Proteomics Facility

Services

PROTEIN IDENTIFICATION FROM DATABASES USING LC-MS/MS OF IN-GEL TRYPTIC DIGEST
Identification of proteins using nano-capillary HPLC ion trap mass spectrometry and database searching. Requires #1A.

MICRO IN-GEL TRYPTIC DIGEST
Trypsin digestion of peptides or proteins for high sensitivity used with #1
and #3.

MATRIX ASSISTED LASER DESORPTION/IONIZATION MS OF PURIFIED PEPTIDES & PROTEINS
Determine masses of purified peptides or proteins in solution. Also used to screen RP-HPLC peaks (#7) prior to Edman sequencing (#8).

MALDI MS FINGERPRINT ID OF IN-GEL DIGEST
Not routinely used due to unreliable and inaccurate protein identifications. LC-MS/MS preferred for identifications. Requires #1A.

ESI ANALYSIS OF INTACT PROTEINS
Determines mass of purified proteins not in high salt buffers.

CHARACTERIZATION OF PROTEIN MODIFICATIONS
Identification of Post-translational Modifications such as phosphorylation, acetylation by services most appropriate to sample submitted. #1 and #2 frequently used.

EDMAN IN-GEL TRYPTIC DIGEST
Trypsin digestion of proteins or peptides in polyacrylamide gels. Used for #7 and/or #8.

RP-HPLC PEPTIDE MAP OF IN-GEL TRYPTIC DIGEST & MALDI MS OF SELECTED PEAKS
Microbore reverse phase HPLC separation of in-gel tryptic peptides. Requires #6 and typically used prior to #2 and #8.

EDMAN SEQUENCE ANALYSIS OF PROTEINS OR PEPTIDES
Determination of amino acids sequences starting at the N-terminal of purified peptides or proteins. N-terminals of proteins are usually performed after blotting 1D or 2D gels to PVDF membranes. Peptides are usually from RP-HPLC (#7) after screening peaks by MALDI-MS (#2).

COMPUTER SEARCH AND ANALYSES
Analyses using specialized programs such as GPMAW or SEQUEST can be performed by facility staff upon request.

TWO-DIMENSIONAL GELS OR ELECTROBLOTTING PROTEINS FROM 1D OR 2D GELS
These services are available upon special arrangements only. Inquire.

1. PROTEIN IDENTIFICATION FROM DATABASES USING LC-MS/MS OF IN-GEL TRYPTIC DIGEST

Purpose: Identification of proteins at high sensitivity or identification of modifications on known proteins utilizing nano-capillary HPLC ion trap mass spectrometry (LC-MS/MS) and database searching. For reliable identifications, the exact protein must be in the database (same species, same isoform). 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 with a minimum of 100 femtomoles desired protein in a single gel band/spot stained with Coomassie blue R250 or Colloidal Coomassie prepared following our "Sequence-quality SDS-gel guidelines". Include a photo 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 1A.

Results: Provided the protein or a close relative is in the NCBI non-redundant (nr) database, identification should be possible. The output from the nr database search using SEQUEST consists of a printout in pdf file format of peptide sequences matched to protein(s) in the database searched by correlated predicted and observed peptide fragmentation patterns, a summary of the quality of the correlations, and the location of the peptides within the identified sequence. If your protein band is a mixture of proteins (very common), positive ID's of at least several proteins present will be obtained. This ID method may be slightly less reliable than Edman sequencing, but it is more reliable than MALDI Mass Fingerprinting. Other databases including EST and IPI databases can be searched if needed and/or de novo sequence interpretations are usually feasible (additional charges apply for more extensive analyses).

Related Info : Micro in-gel tryptic digest | Characterization of protein modifications|  (back to top)

1A. MICRO IN-GEL TRYPTIC DIGEST

Purpose: Enzymatic digestion of proteins in polyacrylamide gels is the preliminary procedure for multiple alternative services including LC-MS/MS (#1), MALDI Fingerprint ID (#3) and frequently Characterization of Post-translational Modifications (#5). The micro digest has been developed to minimize volumes while maximizing the concentration of high sensitivity samples. Porcine Modified Trypsin, the protease of choice, cleaves at the carboxyl side of lysine and arginine residues.

What is necessary: Submit an intact 1D or 2D gel stained with Coomassie blue R250 or Collodial Coomassie that was prepared following our "Sequence-quality SDS-gel guidelines". For protein identification using LC-MS/MS (#1), at least 100 femtomoles of the desired protein in a single mini-gel band or 2D gel spot is needed (band/spot must be detectable by Collodial Coomassie). After staining/destaining gel, rinse it for 1 hr with high purity water, seal in plastic bag, photograph the gel and mark the desired protein band(s) on the photo, and store at 4^o C until the entire gel is delivered to the facility.

Results: Small volume digest solution of tryptic peptides for further characterization via LC-MS/MS (#1) or MALDI Fingerprint (#3).

Related Info: Sequence-Quality SDS Gel | LC-MS/MS of In-Gel Tryptic Digest | MALDI MS Fingerprint ID | Publications | Submission Forms | Gel Shipping Instructions | (Back to Top)

2. MATRIX ASSISTED LASER DESORPTION/IONIZATION MS OF PURIFIED PEPTIDES & PROTEINS

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/ml 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)

3. MALDI MS FINGERPRINT ID of IN-GEL DIGEST (Not Routinely Used - LC-MS/MS preferred for ID's)

Purpose: MALDI MS analysis of in-gel tryptic digest after sample clean-up/concentration. MS analysis of peptide mixture can sometimes identify peptide modifications in a complementary manner to #1 and #8. Data can also be used to identify unknown proteins by searching databases. Major limitations are the exact protein must be in the database (same species, same isoform) and identification is less reliable than with LC-MS/MS or Edman sequencing.

What is necessary: Intact 1D or 2D gel with 200 femtomoles desired protein in a single band/spot stained with Coomassie blue R250 or Colloidal Coomassie, a gel photo with desired bands clearly marked, and a "Mass Fingerprinting Submission Form" with all entries completed including species of origin, MW, gel thickness, etc.

Results: Masses of some, but not all tryptic peptides in the tryptic digest with isotopic resolution and mass accuracy of about 100 ppm. If the protein is in the nr database, it is reasonably pure in the gel band used, and enough masses are produced, a protein ID is usually feasible. Data results are in pdf file format.

4. ESI ANALYSIS 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.

Related Info: Matrix Assisted Laser Desorption/Ionization MS of Purified Peptides & Proteins |Publications |Submission Form | Gel Shipping Instructions |  (Back to Top)

5. CHARACTERIZATION OF PROTEIN MODIFICATIONS

Purpose: Identification, location of post-translational modifications such as phosphorylation, acetylation, etc. This can be accomplished using several services such as in-gel digest (#1A), MALDI MS (#2), LC-MS/MS (#1) and RP-HPLC mapping (#7) 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 (#1).

Results: Print outs of description of post-translational modifications found and supporting data in pdf file format.

Related Info : Micro in-gel Tryptic Digest | LC-MS/MS of In-Gel Tryptic Digest | Matrix Assisted Laser Desorption/Ionization MS of Purified Peptides & Proteins | Sequence-Quality SDS Gel | Publications | Submission Forms |

Useful Web Links: ProFound | Mascot | (Back to Top)

6. EDMAN IN-GEL TRYPTIC DIGEST

Purpose: Enzymatic digestion of proteins in polyacrylamide gels is the preliminary procedure for several alternative subsequent services including comparative HPLC peptide maps (#7), or isolation of internal peptides for Edman sequencing (#7+#2+#8). Porcine Modified Trypsin, the protease of choice, cleaves at the carboxyl side of lysine and arginine residues.

What is necessary: Submit an intact 1D or 2D gel stained with Coomassie blue R250 or Colloidal Coomassie that was prepared following our "Sequence-quality SDS-gel guidelines". For a reliable HPLC peptide map and Edman sequencing, at least 10 pmoles of the desired protein in a total gel volume of <40 mm3 (for example, 3 replicate 2 X 7 mm bands on a 1.0 mm thick mini-gel). After staining/destaining gel, rinse it for 1 hr with high purity water, seal in plastic bag, photograph the gel and mark the desired protein band(s) on the photo, & store at 4° C until entire gel is delivered to the facility.

Results: This procedure results in highly reproducible digested peptides in solution.

7. RP-HPLC PEPTIDE MAP OF IN-GEL TRYPTIC DIGEST & MALDI MS OF SELECTED PEAKS

Purpose: High-resolution, high-sensitivity, reversed-phase chromatographic separation of in-gel tryptic digest utilizing a microbore C-18 silica 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 with 10 pmoles of target protein(s).

Results: Reproducible peptide maps monitored at 215 nm with peaks collected into microcentrifuge tubes for subsequent analysis of selected peaks (minimum of 10) by MALDI MS (#2) and Edman Sequencing (#8).

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 #6+#7+#2 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.

Related Info: Sequence-Quality SDS-Gel | Electroblotting for N-terminal Sequencing | Publications |Gel Shipping Instructions | Submission Forms | Edman in-gel Tryptic Digest |  (Back to Top)

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.COMPUTER SEARCH AND ANALYSES

Purpose: Standard database searches of MS information are included in the services described above (#1 and #3). For other applications, investigators are strongly encouraged to periodically perform their own sequence database searches and analyses since databases are constantly and rapidly expanding. Alternatively, analyses using specialized programs such as GPMAW or SEQUEST can be performed by facility staff upon request.

What is necessary: Digital file containing the sequence/data of interest. Note that at least 20 contiguous amino acids must be queried against the nr database if conformation of identity is desired.

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. Netscape - download the latest version of this web browser. Adobe Acrobat - download this popular web tool for viewing and printing web documents. (Back to Top)

10. TWO-DIMENSIONAL GELS OR ELECTROBLOTTING PROTEINS FROM 1D or 2D GELS

These services are available upon special arrangements only. Inquire.
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