The YCC Proteomics Shared Resource (PSR) brings state-of-the-art technologies to cancer research. These technologies include a wide range of mass spectrometry, protein identification and protein profiling technologies. Biophysical technologies that can be used to quantitatively study protein:protein and protein:ligand interactions complement and greatly extend the range of available proteomic analyses.
- Provide exact/accurate molecular weight information on peptides, proteins, small molecules, oligonucleotides and other biomolecules that may extend past 100,000 daltons.
- Quantitatively profile relative protein expression in multiple complex proteomes.
- Identify and locate protein post-translational modifications.
- Detect and characterize synthetic intermediates (<5 ppm error on <1,000 dalton compounds).
- Identify proteins via MS/MS analysis of in-gel tryptic digests.
- Quantitatively analyze protein:protein and protein:ligand interactions
Sample Submission FormsYale Investigators
Matrix Assisted Laser Desorption Ionization Mass Spectrometry (MALDI MS)
MALDI MS is carried out on a Waters M@LDI-L/R. The analysis is typically done in positive ionization mode and is used to obtain intact protein masses, for comparative tryptic peptide mapping, for screening of RP-HPLC isolated tryptic peptides prior to Edman sequencing, peptide biomarker discovery, and Q/C of peptides made in the Keck Peptide Synthesis Resources. Masses can be obtained on 1-500 fmol of 750-2,500 Da peptides with an average mass accuracy (external calibration) of <150 ppm (parts per million) in linear and 70 ppm in reflectron mode – which provides monoisotopic peptide masses. A variety of biomolecules can be analyzed including oligosaccharides, gangliosides, and proteins that are up to ~250,000 Da. Because of matrix interference, the lower mass limit is about 750 Da. Protein solutions in 10 μM volatile buffers can be analyzed directly while samples with higher salt concentrations require prior C4 ZipTip desalting. Learn More >>
Electrospray Ionization MS
Electrospray ionization mass spectrometry (ESI MS) is typically carried out on a Waters Q-Tof Micro. This approach is amenable to analyzing oligonucleotides, peptides, proteins, lipids, and synthetic molecules containing various functional groups. Analyses are done in either positive or negative ion mode on 100 Da to 100,000 Da compounds with a mass accuracy of ~50 ppm. Molecules such as oligos and peptides with large numbers of dibasic acids often require negative ion analysis. Salt-free samples are submitted dry or in a volatile solvent. Samples in non-volatile buffer/salt solutions cannot be directly analyzed because the buffer/salt suppresses analyte ionization. These samples are first desalted on a C18/C4 ZipTip. Typically, 10 µl of a 1-10 µM protein solution is needed for an analysis. Learn More >>
Fourier Transform-Ion Cyclotron Resonance Mass Spectrometry (FT-ICR MS) is performed on a 9.4 Tesla Bruker Apex-Qe Hybrid Qq-FTMS. The ultra high resolution, high mass accuracy, interchangeable MALDI and ESI sources, and multiple fragmentation modes of this MS platform make it the instrument of choice for exact mass determinations on small molecules, comparative LC/MS proteome profiling, detection and identification of PTMs, de novo peptide sequencing, and determining accurate protein masses (with MW 5000 to ~60,000 Da). Exact mass measurement errors are within typically <2 ppm with external calibration on standards. This allows for the determination of the elemental composition of small molecule (<450 Da). Nanoelectrospray (nESI) or MALDI-FT-ICR MS is used when the sample amount is limited (<20μL at <10μM) and not overly complex (~typically 200-500 components such as peptides). For moderately complex samples (e.g., tryptic digest of 10 proteins), nano-flow HPLC can be coupled to nESI for separation prior to MS or MS/MS analysis of the eluting peptides. With its high mass accuracy and multiple fragmentation techniques (i.e., InfraRed MultiPhoton Dissociation (IRMPD), Electron Captured Dissociation (ECD), and Collision Induced Dissociation (CID)), FT-ICR-MS is well suited for identifying PTMs. Depending upon the PTM, ECD fragmentation often retains the covalently bound modification so the site of modification can be elucidated. IRMPD fragmentation is complementary to ECD in that it often (e.g., phosphorylation) shows loss of a neutral mass corresponding to the PTM. Learn More >>
MS/MS Protein Identification
Protein identifications are carried out using multiple technologies. Proteins from 1D/2D gel electrophoresis, or relatively simple in-solution protein mixtures (<100 proteins) are digested with trypsin followed by LC-MS/MS (Waters Q-Tof API). Data-Dependent Acquisition is performed which switches automatically from MS to MS/MS modes when the total ion current increases by a pre-determined factor. To optimize fragmentation, a collision energy ramp is used which adjusts for the different peptide mass sizes and charge states (e.g., preference is given to doubly and triply charged species over singly charged species because the former fragment better). MS/MS spectra are searched against protein databases using Mascot. A protein is considered identified when Mascot lists its score as significant (P<0.05) and at least 2 peptides match to the same protein. Protein amounts that are less than the minimum 5-10 fmol required for LC-MS/MS or the protein sequence not being in the database searched are common reasons for the inability to identify an unknown protein. Protein identification of gel spots obtained from differential (fluorescence) 2D gel electrophoresis (DIGE) is currently performed on an Applied Biosystems (AB) Model 4700 MALDI-Tof/Tof mass spectrometer. The NCBInr database is routinely searched, with species-specific database searches also available (note that databases other than human, rat and mouse may be subject to the database usage fee). Search parameters include a peptide mass accuracy of 50ppm, a MS/MS fragment accuracy of 0.2Da and variable modifications of carbamidomethyl cysteine (cysteines are reduced and alkylated prior to DIGE using dithiothreitol and iodoacetamide), and methionine oxidation. Mascot database searching is used with a combined peptide mass fingerprint and MS/MS search. DIGE protein identification results are posted to the Yale Protein Expression Database (YPED) for online viewing of identifications relative to spot number and gel image. Learn More >>
Quantitative Protein Profiling
Differential (fluorescence) 2D gel electrophoresis (DIGE)has the ability to globally view changes in protein expression and to separate complex protein mixtures by orthogonally combining ionic charge and molecular size. DIGE overcomes the reproducibility challenges of conventional 2D gel electrophoresis (2DGE) by running the control and experimental samples in the same gel. Up to three mass and charge-matched, spectrally resolvable fluorescent dyes (Cy2, Cy3 and Cy5) are utilized to label a control and two different protein samples in vitro prior to DIGE. Each sample is then imaged separately but can be perfectly overlaid without the usual challenges with regards to spot alignment. The confidence and margin of error with which protein changes between samples can be detected are thus substantially improved so that changes can be detected that are as small as 1.3-fold for large volume spots. The sensitivity of detection is comparable to silver staining (e.g., 0.25 ng to 1 ng/spot). Protein spots found to be differentially expressed are robotically excised, digested with trypsin, and identified as outlined above. Learn More >>
Multiplexed Isobaric Tagging Technology (iTRAQ™)uses a tagging reagent which provides the possibility for both relative and absolute quantitation of a control vs three other samples. The reagent has: 1) a reactive group which uses amine-reactive chemistry to label N-terminal and lysine side-chains, 2) a balance group which changes in concert with the reporter mass to maintain a total mass of 145 and 3) a reporter group which gives strong signature ions in MS/MS spectra that are used to determine ratios between the four different iTRAQ labeled peptides. MS/MS analysis of an iTRAQ-labeled peptide produces one of four possible reporter groups, which have a mass of 114, 115, 116, or 117, depending upon which one of the four iTRAQ reagents was used to modify the parent protein. The relative areas of the reporter ion peaks provide the expression ratios of the parent proteins Protein identifications and quantitation are viewed on line using YPED. Learn More >>
Isotope Coded Affinity Tag (ICAT)protein profiling: the ICAT reagent reacts with free cysteines in proteins in cell lysates and contains an affinity tag (biotin) and an acid cleavage site (for biotin removal). The tag is isotopically coded for a “heavy” (i.e., 9 x 13C = 236 amu) or “light” (i.e., 9 x 12C = 227 amu) tag, with 9 amu mass difference. After reacting 100µg control protein extract with the “light” tag and an equal amount of experimental protein extract with the “heavy” tag; the samples are mixed, trypsin digested, and cysteine-containing peptides are affinity purified on an avidin column. While ICAT has the advantage of simplifying complex extracts, it cannot detect proteins that lack cysteine. The purified, cysteine containing tryptic peptides are cleaved with trifluoroacetic acid to remove the biotin group (thus aiding MS/MS) and parent proteins are identified using our AB Q-STAR LC-MS/MS. Quantitation is carried out with AB ProQuant software with the results then being posted on YPED. Learn More >>
2DLC Protein Profiling
Separates complex protein extracts using chromatofocusing followed by RP HPLC separation on a highly automated Beckman Coulter PF2D system. This approach is suitable for comparative profiling of crude cell lysates, serum, and other samples and has the ability to separate each sample into ~500 fractions. Prior to analyzing sera, highly abundant proteins are depleted with the ProteomeLab IgY12 kit. Because RP-HPLC-isolated proteins are in a volatile solvent, they are well suited for MW analysis and "top-down" protein identification by FT-ICR MS. Samples for 2DLC can be in a variety of buffers, but they must be free of ionic detergents such as SDS. To identify and quantify isolated proteins found to be differentially regulated in two or more samples that have each been subjected to 2DLC, the RP-HPLC fractions of interest from each 2DLC run are digested with trypsin, label with iTRAQ, and analyzed by LC-MS/MS. Learn More >>
Multi-dimensional Protein Identification Technology (MudPIT)
Used to identify the proteins that make up the proteome in a cell or tissue extract. Protein extracts are digested with trypsin prior to cation exchange fractionation into 10-20 fractions. Each fraction is subjected to RP-LC-MS/MS with Mascot database searching. The results are concatenated using Peptide and Protein Prophet software from the Institute for Systems Biology and are viewed using YPED. Quantitative profiling can be done with iTRAQ (see above) or in vivo metabolic labeling with 14N/15N.
Sites of protein phosphorylation can be identified by analyzing [332P]-labeled tryptic peptides isolated from [32P]-proteins, using MALDI-MS mapping techniques, and/or FT-ICR MS analysis. With non-radiolabeled phosphoproteins, we have most often used MALDI-MS mapping techniques followed by LC-MS/MS analysis of the putative phosphopeptides. One approach is to analyze the tryptic digest using both linear and reflectron modes. In "linear" MALDI-MS, the phosphorylated peptide has a mass that is +80 Da compared to that of any tryptic peptide. In "reflectron" MALDI-MS the phosphorylated peptide will be unique in that it often will show characteristic fragmentation resulting from loss of phosphate during post-source decay. By comparing the linear to the reflectron MALDI-MS spectra it is possible to tentatively assign which peptide mass corresponds to the phosphorylated peptide. This identification is confirmed and the site of phosphorylation is determined by MS/MS sequencing. An alternative approach is to compare the MALDI-MS spectra of the tryptic digest before and after treatment with alkaline phosphatase and then look for the characteristic -80 Da loss due to removal of phosphate. If no loss is observed in the MALDI-MS spectrum, or to further confirm the identity of a phosphopeptide and the site of phosphorylation, an InfraRed MultiPhoton Dissociation (IRMPD) or Electron Capture Dissociation (ECD) MS/MS spectrum can be acquired on our FT-ICR MS. With IRMPD a characteristic loss of 98 Da is observed for a serine/threonine phosphorylated peptide. The complementary fragmentation by ECD, where the post-translational modification (PTM) is not cleaved from the peptide backbone, can validate the site of phosphorylation by MS/MS sequencing. Since phosphorylation alters protein isoelectric points, DIGE can be used to separate the often small amounts of phosphoproteins from their non-phosphorylated counterparts. Since other enrichment approaches (e.g., immobilized metal affinity chromatography (IMAC)) are somewhat peptide specific, new phosphopeptide enrichment approaches are being developed in our lab using metal oxides. Other types of post-translational modifications often can be identified using similar approaches of MALDI-MS mapping and FT-ICR analysis. Learn More >>
Biophysics services are aimed at quantitative characterization of interactions between biomolecules and enable determination of the 1) assembly states of each interacting partner, 2) binding affinity, 3) kinetics, 4) stoichiometry, and 5) thermodynamics of the interaction.
Size Exclusion Chromatography coupled with laser light scattering (SEC/LS), refractive index (RI), and UV detection provides a universal approach for determination of the molar mass and oligomeric state in solution of native as well as glycosylated proteins or membrane proteins solubilized in non-ionic detergents. Since glycosylated proteins and protein-detergent complexes show anomalous behavior on SEC, they present a challenge in terms of determining their molar mass and oligomeric state in solution. In the SEC/LS/RI/UV approach, SEC serves solely as a fractionation step while the responses from the three detectors are utilized to calculate the molar mass for the polypeptide portion of the native or modified protein. The amount of sugar, lipid, or detergent bound to the polypeptide chain can also be estimated from the SEC-UV/LS/RI analysis. Learn More >>
Dynamic Light Scattering (DLS)
Measures time fluctuations of light intensity caused by motions of macro-molecules in solution. How rapidly the intensity fluctuates over time is represented by an autocorrelation function. Analysis of the correlation function decay as a function of short time intervals can be used to evaluate the translational diffusion coefficient, D. The hydrodynamic radius can then be calculated from D. Learn More >>
Isothermal Calorimetry (ITC)
Provides a universal tool for monitoring binding because it measures the heat/enthalpy change associated with all binding interactions. During ITC a “ligand” in a syringe is titrated into a cell containing a solution of the "macromolecule". As the two molecules interact, heat is released or absorbed. When the macromolecule becomes saturated with ligand, the heat signal diminishes to only the background heat of dilution. The analysis is performed at equilibrium, in solution phase, and without any labeling or need for a fluorescent or other probe. ITC provides direct information about the thermodynamics of binding. The enthalpy of binding is measured directly and when combined with the equilibrium binding constant (also measured directly) yields the entropy change. ITC can also determine the mass ratio between interacting species; however, the absolute stoichiometry of the final complex needs to be verified by other methods. Learn More >>
Spectrofluorometer: SLM 8000 (FL)
A photon counting, computer controlled spectrometer equipped with a double grating excitation and a single grating emission monochromator.
The state-of-the-art SX.18MV Stopped-Flow Reaction Spectrofluorometer is equipped with a low-volume, stopped-flow cell in which microliter volumes of sample are mixed and the resulting absorbance or fluorescence changes monitored within milliseconds of mixing.
Surface Plasmon Resonance (SPR) Sensor
The BiaCore Biosensor is a universal tool for studying macromolecular interactions in a label-free state. SPR detects binding in real time by monitoring changes in mass concentration at the chip surface; the association and dissociation rate constants are determined directly from the reaction traces. Samples ranging from small molecules to crude extracts, lipid vesicles, viruses, bacteria and eukaryotic cells can be studied in real-time, without labels and with little or no sample preparation. Learn More >>