.W.
      Normal human telomeres are not late replicating.
      ,
      • Smith C.D.
      • Smith D.L.
      • DeRisi J.L.
      • Blackburn E.H.
      Telomeric protein distributions and remodeling through the cell cycle in Saccharomyces cerevisiae.
      ). The Werner helicase is also present at the telomeres during S phase where it plays an important role in lagging strand DNA replication (
      • Crabbe L.
      • Verdun R.E.
      • Haggblom C.I.
      • Karlseder J.
      Defective telomere lagging strand synthesis in cells lacking WRN helicase activity.
      ). Despite the plethora of proteins known to bind to the telomere, many proteins that act in a transient manner and/or are present in substoichiometric quantities remain to be identified.
      To identify novel telomere-binding proteins, we developed a method that involves chemical cross-linking of protein complexes in live cells to capture transient interactions followed by affinity purification of the cross-linked telomere complex with an epitope-tagged telomeric protein, TIN2. Using the affinity-captured protein preparations, we optimized cross-link reversal, sequential endoprotease digestion, and microscale solid phase peptide purification. The peptide pools were analyzed using nano-LC-FTICR-MS. Comparative quantitative analysis of affinity-purified proteins from cells overexpressing the epitope-tagged TIN2 and control cells was performed using the peptide ion currents at accurate m/z values from the aligned LC-MS chromatograms across multiple samples. The proteins were identified using tandem MS with spectral matching against protein databases. Using this approach, we identified the six members of the shelterin complex and other proteins previously reported to bind to the telomere. We also identified a novel group of candidate telomere-binding proteins that were significantly enriched in samples expressing epitope-tagged TIN2 (HA
      The abbreviations used are:
      HA
      hemagglutinin
      DSP
      dithiobis(succinimidyl)propionate
      ChIP
      chromatin immunoprecipitation
      IP
      immunoprecipitation
      TAP
      tandem affinity purification
      FISH
      fluorescent in situ hybridization
      MS1
      parent scan mass spectra
      MS2
      tandem mass spectra
      tr
      retention time
      GFP
      green fluorescent protein
      DAPI
      4′6-diamidino-2-phenylindole
      RT
      room temperature
      TCEP
      tris(2-carboxyethyl)phosphine
      LTQ
      linear trap quadrupole
      hnRNP
      heterogeneous nuclear ribonucleoprotein
      TERT
      reverse transcriptase component of telomerase.
      1The abbreviations used are:HA
      hemagglutinin
      DSP
      dithiobis(succinimidyl)propionate
      ChIP
      chromatin immunoprecipitation
      IP
      immunoprecipitation
      TAP
      tandem affinity purification
      FISH
      fluorescent in situ hybridization
      MS1
      parent scan mass spectra
      MS2
      tandem mass spectra
      tr
      retention time
      GFP
      green fluorescent protein
      DAPI
      4′6-diamidino-2-phenylindole
      RT
      room temperature
      TCEP
      tris(2-carboxyethyl)phosphine
      LTQ
      linear trap quadrupole
      hnRNP
      heterogeneous nuclear ribonucleoprotein
      TERT
      reverse transcriptase component of telomerase.
      -FLAG-TIN2) compared with non-expressing control cells. Importantly, the presence of telomeric DNA in our immunoprecipitants from cells expressing HA-FLAG-TIN2 but not in control cells demonstrates that it is possible to identify proteins bound to DNA by utilizing a protein-protein cross-linking reagent. This strategy will prove versatile for the identification of other proteins found in large protein complexes as well as bound to DNA.

      DISCUSSION

      In this study, we describe a novel in strategy to identify and characterize proteins that comprise the central telomeric complex and other associated proteins. A combination of in vivo cross-linking, tandem affinity purification, and label-free, quantitative, high resolution mass spectrometry was used to identify 62 proteins that were enriched in lysates from cells expressing a HA-FLAG-TIN2 protein construct. Using this approach, we identified the core telomere-binding proteins that constitute the shelterin complex (TRF1, TRF2, TIN2, RAP1, POT1, and TPP1). Although classical biochemical approaches have successfully identified telomere-binding proteins (
      • Zhu X.D.
      • Niedernhofer L.
      • Kuster B.
      • Mann M.
      • Hoeijmakers J.H.
      • de Lange T.
      ERCC1/XPF removes the 3′ overhang from uncapped telomeres and represses formation of telomeric DNA-containing double minute chromosomes.
      ,
      • Li B.
      • Oestreich S.
      • de Lange T.
      Identification of human Rap1: implications for telomere evolution.
      ,
      • Zhong Z.
      • Shiue L.
      • Kaplan S.
      • de Lange T.
      A mammalian factor that binds telomeric TTAGGG repeats in vitro.
      ,
      • Bianchi A.
      • Smith S.
      • Chong L.
      • Elias P.
      • de Lange T.
      TRF1 is a dimer and bends telomeric DNA.
      ,
      • van Overbeek M.
      • de Lange T.
      Apollo, an Artemis-related nuclease, interacts with TRF2 and protects human telomeres in S phase.
      ), we aimed to develop a method in which telomere-interacting proteins from low microgram quantities of highly complex protein mixtures could be identified. In addition, we sought to identify proteins that weakly and/or transiently interacted with the telomere that would have been absent from previous studies. To facilitate these studies, we created an epitope-tagged TIN2 construct that allowed purification of protein complexes using two-tag TAP technology and used the chemical cross-linker DSP to capture telomeric complexes in their in vivo state. Indeed, we found that the TAP method reduced the isolation of nonspecifically bound proteins that tend to increase when cross-linkers are used (data not shown and Ref.
      • Tagwerker C.
      • Flick K.
      • Cui M.
      • Guerrero C.
      • Dou Y.
      • Auer B.
      • Baldi P.
      • Huang L.
      • Kaiser P.
      A tandem affinity tag for two-step purification under fully denaturing conditions: application in ubiquitin profiling and protein complex identification combined with in vivo cross-linking.
      ). We optimized a sequential endoprotease digestion and peptide solid phase extraction method for reproducibility and peptide recovery to use label-free quantitative LC-MS to determine the peptides that were significantly enriched by the cross-linking/tandem affinity protocol. Finally, we used comparative label-free LC-FTCIR-MS to identify peptides that were quantitatively enriched in the samples purified by TAP from epitope-tagged TIN2-expressing cells, bypassing the need for comparative SDS-PAGE. This approach increased the sensitivity of protein identification and led to the discovery of putative telomere-associated proteins.
      To facilitate protein identification within our complex peptide mixtures, we used LC-FTICR-MS to quantify tryptic peptides that were prepared from the tandem affinity-captured proteins. The peptide pools from samples that were isolated from cells expressing the HA-FLAG-TIN2 construct and control cells were subjected to separate LC-MS analyses. The raw MS data from multiple LC-MS analyses were imported directly into software that aligned, identified, normalized, and quantified the ion currents from individual peptides that were measured with high resolution. A total of ∼11,000 signal features were detected that could be concatenated into ∼6000 isotope groups comprising the detected charge states of the individual peptides (from 2+ to 5+). Using this analytical approach and the described software, we readily identified peptides enriched in the experimental samples and contaminants that were increased in the control samples and assessed the reproducibility of peptide digestion and preparation. Importantly, our approach was able to 1) identify the shelterin complex using 10–40-fold less cell lysate than was used in previous experiments, demonstrating that our approach is capable of identifying numerous proteins within a complex mixture regardless of their stoichiometries, and 2) identify proteins known to be present in higher order TIN2 complexes but that do not directly interact with TIN2, demonstrating that our protein cross-linking was effective at capturing important protein interactors.
      Isolation of the shelterin components demonstrates that our approach is able to isolate large protein complexes. In addition, isolation of telomeric DNA indicated that this approach resulted in the isolation of telomeric bound protein complexes. In addition to the known TIN2-interacting proteins, we isolated numerous proteins in samples prepared from cells expressing HA-FLAG-TIN2 but not from control cells that have not been described to interact with the telomeric complex. One concern that could be raised with our approach is that it resulted in nonspecific cross-linking to proteins in the neighborhood of HA-FLAG-TIN2. We find this possibility unlikely for three reasons. First, peptides that represent the shelterin complex were enriched in our samples and were the most predominant peptides present in the samples. This included POT1, which does not directly interact with TIN2 but instead interacts through TPP1. If the cross-linking were random, we would not expect to see POT1 peptides so highly enriched. Second, the shelterin proteins were not observed in lysates from HA-FLAG-GFP-expressing cells. This indicated that the shelterin proteins do not associate with a nonspecific HA-FLAG-tagged protein such as GFP. Third, if treatment with DSP led to significant nonspecific cross-linking, we would expect to see proteins that are present in high molar ratios such as nuclear membrane proteins. Although we did observe both ribosomal and hnRNP proteins in our TIN2 pulldowns, we did not observe these proteins in our HA-FLAG-GFP pulldowns, suggesting that their interaction with TIN2 was specific and not due to their high expression within the cell. Finally, the TIN2-binding protein TRF2 traffics through the nucleolus (
      • Zhang S.
      • Hemmerich P.
      • Grosse F.
      Nucleolar localization of the human telomeric repeat binding factor 2 (TRF2).
      ), and in several instances we isolated nucleolar proteins. However, we did not isolate a significant number of nucleolar proteins, arguing that the proteins that were isolated specifically interact with TIN2 possibly through TRF2. Interestingly, most of the novel proteins identified are involved in the transmission of genetic information, including replication, transcription, mRNA processing, translation, and chromatin structure/remodeling, processes that are linked to telomere biology. Even the process of transcription has been recently linked to telomere biology (
      • Azzalin C.M.
      • Reichenbach P.
      • Khoriauli L.
      • Giulotto E.
      • Lingner J.
      Telomeric repeat containing RNA and RNA surveillance factors at mammalian chromosome ends.
      ) in a study that showed mammalian telomeres are transcribed into telomeric repeat-containing RNA (TERRA). Prior to this report, telomeres were considered to be transcriptionally silent. Thus, it is likely that a number of the proteins identified in our study will prove to be biologically relevant to telomere biology.
      Our approach successfully identified many telomere-binding proteins, but a number of known telomere-binding proteins were not identified in our experiments. Although it is not clear why we failed to identify these proteins, it is possible that 1) their identification required larger amounts of cell lysate or 2) the choice of DSP as a cross-linking reagent might preclude isolation and/or identification of certain proteins. Indeed, cross-linkers add permanent modifications to proteins resulting in alterations in peptide mass, making the detection of some peptides impossible. In addition, the use of a protein cross-linker could alter antibody binding sites, resulting in reduced protein recovery following immunoprecipitation. Alternatively, the method of isolation may impact the proteins identified. Indeed, a recent study utilized sequence-specific nucleic acids to isolate telomere-binding proteins from formaldehyde-cross-linked lysates (referred to as proteomics of isolated chromatin (PICh)) (
      • Déjardin J.
      • Kingston R.E.
      Purification of proteins associated with specific genomic loci.
      ). Using this approach, all of the components of the shelterin complex were identified as well as additional proteins previously reported to bind the telomere. Novel telomere-binding proteins were also identified, and interestingly there was no overlap with our list of novel telomere-interacting proteins. This finding likely reflects differences in the isolation procedures and cross-linking reagent and underscores the need for multiple, complementary approaches to identify components of large multiprotein complexes such as those found at the telomere.

      Acknowledgments

      We are grateful to Petra Gilmore, Alan Davis, Marjorie Case, Jim Malone, and Julia Gross for providing excellent technical support for the proteomics studies. We thank Rekha Meyer and Josh Coats for expert assistance with the database search algorithms, mass spectrometric software, and statistical analyses. We thank Joshua Behlman for preparation of adenoviral stocks, and members of our laboratories for many helpful discussions and advice.

      REFERENCES

        • Ranish J.A.
        • Yi E.C.
        • Leslie D.M.
        • Purvine S.O.
        • Goodlett D.R.
        • Eng J.
        • Aebersold R.
        The study of macromolecular complexes by quantitative proteomics.
        Nat. Genet. 2003; 33: 349-355
        • Blagoev B.
        • Kratchmarova I.
        • Ong S.E.
        • Nielsen M.
        • Foster L.J.
        • Mann M.
        A proteomics strategy to elucidate functional protein-protein interactions applied to EGF signaling.
        Nat. Biotechnol. 2003; 21: 315-318
        • Ross P.L.
        • Huang Y.N.
        • Marchese J.N.
        • Williamson B.
        • Parker K.
        • Hattan S.
        • Khainovski N.
        • Pillai S.
        • Dey S.
        • Daniels S.
        • Purkayastha S.
        • Juhasz P.
        • Martin S.
        • Bartlet-Jones M.
        • He F.
        • Jacobson A.
        • Pappin D.J.
        Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents.
        Mol. Cell. Proteomics. 2004; 3: 1154-1169
        • Schmidt A.
        • Kellermann J.
        • Lottspeich F.
        A novel strategy for quantitative proteomics using isotope-coded protein labels.
        Proteomics. 2005; 5: 4-15
        • Ong S.E.
        • Blagoev B.
        • Kratchmarova I.
        • Kristensen D.B.
        • Steen H.
        • Pandey A.
        • Mann M.
        Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.
        Mol. Cell. Proteomics. 2002; 1: 376-386
        • Himeda C.L.
        • Ranish J.A.
        • Angello J.C.
        • Maire P.
        • Aebersold R.
        • Hauschka S.D.
        Quantitative proteomic identification of six4 as the trex-binding factor in the muscle creatine kinase enhancer.
        Mol. Cell. Biol. 2004; 24: 2132-2143
        • Rinner O.
        • Mueller L.N.
        • Hubálek M.
        • Müller M.
        • Gstaiger M.
        • Aebersold R.
        An integrated mass spectrometric and computational framework for the analysis of protein interaction networks.
        Nat. Biotechnol. 2007; 25: 345-352
        • de Lange T.
        Shelterin: the protein complex that shapes and safeguards human telomeres.
        Genes Dev. 2005; 19: 2100-2110
        • Liu D.
        • O'Connor M.S.
        • Qin J.
        • Songyang Z.
        Telosome, a mammalian telomere-associated complex formed by multiple telomeric proteins.
        J. Biol. Chem. 2004; 279: 51338-51342
        • Blackburn E.H.
        Switching and signaling at the telomere.
        Cell. 2001; 106: 661-673
        • Ferreira M.G.
        • Miller K.M.
        • Cooper J.P.
        Indecent exposure: when telomeres become uncapped.
        Mol. Cell. 2004; 13: 7-18
        • Takai H.
        • Smogorzewska A.
        • de Lange T.
        DNA damage foci at dysfunctional telomeres.
        Curr. Biol. 2003; 13: 1549-1556
        • Hsu H.L.
        • Gilley D.
        • Blackburn E.H.
        • Chen D.J.
        Ku is associated with the telomere in mammals.
        Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 12454-12458
        • Zhu X.D.
        • Niedernhofer L.
        • Kuster B.
        • Mann M.
        • Hoeijmakers J.H.
        • de Lange T.
        ERCC1/XPF removes the 3′ overhang from uncapped telomeres and represses formation of telomeric DNA-containing double minute chromosomes.
        Mol. Cell. 2003; 12: 1489-1498
        • Karlseder J.
        • Hoke K.
        • Mirzoeva O.K.
        • Bakkenist C.
        • Kastan M.B.
        • Petrini J.H.
        • de Lange T.
        The telomeric protein TRF2 binds the ATM kinase and can inhibit the ATM-dependent DNA damage response.
        PLoS Biol. 2004; 2: E240
        • Wright W.E.
        • Tesmer V.M.
        • Liao M.L.
        • Shay J.W.
        Normal human telomeres are not late replicating.
        Exp. Cell Res. 1999; 251: 492-499
        • Smith C.D.
        • Smith D.L.
        • DeRisi J.L.
        • Blackburn E.H.
        Telomeric protein distributions and remodeling through the cell cycle in Saccharomyces cerevisiae.
        Mol. Biol. Cell. 2003; 14: 556-570
        • Crabbe L.
        • Verdun R.E.
        • Haggblom C.I.
        • Karlseder J.
        Defective telomere lagging strand synthesis in cells lacking WRN helicase activity.
        Science. 2004; 306: 1951-1953
        • Loayza D.
        • De Lange T.
        POT1 as a terminal transducer of TRF1 telomere length control.
        Nature. 2003; 423: 1013-1018
        • Adeli K.
        • Wettesten M.
        • Asp L.
        • Mohammadi A.
        • Macri J.
        • Olofsson S.O.
        Intracellular assembly and degradation of apolipoprotein B-100-containing lipoproteins in digitonin-permeabilized HEP G2 cells.
        J. Biol. Chem. 1997; 272: 5031-5039
        • Nakatani Y.
        • Ogryzko V.
        Immunoaffinity purification of mammalian protein complexes.
        Methods Enzymol. 2003; 370: 430-444
        • Washburn M.P.
        Sample preparation and in-solution protease digestion of proteins for chromatography-based proteomic analysis.
        Curr. Protoc. Protein Sci. 2008; (Chapter 23, Unit 23.6.1–23.6.11)
        • Baldwin M.A.
        • Medzihradszky K.F.
        • Lock C.M.
        • Fisher B.
        • Settineri T.A.
        • Burlingame A.L.
        Matrix-assisted laser desorption/ionization coupled with quadrupole/orthogonal acceleration time-of-flight mass spectrometry for protein discovery, identification, and structural analysis.
        Anal. Chem. 2001; 73: 1707-1720
        • Neubert H.
        • Bonnert T.P.
        • Rumpel K.
        • Hunt B.T.
        • Henle E.S.
        • James I.T.
        Label-free detection of differential protein expression by LC/MALDI mass spectrometry.
        J. Proteome Res. 2008; 7: 2270-2279
        • Ye J.Z.
        • Hockemeyer D.
        • Krutchinsky A.N.
        • Loayza D.
        • Hooper S.M.
        • Chait B.T.
        • de Lange T.
        POT1-interacting protein PIP1: a telomere length regulator that recruits POT1 to the TIN2/TRF1 complex.
        Genes Dev. 2004; 18: 1649-1654
        • O'Connor M.S.
        • Safari A.
        • Xin H.
        • Liu D.
        • Songyang Z.
        A critical role for TPP1 and TIN2 interaction in high-order telomeric complex assembly.
        Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 11874-11879
        • Vasilescu J.
        • Guo X.
        • Kast J.
        Identification of protein-protein interactions using in vivo cross-linking and mass spectrometry.
        Proteomics. 2004; 4: 3845-3854
        • Li B.
        • Oestreich S.
        • de Lange T.
        Identification of human Rap1: implications for telomere evolution.
        Cell. 2000; 101: 471-483
        • Metz B.
        • Kersten G.F.
        • Hoogerhout P.
        • Brugghe H.F.
        • Timmermans H.A.
        • de Jong A.
        • Meiring H.
        • ten Hove J.
        • Hennink W.E.
        • Crommelin D.J.
        • Jiskoot W.
        Identification of formaldehyde-induced modifications in proteins: reactions with model peptides.
        J. Biol. Chem. 2004; 279: 6235-6243
        • Lomant A.J.
        • Fairbanks G.
        Chemical probes of extended biological structures: synthesis and properties of the cleavable protein cross-linking reagent [35S]dithiobis(succinimidyl propionate).
        J. Mol. Biol. 1976; 104: 243-261
        • Davidson W.S.
        • Hilliard G.M.
        The spatial organization of apolipoprotein A-I on the edge of discoidal high density lipoprotein particles: a mass spectrometry study.
        J. Biol. Chem. 2003; 278: 27199-27207
        • Zang X.
        • Komatsu S.
        A proteomics approach for identifying osmotic-stress-related proteins in rice.
        Phytochemistry. 2007; 68: 426-437
        • Greenberg R.A.
        • Sobhian B.
        • Pathania S.
        • Cantor S.B.
        • Nakatani Y.
        • Livingston D.M.
        Multifactorial contributions to an acute DNA damage response by BRCA1/BARD1-containing complexes.
        Genes Dev. 2006; 20: 34-46
        • America A.H.
        • Cordewener J.H.
        Comparative LC-MS: a landscape of peaks and valleys.
        Proteomics. 2008; 8: 731-749
        • Khurts S.
        • Masutomi K.
        • Delgermaa L.
        • Arai K.
        • Oishi N.
        • Mizuno H.
        • Hayashi N.
        • Hahn W.C.
        • Murakami S.
        Nucleolin interacts with telomerase.
        J. Biol. Chem. 2004; 279: 51508-51515
        • Pollice A.
        • Zibella M.P.
        • Bilaud T.
        • Laroche T.
        • Pulitzer J.F.
        • Gilson E.
        In vitro binding of nucleolin to double-stranded telomeric DNA.
        Biochem. Biophys. Res. Commun. 2000; 268: 909-915
        • Ishikawa F.
        • Matunis M.J.
        • Dreyfuss G.
        • Cech T.R.
        Nuclear proteins that bind the pre-mRNA 3′ splice site sequence r(UUAG/G) and the human telomeric DNA sequence d(TTAGGG)n.
        Mol. Cell. Biol. 1993; 13: 4301-4310
        • Tanaka E.
        • Fukuda H.
        • Nakashima K.
        • Tsuchiya N.
        • Seimiya H.
        • Nakagama H.
        HnRNP A3 binds to and protects mammalian telomeric repeats in vitro.
        Biochem. Biophys. Res. Commun. 2007; 358: 608-614
        • LaBranche H.
        • Dupuis S.
        • Ben-David Y.
        • Bani M.R.
        • Wellinger R.J.
        • Chabot B.
        Telomere elongation by hnRNP A1 and a derivative that interacts with telomeric repeats and telomerase.
        Nat. Genet. 1998; 19: 199-202
        • Ford L.P.
        • Suh J.M.
        • Wright W.E.
        • Shay J.W.
        Heterogeneous nuclear ribonucleoproteins C1 and C2 associate with the RNA component of human telomerase.
        Mol. Cell. Biol. 2000; 20: 9084-9091
        • Eversole A.
        • Maizels N.
        In vitro properties of the conserved mammalian protein hnRNP D suggest a role in telomere maintenance.
        Mol. Cell. Biol. 2000; 20: 5425-5432
        • Seimiya H.
        • Sawada H.
        • Muramatsu Y.
        • Shimizu M.
        • Ohko K.
        • Yamane K.
        • Tsuruo T.
        Involvement of 14-3-3 proteins in nuclear localization of telomerase.
        EMBO J. 2000; 19: 2652-2661
        • Forsythe H.L.
        • Jarvis J.L.
        • Turner J.W.
        • Elmore L.W.
        • Holt S.E.
        Stable association of hsp90 and p23, but Not hsp70, with active human telomerase.
        J. Biol. Chem. 2001; 276: 15571-15574
        • Holt S.E.
        • Aisner D.L.
        • Baur J.
        • Tesmer V.M.
        • Dy M.
        • Ouellette M.
        • Trager J.B.
        • Morin G.B.
        • Toft D.O.
        • Shay J.W.
        • Wright W.E.
        • White M.A.
        Functional requirement of p23 and Hsp90 in telomerase complexes.
        Genes Dev. 1999; 13: 817-826
        • Azzalin C.M.
        • Reichenbach P.
        • Khoriauli L.
        • Giulotto E.
        • Lingner J.
        Telomeric repeat containing RNA and RNA surveillance factors at mammalian chromosome ends.
        Science. 2007; 318: 798-801
        • Zhong Z.
        • Shiue L.
        • Kaplan S.
        • de Lange T.
        A mammalian factor that binds telomeric TTAGGG repeats in vitro.
        Mol. Cell. Biol. 1992; 12: 4834-4843
        • Bianchi A.
        • Smith S.
        • Chong L.
        • Elias P.
        • de Lange T.
        TRF1 is a dimer and bends telomeric DNA.
        EMBO J. 1997; 16: 1785-1794
        • van Overbeek M.
        • de Lange T.
        Apollo, an Artemis-related nuclease, interacts with TRF2 and protects human telomeres in S phase.
        Curr. Biol. 2006; 16: 1295-1302
        • Tagwerker C.
        • Flick K.
        • Cui M.
        • Guerrero C.
        • Dou Y.
        • Auer B.
        • Baldi P.
        • Huang L.
        • Kaiser P.
        A tandem affinity tag for two-step purification under fully denaturing conditions: application in ubiquitin profiling and protein complex identification combined with in vivo cross-linking.
        Mol. Cell. Proteomics. 2006; 5: 737-748
        • Zhang S.
        • Hemmerich P.
        • Grosse F.
        Nucleolar localization of the human telomeric repeat binding factor 2 (TRF2).
        J. Cell Sci. 2004; 117: 3935-3945
        • Déjardin J.
        • Kingston R.E.
        Purification of proteins associated with specific genomic loci.
        Cell. 2009; 136: 175-186