Proteomics Facility

Helpful Information

1.Gel Shipping Instructions guidelines

2. Sequence-Quality SDS-Gel Guidelines

3. Electroblotting for N-terminal Sequencing of the Intact Protein

4. MALDI Mass Fingerprint ID

5. Web Links

6. Publications

1. GEL SHIPPING INSTRUCTIONS

Please note that we require the entire gel be sent to us. We strongly recommend the gel be packaged and shipped per the instructions noted below. Gels that are improperly packaged will break in transit and cannot be used for analysis.

Gel(s) that are not securely packaged prior to shipment usually are severely damaged or destroyed during shipment due to abuse of the package by the shipper. We have found the following packaging technique to be successful (gel arrives intact).

1. Take a picture or make a photocopy of the gel, mark on the copy the bands of interest and include this with the submission sheet in a separate plastic bag in the shipping box but packed outside the ice (see further instructions regarding the use of ice).
   
   
2. Place the gel in a watertight ziplock plastic bag after draining all excess water from the gel. The gel should remain moist with only a few drops of water in the bag. The gel should not be floating in the bag.
   
   
3. Cut 2 pieces of cardboard slightly larger than the size of the bag containing the gel. Place the gel and bag between the cardboard covers and tape all sides.
   
   
4. Securely wrap the cardboard/gel+bag/cardboard sandwich with bubblewrap.
   
   
5. Place the whole bubble-wrapped sample on top of or near the top of loose wet ice in a styrofoam shipping container and send to us by the overnight courier of your choice. DO NOT use icepacks or pack bubble-wrapped sample between layers of wet ice as this may freeze the gel resulting in breakage.
   
   
6. Send an email message to either dreim@wistar.org or kspeicher@wistar.org (not the website) the day the package is shipped. We will alert you if we do not receive the package by 4pm the following day. Do not ship packages on a Friday afternoon for Saturday or other weekend/holiday delivery. SHIP TO:

Wistar Proteomics Facility
Attn: Kaye D. Speicher
3601 Spruce St., Room 154
Philadelphia, Pa. 19104-4268

PVDF bound samples (for N-terminal sequence only) can be shipped at room temperature using the typical cardboard FedEx letter envelop after the membrane has been placed in a plastic ziplock bag following complete drying. Enclose a copy of the membrane with the bands of interest marked and a completed "N-TERMINAL SEQUENCE SAMPLE SUBMISSION FORM".

2. SEQUENCE-QUALITY SDS-GEL GUIDELINES

Special Precautions and Changes to Laemmli Gels for MS or Sequence Analysis

Preparing Samples and Gels
1. Maximize protein concentration in the gel. Mini Gels (1.0 mm thick) are preferred unless the higher resolution of a full size gel is needed.

2. Select a gel concentration that will give a sharp, tight band for the protein of interest, preferably with an Rf between 0.3 and 0.7. Use either high quality pre-made gels such as Invitrogen NuPAGE gels following the manufacturer guidelines or follow the guidelines below.

3. Use electrophoresis reagents and solvents of highest purity (we use BioRad).

4. Filter gel solutions (0.2 mM), except running buffer, and store at 4ºC, except SDS (R.T). Store solutions no more than one month.

5. Solubilize samples using 2X or 5X solubilizing buffer containing sucrose or glycerol. DO NOT USE UREA!

6. Do not heat samples excessively. If higher temperatures are needed to properly solubilize sample, minimize as much as possible; e.g., 1-2 min at 80-90ºC.

7. If the gel has been made in-house, let the completely cast gel including the polymerized stacker stand submerged in MilliQ water for at least 24 hrs but, no more than 48 hrs at room temperature prior to use.

8. Add 11.4 mg/L (0.1 mM) thioglycolate to the upper chamber buffer prior to electrophoresis, unless non-reducing conditions are needed.

9. Follow manufacturers' guidelines when using pre-cast gels.

Separation and Staining of Gels for In-Gel Digestions

1. Avoid excessive heating (>25ºC) during electrophoresis.

2. DO NOT run Bromophenol Blue dye front off the bottom of the gel.This will not improve the separation.

3. After electrophoresis immediately stain with either a) or b): 

a) Coomassie Blue R-250 30-60 minutes.Destain 1-4 hours maximum (background does not need to be completely clear).Rinse in high quality water (such as MilliQ) for 1 hour.

 b) Invitrogen Colloidal Coomassie Blue G-250 (follow manufacture's guidelines for staining and destaining).

4. Seal in ziploc bag and store at 4ºC (add only a few drops of water, gel should not be floating).

5. The entire gel must be sent to us; follow "Gel Shipping Instructions".

6. Include xerox or photo of gel with desired band(s) for digestion clearly marked on the xerox or photo.

General Gel Comments

- Urea decomposes readily to form cyanate, which will modify your protein at higher temperatures and pH's above 7. Eliminate urea from your protocol when doing gels for sequence. The use of urea for isoelectrofocusing gels as a first step in 2D gels requires extra care to minimize protein modifications and proteins with pI's > 7 will probably be blocked during isofocusing.

- Heating during sample prep or electrophoresis may contribute to chemical modifications of your protein.

- Conditioning the gel, including the stacking gel, for 24-48 hours at room temperature prior to electrophoresis helps to eliminate free radicals.

- Thioglycolate scavenges free radicals and oxidants left in gel.

- DO NOT run the tracking dye off the gel! Instead, change the gel % if needed for optimal results.

3. ELECTROBLOTTING FOR N-TERMINAL SEQUENCING OF THE INTACT PROTEIN

Note: Gel must be electrotransferred immediately after electrophoresis. Do not use CAPS as a transfer buffer. Do NOT stain gel prior to transfer.

1. Use a 10 mM Tris, 100 mM glycine, 10% MeOH transfer buffer for most proteins. Do not add any detergents to transfer buffer.

2. For transfer, use a high retention PVDF membrane (not nitrocellulose). Only the following PVDF membranes will be accepted: BioRad Sequi-Blot, Applied Biosystems ProBlott, or Millipore Immobilon-Psq (Do NOT use Immobilon P.)

3. Pre-wet PVDF membrane in 100% MeOH for 10+ seconds, then in transfer buffer for 5+ minutes. Keep submerged.

4. When preparing transfer "sandwich" make sure that there are no air bubbles between the membranes and the gel, and do not allow the PVDF to dry out.

5. Electrotransfer 3 hrs for sequencing. After transfer, rinse membrane with a large volume (approx 500mls) of MilliQ water three times for five minutes each.

6. Stain PVDF membrane with Amido Black for 60 seconds, destain with 5% Acetic Acid about 5 minutes and rinse thoroughly (about three times) with MilliQ water. Air dry then seal in plastic in plastic bag. Store at -20ºC.

7. Include Xerox or Photo of blot with desired band clearly marked on the Xerox or Photo. Do not write directly on the Blott.

8. Stain gel after transfer with Coomassie Blue to detect proteins that did not transfer.

Electroblotting Comments

- We found 10 mM Tris, 100 mM glycine, 10% MeOH (pH is about 8.3 without adjustment) transfer buffer to be the most effective in terms of transfer yield and sequence quality. Most other common transfer buffers produce similar results. Use of high pH buffers such as CAPS buffer may contribute to deamidation of asparagines and other chemical modifications and are not recommended.

- High retention PVDF membranes from BioRad, ABI, and Millipore have higher protein binding than Immobilin P and can dramatically improve recovery of some proteins as well as sequence performance. There may be other membranes that work just as well but we know the above mentioned membranes will work without causing instrumentation problems. Therefore, only the following membranes will be accepted for sample submission: BioRad Sequi-Blot, Applied Biosystems ProBlott, or Millipore Immobilon-Psq. For example: BioRad Sequi-Blot - catalog #162-0182, Applied Biosystems ProBlott - catalog #400994 or Millipore Immobilon-Psq - catalog #ISEQ20200.

- PVDF membranes are very hydrophobic and must be pre-wetted in MeOH for several seconds. Equilibration in transfer buffer is necessary and requires several minutes.

- Even very small air bubbles may produce large artifacts on the membrane. Keep membranes wet during the entire assembly of the transfer sandwich. Local drying of the membrane will cause artifacts and the hydrophobic PVDF membranes dry out quickly.

- Amido Black is the preferred PVDF stain. It is slightly more sensitive than Coomassie Blue and does not cause undesired artifacts during sequencing

4. MALDI MASS FINGERPRINT ID

Background MALDI mass fingerprinting is a protein identification method from in-gel tryptic digestions. This differs in several ways from typical in-gel digestion/microbore HPLC/Edman sequencing identifications.The digestion is still done under the same conditions. For mass fingerprinting, a small aliquot of the digest (5-10%) is usually cleaned up and concentrated. Alternatively, satisfactory signals can sometimes be obtained from very low level samples (< 1 pmol protein in the gel) by using the entire sample. MALDI MS analysis is then performed on the sample, preferably using an internal standard unless the signals are too weak (not enough sample). Mass analysis must be performed on a mass spectrometer with delayed extraction capacity in reflectron mode so that the monoisotopic peptide masses can be resolved and measured with an accuracy better than 200 ppm. The resulting masses are then entered into a database search program (either PROFOUND or MS-FIT or MASCOT). Initially a broad search encompassing the whole database (all species) and the entire mass range is performed. The search is then narrowed based on the species and MW information given by the investigator.

Interpretation and Use of Database Search Results

Protein identifications based entirely on a mass fingerprint ID should be used with caution. Due to the nature of this analysis method, false positive and false negative identifications are possible. If sufficient sample is available, we strongly recommend sequencing at least one peptide by conventional Edman sequencing to verify the ID.

We have indicated a confidence level on the report based strictly on quality and characteristics of mass data, and search results in comparison to other ID’s performed in our lab. However, you, the research investigator can best evaluate whether a positive ID really makes sense, or alternatively if a "non-significant" hit in the results list is highly likely and hence worth verifying by alternative means such as Western blotting.

The report includes a list of possible proteins and their probability scores. It also gives the % of the protein covered by matched peptide masses. The PROFOUND program reports a Z value, which is a probability parameter; a Z value > 1.65 is considered significant at a 95% confidence level as determined by the software developers, i.e., the identified protein match is most likely not random. The lower the Z score, the higher the chance that the match could occur randomly.

To supplement the computer prediction of significance, (Z value > 1.65 for p > 0.05) we have added our overall assessment of protein ID confidence based on additional criteria and our experience with other samples. Our evaluation considers the degree of protein coverage relative to protein size and MS spectra quality, effects of search parameter changes on search results, protein mass fit and pI fit if known, number of modifications and incomplete cleavages in matched data, etc.

Confidence level and overall assessment of probability - i.e., likelihood that the experimental sample matches the identified protein or an alternatively spliced form of this protein or a proteolytic product of this protein or a very highly homologous form of this protein;

5.USEFUL WEB LINKS

BLAST
Entrez
ExPaSy
ProFound
Netscape
Adobe
MASCOT
MS-FIT

6. PUBLICATIONS

1. Speicher, D.W., Mozdzanowski, J., Beam, K., and Chen, D. 1990. Electrotransfer and sequence enhancements improve low picomole sequence analysis using PVDF membranes without glass fiber filters. J. Prot. Chem. 9:254-255.

2. Speicher, D.W., Grant, G.A., Niece, R.L., Blacher, R.W., Fowler, A.V., and Williams, K.R. 1990. Design, characterization, and results of ABRF-89SEQ: A test sample for evaluating protein sequencer performance in protein microchemistry facilities. In: Current Research in Protein Chemistry (J. Villafranca, ed.), Academic Press, pp. 159-166.

3. Mozdzanowski, J. and Speicher, D. W. 1990. Quantitative electrotransfer of proteins from polyacrylamide gels onto PVDF membranes. In: Current Research in Protein Chemistry (J. Villafranca, ed.), Academic Press, pp. 87-94.

4. Yuksel, K.U., Grant, G.A., Mende-Mueller, L.M., Niece, R.L., Williams, K.R., and Speicher, D.W. 1991. Protein Sequencing from Polyvinylidenedifluoride Membranes: Design and characterization of a test sample (ABRF-90SEQ) and evaluation of results. In: Techniques in Protein Chemistry II (J. Villafranca, ed.). Academic Press, NY, pp. 151-162.

5. Niece, R., Ericsson, L., Fowler, A., Smith, A., Speicher, D., Crabb, J., and Williams, K. 1991. Amino acid analysis and sequencing - What is state-of-the-art? In: Methods in Protein Sequence Analysis 1990 (H. Jornvall and J.-O. Hoog, eds.). Birkhauser Verlag, Basel, Berlin, pp. 133-141.

6. Reim, D.F., Hembach, P., and Speicher, D.W. 1992. Evaluation of the Blott cartridge for enhanced gas phase sequencing at maximum sensitivity. In: Techniques in Protein Chemistry III (R. Angeletti, ed.). Academic Press, NY, pp. 53-60.

7. Crimmins, D.L., Grant, G.A., Mende-Mueller, L.M., Niece, R.L., Slaughter, C., Speicher, D.W. and Yuksel, K.U. 1992. Evaluation of protein sequencing core facilities: design, characterization, and results from a test sample (ABRF-91SEQ). In: Techniques in Protein Chemistry III (R. Angeletti, ed.). Academic Press, NY, pp. 35-51.

8. Mozdzanowski, J., Hembach, P., and Speicher, D.W. 1992. High yield electroblotting onto PVDF membranes from polyacrylamide gels. Electrophoresis, 13:59-64.

9. Mozdzanowski, J. and Speicher, D.W. 1992. Microsequence analysis of electroblotted proteins. I. Comparison of electroblotting recoveries using different types of PVDF membranes. Anal. Biochem. 207:11-18.

10. Reim, D.F., and Speicher, D.W. 1992. Microsequence analysis of electroblotted proteins. II. Comparison of sequence performance on different types of PVDF membranes. Anal. Biochem. 207:19-23.

11. Reim, D.F. and Speicher, D.W. 1993. High-sensitivity gas phase sequence analysis of proteins on PVDF membranes using short cycle times. Anal. Biochem. 214:87-95.

12. Best, S., Reim, D.F., Mozdzanowski, J., and Speicher D.W. 1993. High sensitivity peptide sequence analysis using in situ proteolysis on high retention PVDF membranes and a biphasic reaction column sequencer. In: Techniques in Protein Chemistry V (J. Crabb, ed.) Academic Press, NY. pp.205-213.

13. Reim, D.F. and Speicher, D.W. 1994. A method for high performance sequence analysis using PVDF membranes with a biphasic reaction column sequencer. Anal. Biochem. 216:213-222.

14. Speicher. D. W. 1994. Methods and strategies for the sequence analysis of proteins on PVDF membranes. Methods 6:262-273.

15. Rush, J., Andrews, P.C., Crimmins, D.L., Gambee, J., Grant, G.A., Mische, S.M., and Speicher, D.W. 1994. A synthetic peptide for evaluating protein sequencing capabilities: Design of ABRF-93SEQ and results. In:
Techniques in Protein Chemistry V (J. Crabb, ed.) Academic Press, NY. pp. 133-141.

16. Ursitti, J., Mozdzanowski, J., and Speicher, D.W. 1995. Electroblotting from 1D and 2D gels. In: Current Protocols in Protein Science. John Wiley & Sons, Inc. New York. pp. 10.7.1-14.

17. DeSilva, T.M., Ursitti, J.A., and Speicher, D.W. 1995. Protein detection in gels using fixation. In: Current Protocols in Protein Science. John Wiley & Sons, Inc. New York. pp. 10.5.1-12.

18. Ursitti, J.A., DeSilva, T.M., and Speicher, D.W. 1995. Protein detection in gels without fixation. In: Current Protocols in Protein Science. John Wiley & Sons, Inc. New York. pp. 10.6.1-14.

19. Best, S. and Speicher, D.W. 1995. Detection of proteins on blot membranes. In: Current Protocols in Protein Science. John Wiley & Sons, Inc. New York. pp. 10.8.1-7.

20. Mozdzanowski, J., Best, S., and Speicher, D.W. 1995. Two dimensional gel electrophoresis methods. In: Current Protocols in Protein Science. John Wiley & Sons, Inc. New York. pp. 10.4.1-30

21. Speicher, D.W., Reim, D.F., and Speicher, K.D. 1995. High sensitivity protein sequence analysis using in situ protease digestion on PVDF membranes, biphasic cartridge sequencing and MALDI mass spectrometry. In: Molecular Biology: Current Innovations and Future Trends, Part 2. (H. Griffin, ed.). Horizon Scientific Press, Wymondham, UK, pp. 19-37.

22. Williams, K., Hellman, U., Kobayashi, R., Lane, W. Mische, S., and Speicher, D. 1997. Internal protein sequencing of SDS PAGE-separated proteins: A collaborative ABRF study. In Techniques in Protein Chemistry VIII (D.R. Marshak, ed.) Academic Press, NY, pp. 99-109.

23. Speicher, D.W. and Reim, D. 1997. N-terminal sequence analysis. In: Current Protocols in Protein Science. John Wiley & Sons, Inc. New York. pp. 11.10.1-38.

24. Harper, S.L., and Speicher, D.W. 1997. Expression, isolation and protease cleavage of GST fusion proteins in E. coli. In: Current Protocols in Protein Science. John Wiley & Sons, Inc. New York. pp. 6.6.1-9.

25. Harper, S., Mozdzanowski, J., and Speicher, D.W. 1998. Two-dimensional gel electrophoresis. In: Current Protocols in Protein Science. John Wiley & Sons, Inc. New York, NY. pp. 10.4.1-36.

26. Speicher, D.W. 1998. Characterization of protein primary structure. In: Characterization of Biotechnology Pharmaceutical Products. Development of Biological Standards. Karger. Basel Vol. 96:25-26.

27. Mische, S., Hellman, U., Speicher, D., and Williams, K. 1999. Internal protein sequencing. In: Encyclopedia of Bioprocess Technology. John Wiley & Sons, Inc. New York. pp. 2099-2102.

28. Begg, G.E, Harper, S.L., and Speicher, D.W. 1999. Characterizing recombinant proteins using HPLC gel filtration and MALDI mass spectrometry. In: Current Protocols in Protein Science. John Wiley & Sons, Inc. New York, NY. pp. 7.10.1-15.

29. Begg, G.E. and Speicher, D.W. 1999. Mass spectrometry detection and reduction of disulfide adducts between reducing agents and recombinant proteins with highly reactive cysteines. J. Biomolecular Techniques 10:17-20.

30. Zuo, X. and Speicher, D.W. 2000. Quantitative evaluation of protein recoveries in two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 21:3035-3047.

31. Zuo, X. and Speicher, D.W. 2000. A method for global analysis of complex proteomes using sample prefractionation by solution isoelectrofocusing prior to two-dimensional electrophoresis. Anal. Biochem. 284 (2):266-278.