Peptides and Proteomics Core
Charges for U-M-MGPRC investigators for all services are reduced by 50%.
The two major components of this program are the Proteomics Core and the Protein Structure Core.
 Contact
Philip Andrews, Ph.D.
Director
734-763-3130
Henriette Remmer, Ph.D.
Co-Director
734-763-6285
Services
Two-dimensional gel electrophoresis
Two-dimensional gel electrophoresis is provided in both large and small formats using immobilized pH gradient (IPG) gels for the first dimension. The core has experience with a broad range of cell extracts, including difficult ones like membrane proteins (1). A variety of staining methods are provided, including silver stain and a variety of fluorescent stains. Image analysis is available by trained Core technicians or investigators may use our workstations themselves. Training in image analysis is provided. A variety of one-dimension gels are also run as needed by investigators as well as customized gel formats.
Analysis of intact proteins
Matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) and electrospray ionization interfaced to a quadrupole or an ion-trap mass spectrometer (ESI MS) will be used to perform the analyses of intact proteins and synthetic peptides. Electrospray ionization is uniquely distinguished by the production of multiply charged ions that provide independent confirmation of molecular mass [29]. Any species that acquires enough charge during ionization to bring the mass-to-charge ratio (m/z) less than 2000 can be analyzed using the ion trap (LCQ). In practice, the upper mass limit is on the order of 70 kDa. Deconvolution algorithms (transform, maximum entropy) are applied to ESI mass spectra to generate true molecular mass values with enhanced signal-to-noise. The LCQ mass analyzer is calibrated across the entire scan range with mixtures of standard compounds. For routine analysis, a mass accuracy of 0.05% or better is achieved. Higher mass accuracy can be attained with the LCQ in the zoom scan mode.
Proteome mapping
Proteome mapping is provided directly from polyacrylamide gels using robotic workstations for spot picking, proteolytic digestions, sample cleanup and spotting. Digests are analyzed by tandem mass spectrometry by facility personnel on the ABI MALDI TOFTOF mass spectrometer or investigators may run their own analyses on our MALDI QTOF or MALDI TOF mass spectrometers. Several search engines are supported in house and searches are queued automatically or may be manually submitted. Proteome mapping by LC MSMS or 2D-LC MSMS is also provided both through our capillary HPLC coupled to the Finnegan LCQ or via collection on the Agilent MALDI plate fraction collector followed by analysis on the TOFTOF mass spectrometer. Sensitivity is generally in the low femtomole range.
Protein expression profiling
Protein expression profiling is provided by 2D gel electrophoresis and through the isobaric tag technology (Applied Biosystems iTRAQ) using LC MSMS (2). This new approach to quantitation allows up to four samples to be multiplexed for relative quantitation and provides errors of less than 20%. We were one of the beta test sites for this reagent, gaining valuable experience with this reagent. The core has already successfully applied this technology to a variety of biological problems.
Post-translational modifications
Mapping the sites of post-translational modifications using tandem mass spectrometry is routinely provided for investigators. This work is performed on our model 4700 TOFTOF mass spectrometer, our Finnegan LCQ, or through our spoke laboratory at MSU, using the Finnegan LTQ FTMS.
Protein interactions
Protein interactions are mapped through analysis of protein complexes in pull-down experiments typically through LCMSMS analysis or after separation on gel electrophoresis. The iTRAQ reagent has proven to be particularly helpful for analysis of multiple pull-down experiments. Additional resources are available to investigators through the high-throughput yeast two-hybrid laboratory associated with the Michigan Proteome Consortium.
Peptide Synthesis
Peptide Synthesis is the most practical approach for creating biomolecules with tailored characteristics. In addition to assembly of custom peptide sequences containing the natural amino acids, the versatile methodology of peptide synthesis allows for construction of peptide molecules containing modified and unnatural structures. Synthesis of those special peptides may include posttranslational modifications such as hydroxylation, phosphorylation, sulfation and disulfide bridges as well as unnatural structures such as modified peptide bonds and cyclic conformations produced by lactame formation. A recent review focusing on synthesis of modified peptides for drug design has been published by Remmer and Fields [4]. Synthetic peptides are widely used for antibody production and as substrates for structure-function studies. For example cyclic peptides with constrained conformations may exhibit diminished enzyme susceptibility or increased potency. Labeled peptides are used in immunological assays, to study cellular uptake and localization, to determine substrate specificity, and for receptor cross-linking studies. Thus peptide synthesis is an essential tool in research on gastrointestinal peptides. Peptides are assembled by the methodology of Fmoc-Solid Phase Synthesis (Fmoc-SPPS) [5]. This synthesis methodology utilizes an orthogonal protection strategy as the base labile and acid stabile N-terminal Fluorenylmethoxycarbonyl (Fmoc; [6]) protecting group is used in combination with acid labile and base stable side-chain protecting groups on tertiary butyl (tBu)/ trityl (Trt) basis. For synthesis of linear peptides the Core uses current standard protocols based on Fmoc/tButyl solid phase synthesis. Polyethyleneglycol-polystyrene (PEG-PS) resins are used as solid supports and 20% piperidine in dimethylformamide (DMF) containing 0.1M 1-Hydroxybenzotriazole (HOBt; [7]) for Fmoc deprotection. Amino acids are incorporated using the coupling reagent 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU; [8]). DMF is used as solvent. Protocols for routine synthesis include a 15-min. Fmoc-deprotection step and two 30-min. coupling steps per amino acid addition. Following peptide chain assembly and final N-terminal Fmoc-deprotection, removal of side-chain protecting groups and release of the peptide from the resin is achieved in a single step with a cleavage mixture containing trifluoroacetic acid (TFA) and other scavenger molecules to prevent side-reactions. The concept of stepwise Fmoc-SPPS allows for incorporation of amino acids carrying special side chain protecting groups which are removed selectively in order to perform a desired reaction on solid phase without affecting the remainder of the resin-bound peptide. This is the general strategy applied to produce labeled and cyclic peptides.
Multiply labeled peptides
The most commonly used peptide labels are fluorophores and biotin. Both labels can either be introduced as prelabeled amino acid building blocks or post-synthesis on-resin with the latter approach yielding higher quality results on average [9,10]. Both labels are usually attached to peptides at the N-terminus or at the amino group of the Lysine side chain. Amino terminal labeling is performed on-resin using preformed fluorophore-succinimide esters. Side-chain labeling requires the presence of Lysine residues in the peptide sequence which carry selectively removable protecting groups e.g. Fmoc-Lys (4,4-dimethyl-2,6dioxocyclohex-1-ylidenethyl (Dde))-OH [11] Fmoc-Lys(4-Methyltrityl(Mtt))-OH [12] and Fmoc-Lys(Allyloxycarbonyl (Alloc))-OH [13]. These protecting groups are compatible with the Fmoc/tBu strategy and can also be removed independently from each other. This allows for combined use of these different Lysine derivatives in one peptide thus creating a total of four labeling sites per peptide. Steric hindrance may limit the applicability of this approach. Fluorophore-labels are introduced most efficiently using preformed succinimide esters. Biotin labeling is usually performed using standard coupling protocols.
Cyclization via lactam formation
Peptide cyclization via lactame formation can be performed in three different ways: (1) ring closure between the N-and C-termini: “head-to-tail” cyclization; (2) ring closure between the side chains of Lys/Orn and Asp/Glu: “side-chain-to-side-chain” cyclization, or (3) cycle formation of one of the side chains linked to the N-or C-terminus: “side-chain-to-end” cyclization. In all cases cyclization is performed on-resin and the amino and carboxyl groups, which form the cycle, carry compatible protecting groups to be removed in one step after assembly of the linear peptide. To achieve the side-chain-to-side-chain cyclization, Lys/Asp (Lys/Glu) pairs are incorporated into the peptide, bearing protecting groups which can be removed simultaneously by the same reagent but independently from the rest of the peptide. Possible protecting group combinations are Lys/Orn(Dde) and Asp (alpha-4-{N-[1-(4,4-dimethyl-2,6dioxocyclohexylidene)-3-methylbutyl]amino}benzyl ester (ODmab)) [14a, 14b] as well as Lys(Alloc) and Asp(Allyl(OAl)) [15] respectively. After protecting group removal, cyclization is performed on-resin using standard coupling procedures followed by standard cleavage from the resin. Head-to-tail or head to side-chain cyclization requires the C-terminal alpha-carboxyl-group, which usually links the peptide to the resin, to be available for cycle formation. This problem can be solved by positioning one of the acidic amino acids (Asp or Glu) at the C-terminus of the peptide, so the linkage to the resin is conveniently provided via the side-chain carboxyl group. Since preloaded resins with Asp/Glu side-chain linkage are not commercially available, the Fmoc-Asp/Glu derivative with unprotected side-chain carboxyl group and Dmab-protected alpha-carboxyl-group is used as the C-terminal amino acid and coupled to an amide resin using standard coupling procedures. After peptide chain assembly, Dmab removal and cyclization, the peptide is released from the amide resin. The Asp/Glu residue is converted to Asn/Gln during this step. The use of Dmab is the most practical approach. However, 4-aminobenzylester formation can occur as side reaction when using this protecting group [16].
Cyclization via disulfide bond formation
Disulfide bond formation is either performed post-assembly on-resin or post synthesis in solution using the deprotected and purified peptide as reviewed recently by Andreu and Annis [17,18]. The in-solution approach provides the opportunity to use a purified and defined starting material thus affording less oxidation by-products. The first (or only) disulfide bridge is most easily created by air oxidation of the fully deprotected peptide in aqueous basic buffers at room temperature. This reaction can be monitored by HPLC, Ellman test [19] and mass spectrometry and is usually complete in 12-36 hours. Oxidized and reduced states of one peptide usually differ in retention time, although that difference may be minimal in case of intramolecular oxidation. The preferred technique for reaction monitoring and quality control is mass spectrometry, where a mass difference of 2 amu (intramolecular oxidation) or peptide mass minus 2 amu (intermolecular oxidation) can unambiguously be detected. If intramolecular oxidation of a Cysteine-pair is desired, it is necessary to minimize the formation of side-products from intermolecular oxidation e.g. by choosing a low peptide concentration. Oligomeric side products can be separated by HPLC. If more than one disulfide bridge is to be formed, the additional Cysteine-pair is incorporated during solid phase synthesis carrying an independently removable protecting group, stable to the conditions of synthesis and resin cleavage. Thus, the second Cysteine-pair stays protected during air oxidation of the peptide and the second disulfide bond can be formed selectively. The acetamidomethyl (Acm) protecting group [20] has proven to be an excellent choice for the second Cysteine-pair, since Acm removal and Cystein oxidation occur simultaneously when access Iodine in acetic acid is reacted with the peptide in solution. The already formed first disulfide bridge stays unaffected under these conditions.
Peptides with posttranslational modifications
Posttranslational modifications are introduced into a peptide by incorporating the modified protected amino acid as preformed, protected building block in the peptide chain using standard Fmoc-SPPS protocols. These building blocks are commercially available for most applications. For synthesis of phosphorylated peptides Fmoc-Ser/Thr/Tyr(PO(OBzl)OH)-OH [21] is the amino acid derivative of choice. Protection of the side-chain phosphate moiety for Serine and Threonine prevents beta-elimination during synthesis possibly caused by the repetitive base treatments during Fmoc-deprotection. Alternatively, phosphorylation can be achieved by incorporating the selectively protected hydroxyl-amino acids (e.g. Fmoc-Ser (tert-butyldimethylsilyl (TBDMS))-OH; [22]) in the peptide with subsequent post synthesis phosphorylation on-resin after the protecting group has been removed selectively. In this post assembly approach the free resin-bound hydroxyl groups are functionalized using phosphochloridates, H-phosphonates or phosphoamidites. A recent practical review on phospho peptide synthesis including several protocols for use of these reagents has been published by Garcia-Echeverria [23].
N-terminal sequence analysis
N-terminal sequence analysis is performed on a fully automated instrument (494 HT Procise sequencer). This technique provides the N-terminal sequence tag of proteins used for protein identification. N-terminal sequencing is often used for protein tracking within a protein isolation process. Required sample amounts are in the low pico mol range and are thus 100 to 1000 –fold higher compared to mass spectrometry.
Circular Dichroism Spectroscopy
Circular Dichroism Spectroscopy (CD) is the method of choice to investigate the secondary structure of proteins in solution. This technique can also used to study conformational changes of fluorescence labeled synthetic peptides in comparison to the unlabeled peptide. CD data can provide important information for ligand -receptor binding in structure –function studies.
Consultation, proteome informatics, and training
Other services offered include consultation on experimental design and interpretation of results, training in specific techniques, instrumentation, or in proteome informatics and customized searches. The Core also provides a monthly half-day introductory workshop on proteomics and proteome informatics. This workshop, as well as our experimental protocols and SOPs can be accessed through our Web site: www.proteomeconsortium.org. A three day summer short course in proteome informatics is also offered by the Core.
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