The Wistar Institute :: Proteomics Facility

Overview

Research Programs

•	Gene Expression and Regulation
•	Immunology
•	Molecular and Cellular Oncogenesis

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Shared Facilities

Proteomics Facility

Services

1. IN-GEL PROTEASE DIGESTION
Trypsin digestion of peptides or proteins for high sensitivity
protein identifications (used for #2, #3, and #5).

2. LC-MS/MS PROTEIN IDENTIFICATIONS
Identification of proteins from tryptic peptides using nano-capillary
LC-ion trap MS and database searching. Requires #1.

3. GEL/LC-MS/MS – COMPREHENSIVE ANALYSIS OF A
SUBPROTEOME OR PROTEOME.
Identification (and potentially quantitation) of large numbers of proteins
by analyzing an entire gel lane using #1 and #2.

4. MudPIT (LC/LC-MS/MS) ANALYSIS – COMPREHENSIVE ANALYSIS
OF A SUBPROTEOME OR PROTEOME.
Identification of large numbers of proteins from in-solution protease digests.

5. REVERSE PHASE MICROBORE HPLC PEPTIDE MAPPING
Reverse phase HPLC separation of tryptic peptides with UV detection.
Typically used prior to #6 and #8.

6. MALDI MS
Determine masses of purified peptides or proteins in solution including
screening HPLC peaks (#5) prior to Edman sequencing (#8).

7. ESI OF INTACT PROTEINS
High resolution, highly accurate mass determination of purified proteins
that are less than 40 to 80 kDa.

8. EDMAN SEQUENCE ANALYSIS OF PROTEINS OR PEPTIDES
N-terminal amino acid sequencing of purified peptides or proteins in solution
or proteins that have been electroblotted from 1D or 2D gels onto PVDF membranes. Peptides are usually from RP-HPLC (#5) after screening peaks by MALDI-MS (#6).

9. POST-TRANSLATIONAL MODIFICATION (PTM) IDENTIFICATIONS
Identification of PTMs such as phosphorylation, methylation, and acetylation.
Multiple combinations of above services and specialized methods are typically required.

10. CUSTOM DATABASES
Create custom project-specific databases.

11. CUSTOM DATA ANALYSIS
Project specific, specialized computational analyses such as analysis of PTMs, quantitative comparisons, etc. .

Services - Details
1. IN-GEL PROTEASE DIGESTION

Purpose: Enzymatic digestion of proteins in polyacrylamide gels is the preliminary procedure for multiple alternative services including LC-MS/MS (#2), Gel/LC-MS/MS (#3), Peptide mapping (#5), and PTM analysis (#9). The micro digest used here has been developed to minimize volumes and maximize peptide yields which are typically >80%. Porcine Modified Trypsin, the protease of choice, cleaves at the carboxyl side of lysine and arginine residues. For special applications such as analysis of PTMs, other proteases can be used as well.

What is necessary: Submit an intact 1D or 2D gel stained with Colloidal Coomassie (preferred) or MS-compatible silver stain that was prepared following our "Sequence-quality SDS-gel guidelines". For simple protein identification using LC-MS/MS (#2), as little as 5 femtomoles of the desired protein in a single mini-gel band or 2D gel spot will typically yield identification of at least the most abundant protein present; that is, even barely detectable silver stained bands will result in identifications, although sequence coverage will be quite limited when near the limit of detection. After staining/destaining gel, rinse it for 1 hr with high purity water, seal in plastic bag, photograph or scan the gel and mark the desired protein band(s) on the gel image (annotated image in powerpoint preferred). Store gel at 4^o C until the entire gel is delivered to the facility.

Results: Small volume tryptic digest of purified protein or complex protein mixtures for further characterization via LC-MS/MS (#2), etc. Purified proteins can invariably be identified from barely detectable Colloidal Commassie bands/spots and most barely detectable silver stained bands/spots can usually be identified with limited sequence coverage. Gel slices with intense Collodial Coomassie staining from complex proteomes can yield confident identification of >100 proteins for each slice and up to several thousand proteins from an entire gel lane.

Related Info: Sequence-Quality SDS Gel | LC-MS/MS Protein Identifications | Publications | Submission Forms | Gel Shipping Instructions | (Back to Top)

2. LC-MS/MS PROTEIN IDENTIFICATIONS

Purpose: Identification of proteins at high sensitivity from tryptic peptides (or peptides produced by alternative proteases) or identification of locations of chemical modifications or PTMs on purified proteins. Nano-capillary reverse phase HPLC is interfaced directly with hybrid linear ion trap mass spectrometers (LC-MS/MS) for maximum sensitivity. Proteins are identified by comparing MS/MS spectra to sequence databases, typically using SEQUEST. For reliable identifications, the exact protein sequence must be in the database. Highly homologous proteins can sometimes be identified if a substantial portion of the tryptic peptides have identical sequences.

What is Necessary: An intact 1D or 2D gel (see #1 above for details and detection limits/expected results). Include an image of the gel with bands/spots of interest clearly marked and a completed "MS/MS Submission Form". Complete all entries on the submission form including the species of sample origin, MW, gel thickness, type, etc. Requires service #1.

Results: High confidence (>99%) identifications are expected for proteins identified by two or more peptides provided that the protein or a very close relative is in the sequence database used for the search. However, it should be noted that a substantial number of cases, the MS/MS matches to peptide sequences for an identified protein actually only define a family of homologous proteins. That is, there may be insufficient sequence coverage to unambiguously define a single protein. Hence, in a multi-species search, the protein from an incorrect species may be listed or an isoform or splice variant may be listed that is homologous but not identical to the isoform present in the analyzed sample. The SEQUEST output from the database search consists of a list in pdf file format of peptide sequences matched to protein(s). For complex protein mixtures, the list of peptide identifications may not be listed to reduce report length, but a full list of peptides can be readily provided. It should be noted that all identifications of peptides and proteins based on MS/MS data are identified based correlation of observed peptide masses and fragmentation patterns with those predicted from sequences in the database. Even when data is stringently filtered, there is very small but finite possibility that any individual peptide identification is incorrect.

3. GEL/LC-MS/MS – COMPREHENSIVE ANALYSIS OF A SUBPROTEOME OR PROTEOME

Purpose: Identify as many proteins as possible in a subproteome or an entire proteome. Examples of subproteomes include: analysis of a purified large macromolecular complex, a highly purified organelle, a phosphotyrosine sub-proteome, etc. Examples of proteomes include: a cell line, the secretome of a cell line grown in serum-free media, etc.

What is necessary: An intact 1D SDS mini-gel (see #1 above for details and detection limits/expected results). Depending upon the number of fractions that are to be analyzed and the complexity of the sample, the optimal electrophoresis distance will vary. For example, if the sample is relatively simple (<100 proteins) the SDS gel should only be run until the bromophenol tracking dye reaches either 1.0 or 2.0 cm. It is strongly recommended that you consult with the Facility Manager before preparing the sample to be submitted. Include a protein standard on the gel that is separated from the sample lane by one to two blank lanes. Submit an image of the gel with bands/spots of interest and standard M.W.s clearly marked and a completed "MS/MS Submission Form". Complete all entries on the submission form including the species of sample origin, MW, gel thickness, type, etc.

Results: A large number of high confidence (>99%) identifications are expected where the proteins have been identified by two or more peptides provided that the genome for the species analyzed is essentially complete. Integrating the protein identifications from multiple LC-MS/MS analyses requires extensive specialized computational analysis which will vary depending upon proteome complexity. Further extensive critical evaluation and data refinement by the investigator is generally recommended prior to publication of results.

4. MudPIT (LC/LC-MS/MS ANALYSIS – COMPREHENSIVE ANALYSIS OF A SUBPROTEOME OR PROTEOME

This service is analogous to Gel/LC-MS/MS expect that the subproteome or proteome is digested with a protease in solution rather than separation on a gel and the digest is then fractionated by ion exchange chromatography prior to LC-MS/MS analysis as described above. This method typically yields less protein identifications and sequence coverage compared with Gel/LC-MS/MS when the same number of fractions and LC-MS/MS runs are used. In addition, there is no information concerning approximate M.W. of the identified proteins. Hence, Gel/LC-MS/MS is recommended for most subproteome and proteome analyses. Nonetheless, MudPIT can be used as needed in special situations.

5. REVERSE PHASE MICRO BORE HPLC PEPTIDE MAPPING

Purpose: High-resolution, reversed-phase chromatographic separation of in-gel tryptic digest utilizing a microbore C-18 column prior to Edman Sequencing (#8) or for comparative mapping of two or more gel bands. Comparative mapping is the most quantitative method for detecting differences and similarities between different bands on a gel such as: chemical or post-translational modifications, proteolytic processing, etc.

What is necessary: Tryptic digest performed in this facility of gel bands containing at least 10 pmoles of target protein(s).

Results: Reproducible peptide maps monitored at 215 nm with peaks collected into pre-cleaned microcentrifuge tubes for subsequent analysis of selected peaks (minimum of 10) by MALDI MS (#6) and Edman Sequencing (#8).

6. MALDI MASS SPECTROMETRY

Purpose: Determine masses of purified peptides or proteins in solution. MALDI mass analysis is feasible over a wide mass range and can help identify the extent of post-translational modifications such as oxidation, glycosylation and phosphorylation. It is the method of choice to check integrity of synthetic peptides, peptides from fractions of HPLC separations and integrity of recombinant proteins. While some salts and buffers are tolerated, it may be necessary to 'cleanup' the sample prior to analysis if buffer components suppress the mass signal.

What is necessary: Purified peptides for routine analysis should be between 0.5-5 pmoles/µl in 0.1% TFA (sample may also contain up to 50% acetonitrile). Proteins should be 10 pmoles/µl, preferably in 20 mM NH4HCO3.It is especially important to avoid the following: Phosphate buffer >20 mM, most detergents >0.1% or SDS >0.01%, alkali metal salts >1M, glycerol >1%, Tris buffer >10 mM, guanidine or urea >1 M, or sodium azide >1 mM.

Results: Printouts of the masses found and a print out of the standard used for the calibration of the instrument in pdf file format.

Related Info : Publications | Submission Forms | (Back to Top)

7. ESI OF INTACT PROTEINS

Purpose: Determine the mass of a purified protein in solution.

What is necessary: Purified intact protein in ESI compatible buffer such as 0.05% Acetic acid. It must be free of detergents such as SDS, any salts, etc. Concentration of 10 pmol/µl preferred. Samples can be cleaned up and/or concentrated if needed for an additional charge.

Results: Print out of convoluted/deconvoluted spectra with mass of protein found and controls in pdf file format.

8. EDMAN SEQUENCE ANALYSIS OF PROTEINS OR PEPTIDES

Purpose: Determination of amino acid sequences starting at the N-terminal of purified peptides or proteins.

What is necessary: About 10-50 pmoles is preferred, although 1 pmol of a protein or peptide can yield useful information. Internal sequences from proteins (minimum 10 pmoles protein in gel) can be obtained from either 1D or 2D SDS polyacrylamide gels by in-gel trypsin digestion, HPLC separation and MS analysis of selected fractions by this facility (services #1, #5 + #6 above). For N-terminal sequencing of intact proteins, the proteins should be electrotransferred to a PVDF membrane from 1D or 2D gels and stained with amido black.

Results: If a protein or peptide N-terminal is postranslationally or artifactually modified, no sequence will be obtained. The entire sequence of most tryptic peptides can be determined if > 1 pmole is analyzed. Typically, 10-40 residues of proteins or large fragments with unmodified N-terminals can be determined depending upon protein size and amount sequenced. Major applications include: 1) determine the N-terminal of recombinant proteins and fragments of natural or recombinant proteins used in structure-function studies; 2) determine the sites and nature of post-translational modifications; and 3) identify an unknown protein by searching sequence databases. Data results are in pdf file format.

Useful Web Links: BLAST - Search protein and nucleotide databases to identify sequence similarities. EMBL - an alternative website that enables searching of a non-redundant database to identify sequence similarities.Entrez - integrated access to biomedical literature, nucleotide and protein sequences, 3-D protein structures, complete genomes and population study data.

9. POST-TRANSLATIONAL MODIFICATION IDENTIFICATIONS

Purpose: Identification, location of post-translational modifications such as phosphorylation, acetylation, etc. This can be accomplished using several services such as in-gel digest (#1), MALDI MS (#6), LC-MS/MS (#2) and RP-HPLC mapping (#5) depending on the type of project.

What is necessary: Project dependent (inquire). Typically, an intact 1D or 2D gel stained with Coomassie blue R250 or Collodial Coomassie that was prepared following our "Sequence-quality SDS-gel guidelines", same as for LC-MS/MS (#2).

10. CUSTOM DATABASES

Purpose: Some projects require specialized databases such as multiple isoforms of a protein of interest or a recombinant protein that does not exactly match the sequence in the database due to cloning related changes. These user provided sequences are combined with sequences of expected or likely contaminants, some of which might be experiment specific. Such sequences are used when searching for PTMs, etc.

What is necessary: Digital file containing the sequence/data of interest.

11. CUSTOM DATA ANALYSIS

Purpose: Standard database searches from individual LC-MS/MS runs and filtering out low confidence hits are included in the services described in #2 above. More extensive data analyses including searching for PTMs or other non-standard modifications, combining multiple datasets from subproteome/proteome analyses, and quantitative comparisons of multiple samples either globally or for specific peptides are available from the facility upon request. Other specialized applications include using programs such as GPMAW can be performed by facility staff upon request.

What is necessary: Digital file(s) containing the sequence/data of interest.

Results: Dependent upon program used and purpose of analysis.

Useful Web Links: BLAST - Search protein and nucleotide databases to identify sequence similarities. EMBL - An alternative website that enables searching of a non-redundant database to identify sequence similarities. Mascot - a search engine that uses mass spectrometry data to identify proteins from sequence databases. Entrez - integrated access to biomedical literature, nucleotide and protein sequences, 3-D protein structures, complete genomes and population study data. ExPaSy - a list of tools (web sites) for proteomic type analysis. Adobe Acrobat - download this popular web tool for viewing and printing web documents. (Back to Top)

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