Proteomics - Approaches
The proteomics arm of the Protein Discovery Initiative aims to develop a census of the migration proteome by identifying novel molecules involved in migration. The interactions and post translational modifications, e.g., phosphorylations, will be examined with a subset of major molecules. Finally, changes in associations and phosphorylations will be measured on cells stimulated or otherwise treated by a variety of different conditions. We are approaching these objectives primarily by using tandem mass spectrometry on immunoprecipitated proteins and protein complexes or isolated migration-related organelles, e.g., lamellipodia, filopodia, and adhesions.
Isolation of Proteins & Complexes
Several strategies are being used for the isolation of molecules and their complexes. They include affinity columns, immunoprecipitation of native and TAP- or FLAG-tagged molecules, and isolation of migration organelles.
Affinity columns
This approach seems particularly promising for isolating molecules that bind integrin cytoplasmic domains. The cytoplasmic domain of integrins are directly accessible to the intracellular environment and link these central migration related receptors to the cytoskeletal and cellular signaling machinery responsible for cell movement (Liu et al., 2000). The approach is to use model protein systems designed to mimic the cytoplasmic face of clustered integrins as baits (Liu et al., 1999). These systems accommodate the heterodimeric configuration of integrin cytoplasmic domains and are being used to identify novel proteins that bind to integrin cytoplasmic domains.
Affinity tags
Two related strategies are being used for the isolation of migration molecules and their binding partners. Both involve construction of a fusion protein; one with a FLAG affinity tag and the other with a TAP affinity tag. While the isolation of endogenous molecules by immunoprecipitation has advantages it also presents challenges since an immunoprecipitation protocols for each molecule to be studied would need to be developed individually in the context of available antibodies. In addition the antibody might mask sites or otherwise interfere with the binding interactions that are to be measured. The fusion proteins on the other hand, will usually need to be expressed at levels close to endogenous and characterized to show that the TAG does not interfere with the interactions and function of the protein.
The FLAG purification is relatively simple and rapid (Einhauer & Jungbauer, 2001). A small "FLAG" sequence is cloned into the protein under study. The sequence binds to an immobilized antibody directed against the FLAG sequence. After some washing steps, elution is effected with a peptide that competes for the binding site on an anti-FLAG antibody. A variant of this approach is to use a FLAG-GFP tag; this allows estimates of expression while still allowing simple IP.
The TAP purification is based on a tandem affinity approach (Puig, et al., 2001; Rigaut et al., 1999). While it has more steps, it can provide high protein purity. The TAP tag is comprised of protein A, a rare, TEV protease sensitive site, and a calmodulin binding peptide. The tagged (fusion) protein binds to immobilized IgG via the protein A. It is then cleaved by the TEV protease and the eluent immobilized on calmodulin-coated beads in the presence of calcium. The protein is then eluted with EGTA.
Migration related organelles; The migration machinery is composed of a number of highly organized cytoskeletal and adhesive structures including: focal adhesion, filopodia, membrane ruffle and lamellipodia. We are trying to isolate these for analyses of their contents and how they change in response to different stimuli and perturbations.
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Identification of Protein Components
Samples collected using the purification techniques described above are analysed by mass spectrometry. Tandem mass spectrometry is the major analytic tool used for evaluating proteins and protein complexes in the Consortium. With this approach, protein samples are first digested with proteases into a complex mixture of peptides and analyzed via LC-MS (HPLC separation interfaced to tandem mass spectrometry). Peptide ions are fragmented during the mass spectrometric analysis, yielding structural information about the peptide in the form of an MS/MS spectrum. The amino acid sequence of the peptide is determined by subtracting fragment ion masses from each other, yielding the residue mass of a particular amino acid. The subtraction process is repeated until the sequence has been resolved. For details on sample preparation, LC-MS analysis, peptide sequence identification and IMAC phosphopeptide enrichment please click here.
Electron Transfer Dissociation Mass Spectrometry : Improved Instrumentation
We have recently developed and implemented the use of electron transfer dissociation (ETD) to obtain MS/MS spectra of phosphorylated peptides that is of optimal quality (Syka et al, 2004). The fragmentation pattern obtained from ETD is such that the resulting phosphopeptide spectra are much more easily interpreted by the mass spec analyst and automated tools such as SEQUEST. In this manner, more sequence information will be obtained from phosphorylated (and other post-translationally modified) peptides--something that had previously been an obstacle using traditional collisionally activated dissociation. Work on this technology is on-going and will be used more frequently in the future to sequence phosphopeptides from the experiments described above.
Differential Analysis of Phosphopeptide Expression
Relative quantitation of binding partners and phosphopeptides from cells will be determined by differential mass spectrometric analysis. Cells that have been transfected with the protein of interest are grown up in the presence and absence of phosphatase inhibitors (see example in Figure below).
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| Differential sample preparation - Paxillin +/- phosphatase inhibitors |
The protein of interest from each sample preparation is isolated via immunoprecipitation and then approximately 15% (low pmoles of protein) of each sample prep is subjected to proteolytic digestion. Approximately 1% (~100fmol) of each digest is then spiked with known amounts of phosphopeptide standards and analyzed via mass spectrometry to normalize ion counts with respect to quantity of peptide loaded. Once the quantity of sample has been normalized, the remainder of one digest is labeled with deuterated methanolic HCl (adds 3 amu per carboxylic acid group) while the remainder of the other digest is unlabeled. The samples are then combined, phosphopeptide standards again spiked in, and the mixture is subjected to IMAC separation. All phosphopeptides are retained on the IMAC column, while the non-phosphorylated peptides flow through the column and are collected. The phosphopeptides are then eluted onto a reversed-phase HPLC column and eluted into the mass spectrometer for LC-MS/MS analysis (see Figure below).
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| Enrichment of phosphopeptides with IMAC |
In a separate analysis, the non-phosphorylated peptides are eluted into the mass spectrometer for LC-MS/MS analysis. Pairs of peptides from the inhibitor vs. no inhibitor sample prep conditions will be observed by using data analysis tools to search for co-eluting peptides that differ in mass by 3 amu. This mass difference represents the difference of labeled (no phosphatase inhibitors) versus unlabeled (with phosphatase inhibitors) sample (see Figure below).
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| Finnigan LTQ-FTMS: accurate mass and MS/MS spectra IMAC (phosphorylated) |
Ratios of ion abundance between the phosphorylated and non-phosphorylated peptides can then be calculated by standardizing ion counts between the two analyses (using the phosphopeptide standards) and then normalizing the ion counts for the phosphorylated and unphosphorylated versions of the peptide. Using this technique, relative amounts of increased and decreased phosphopeptide expression can be determined.
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Proteomic Data Analysis
Raw data is extracted for individual spectra with filters applied to remove obvious background ions and extremely low quality spectra. The final set of extracted spectra is searched agains the NCBI NR protein database. The resulting peptide hits are grouped by GI number and sorted. Those GI numbers that have at least one peptide hit with an xcorr >1.5 are extracted into a custom database. The spectra are re-searched against this custom database and grouped by GI number again. GI numbers resulting from at least one peptide with an xcorr >1.5 and >20% abundant ions and ion current of the spectrum explained are grouped into a list. The list is filtered to remove non-mammal species (except in the case of proteins that have high identity across all species for example actin, HSPs, etc). The data is re-searched against a custom database made from these remaining proteins. Results are grouped by GI number after using the above xcorr and ion abundance constraints. The list of peptides that results is placed into an Excel sheet and sorted by GI number (ascending) and then xcorr (descending). This list is manually examined to determine if entries with a high likelihood of being a false positive are present. This step is based on the MW and charge state of the peptide and the number of missed cleavages requiring experience. Non-mammal hits are examined more closely. The peptide list that passes this final check is uploaded. For FT/ICR data, the basic procedure is the same except the mass tolerance for the final search is set to 0.020 Da (3.0 Da for LCQ/LTQ).
Bioinformatics for Protein Identification
Sets of peptides identified as described above are then combined based on shared GI numbers, and the peptide sets are searched against the Human, Mouse, or Rat IPI/ database using the FASTS algorithm (Mackey et al., 2003). Sets of peptides that hit the same protein sequence are combined, and re-searched against the IPI database. Proteins identified are ranked either by expectation value (E()-value) or by peptide coverage. E()-values provide a very accurate estimate of how often a peptide alignment score would occur by chance; proteins identified with E()<10-6 are almost certainly present in the sample.
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Characterization of the Pseudopodium Phosphoprotein Proteome
The overall goal of this project is to quantitatively measure and compare phosphorylation events in the front (pseudopodium) and the back (cell body) of chemotactic cells. Chemotaxis is driven by the reorganization of actin-cytoskeleton and regulated by localized amplification of signals in the side facing the chemoattractant. Establishment of a pseudopodium is a necessary morphological change for cells during chemotaxis.
Purification of pseudopodia from directionally polarized migratory cells
Pseudopodia are isolated from cell bodies as previously described (Cho & Klemke, 2002). Briefly, migratory cells are serum-starved overnight and then allowed to attach to the top of a 3-μm porous filter coated on both sides with matrix proteins in a 6-well Transwell plate. Pseudopodia growth is stimulated by adding chemoattractant to the lower chamber of the well. Cells are then fixed with ice-cold methanol. Cell bodies are wiped off the top of the filter with a cotton swab, and pseudopodia are harvested from the bottom of the filter with buffer. Similarly, cell bodies are harvested with buffer after pseudopodia are wiped off the bottom of the filter. Alternately, chemoattractant is removed from the lower chamber before fixation, causing pseudopodia to retract towards the bulk of the cell body on top of the filter. Thus, pseudopodia are harvested in both growth and retraction phase.
Quantitative identification of total phosphorylation in pseudopodium and cell body (Project 1 in Figure)
The same volume of proteins from pseudopodia and from cell bodies are labeled with 18O and 16O, respectively, using trypsin catalyzed 16O/ 18O labeling procedure. Trypsin catalyzed 16O/ 18O labeling is an effective, quantitative approach towards identifying proteins from a mixture (Liu et al., 2004; Qian et al., 2005). The two fractions are combined and phosphopeptides are purified by immobilized metal affinity chromatography (IMAC) purification. The purified phosphopeptides are identified by LC-MS/MS, and quantitative information is calculated from the ratio of the intensity of the 18O and 16O labeled peptides. Using this approach, we have quantitatively identified 228 phosphopeptides, including some proteins that are expected to be highly phosphorylated in the pseudopodium, such as Erk (Brahmbhatt & Klemke, 2003).
Identification of phosphotyrosine (pY) proteins (Project 2 in Figure)
Tyrosine phosphorylation is much less common and, hence, more difficult to identify than serine or threonine phosphorylation. To overcome this problem, pY proteins are purified from pseudopodia and cell bodies by anti-pY antibody immunoprecipitation, and proteins from each fraction are identified using LC-MS/MS. Using this approach, we have successfully identified more than 200 pseudopodium-unique proteins, including cytoskeletal proteins, signaling proteins, and hypothetical proteins. The major goal of this project is to combine immunoaffinity pY purification with 16O/ 18O labeling followed by IMAC enrichment in order to obtain a quantitative profile of pY proteins in pseudopodia and cell bodies of migratory cells.
Quantitative identification of total proteins from growing or retracting pseudopodia (Project 3 in Figure)
Pseudopodium retraction is not simply a reversal of the protrusion process; it involves distinct proteins and signaling events working together to reorganize the cytoskeleton. Therefore, it is important to identify quantitatively proteins that differentially regulate pseudopodium extension and retraction. Pseudopodium proteins harvested during growth and retraction phase are 16O/ 18O labeled, respectively, before being combined and separated into 25 fractions by strong cation exchange chromatography. Proteins from each fraction are then quantitatively identified with LC-MS/MS.
Bioinformatics and functional testing of phosphoproteins
Large-scale genomic and proteomic analysis provides a wealth of information on biologically relevant systems, and the ability to analyze this information is crucial to uncovering important biological relationships. We developed a fully automated blast program (BlastPro) that facilitates rapid comparison of large protein and/or nucleotide datasets from numerous, independent studies (Wang et al., 2006). Using this system, we compared several published genomic and proteomic databases and have found that the cytoskeletal-associated protein alpha-actinin is increased at both the mRNA and protein level in metastatic breast, prostate, and skin cancer cells. Spatial analysis revealed that alpha-actinin expression is amplified 8 fold in the leading pseudopodium compared to the cell body. These findings indicate that amplification of alpha-actinin and its localization to the leading pseudopodium are potential biomarkers of cancer progression to a more metastatic phenotype. Our ultimate goal is to use BlastPro and other bioinformatics tools to mine for proteins of interest identified in projects 1 – 3 and to perform functional, cell-based assays to further characterize the role of these proteins in chemotaxis.
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Text References
Brahmbhatt AA, Klemke RL. ERK and RhoA differentially regulate pseudopodia growth and retraction during chemotaxis. J Biol Chem. Apr 11 2003;278(15):13016-13025. PubMed
Cho SY, Klemke RL. Purification of pseudopodia from polarized cells reveals redistribution and activation of Rac through assembly of a CAS/Crk scaffold. J Cell Biol. Feb 18 2002;156(4):725-736. PubMed
Einhauer A, Jungbauer A. 2001. The FLAG peptide, a versatile fusion tag for the purification of recombinant proteins. J Biochem Biophys Methods. 49(1-3):455-65. PubMed
Katoh K, Kano Y, Fujiwara K. 2000. Isolation and in vitro contraction of stress fibers. Methods Enzymol. 325:369-80. PubMed
Liu S. Calderwood DA, Ginsberg MH. 2000. Integrin cytoplasmic domain-binding proteins. J. Cell Sci. 113:3563-71. PubMed
Liu S, Thomas SM, Woodside DG, Rose DM, Kiosses WB, Pfaff M, Ginsberg MH. 1999. Binding of paxillin to alpha4- integrins modifies integrin-dependend biological responses. Nature, 402:676-81. PubMed
Liu T, Qian WJ, Strittmatter EF, Camp DG 2nd, Anderson GA, Thrall BD, Smith RD. High-throughput comparative proteome analysis using a quantitative cysteinyl-peptide enrichment technology. Anal Chem. Sep 15 2004;76(18):5345-5353. PubMed
Puig O, Caspary F, Rigaut G, Rutz B, Bouveret E, Bragado-Nilsson E, Wilm M, Seraphin B. 2001. The tandem affinity purification (TAP) method: a general procedure of protein complex purification. Methods. 24(3):218-29. PubMed
Qian WJ, Monroe ME, Liu T, et al. Quantitative proteome analysis of human plasma following in vivo lipopolysaccharide administration using 16O/18O labeling and the accurate mass and time tag approach. Mol Cell Proteomics. May 2005;4(5):700-709. PubMed
Rigaut G, Shevchenko A, Rutz B, Wilm M, Mann M, Seraphin B. 1999. A generic protein purification method for protein complex characterization and proteome exploration. Nat Biotechnol. 1999 Oct;17(10):1030-2. PubMed
Syka JEP,. Coon JJ, Schroeder MJ, Shabanowitz J, Hunt DF. 2004. Peptide and protein sequence analysis by electron transfer dissociation mass spectrometry. PNAS 101(26) 9528-9533. PubMed
Wang Y, Hanley R, Klemke RL. 2006. Computational methods for comparison of large genomic and proteomic datasets reveal protein markers of metastatic cancer. J Proteome Res. 5(4):907-15. PubMed
